CN117098542A - Method for storing stem cells - Google Patents
Method for storing stem cells Download PDFInfo
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- CN117098542A CN117098542A CN202180094235.XA CN202180094235A CN117098542A CN 117098542 A CN117098542 A CN 117098542A CN 202180094235 A CN202180094235 A CN 202180094235A CN 117098542 A CN117098542 A CN 117098542A
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- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
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Abstract
Described herein are methods and systems for preparing a sample of stem cells for direct infusion. Some of the methods and systems described herein include low temperature storage of stem cells. The methods and systems described herein can supplement transport of stem cells across long distances, thereby preparing stem cells for direct infusion upon receiving the cells.
Description
Cross reference
The present application claims the benefit of U.S. provisional application No. 63/128,780, filed on 12/21/2020, which is incorporated herein by reference in its entirety.
Background
Induction of immune tolerance with solid organ and vascularized composite allograft (vascular composite allograft) is a gold standard for transplantation medicine. Inducing immune tolerance to mismatched grafts would eliminate the need for lifelong immunosuppression associated with serious adverse consequences such as renal failure, cancer, and infection. Currently, the most promising tolerance-inducing means is by establishing a mixed chimeric state by transplanting donor hematopoietic stem cells; however, the hybrid chimera approach has not achieved long-lasting immune tolerance in preclinical or clinical trials using most solid organs or vascularized composite allografts (vascular composite allotransplant) (VCA) except tolerogenic organs such as kidneys.
Mesenchymal stem (stromal) cells (MSCs) have been identified as potentially useful adjuvants for Stem Cell Transplantation (SCT) for promoting hybrid chimeras and complementary peripheral immunomodulatory functions, as well as Solid Organ Transplantation (SOT). However, prior to clinical transformation of these promising therapeutic cells, there are a number of unresolved problems to be solved. The major obstacle is the source of MSCs, which are rare in all organizations and require invasive procedures for acquisition. Low abundance requires extensive expansion in culture to produce sufficient numbers for human administration. It has been observed in a clinical setting that the extent of amplification is inversely related to the outcome. It has further been observed that cryopreservation (cryopreservation) of MSCs is also inversely related to the results. Thus, there is a need for a low temperature (hypothermic) storage method for MSC populations that preserves their viability to enable their distribution for clinically useful applications.
Disclosure of Invention
In one aspect, provided herein is a method for preparing stem cells for infusion, the method comprising: (a) Providing a cryopreserved population of cells comprising the stem cells; (b) Warming the stem cells to a first temperature and maintaining the stem cells at the first temperature for a first period of time; and (c) changing the first temperature to a second temperature and maintaining the stem cells at the second temperature for a period of time.
One aspect of the present disclosure is a method for preparing stem cells for infusion, the method comprising: providing a cryopreserved population of cells comprising the stem cells; heating the stem cells to a first temperature and maintaining the stem cells at the first temperature for a first period of time; and changing the first temperature to a second temperature and maintaining the stem cells at the second temperature for a period of time. In some embodiments, the first temperature is greater than 0 ℃. In some embodiments, the second temperature is less than 40 ℃. In some embodiments, the second temperature is low temperature. In some embodiments, the first period of time or the period of time is less than one week. In some embodiments, the first period of time or the period of time is less than 5 days. In some embodiments, the first period of time or the period of time is less than 2 days. In some embodiments, the first period of time or the period of time is less than 1 day. In some embodiments, the first period of time or the period of time is less than 12 hours. In some embodiments, the first period of time or the period of time is less than 6 hours. In some embodiments, the first period of time or the period of time is less than 2 hours. In some embodiments, the method further comprises passaging the stem cells prior to providing a cryopreserved population of cells comprising the stem cells. In some embodiments, the method further comprises passaging the stem cells at least once prior to providing a cryopreserved population of cells comprising the stem cells. In some embodiments, the method further comprises passaging the stem cells at least 2 times prior to providing a cryopreserved cell population comprising the stem cells. In some embodiments, the method further comprises passaging the stem cells at least 4 times prior to providing a cryopreserved cell population comprising the stem cells. In some embodiments, the method further comprises culturing the stem cells at the first temperature at about 10,000 cells/cm 2 to about 50,000 cells/cm 2 before changing the first temperature to the second temperature and maintaining the stem cells at the second temperature for a period of time. In some embodiments, the method further comprises culturing the stem cells at the first temperature at about 32,000 cells/cm 2 before changing the first temperature to the second temperature and maintaining the stem cells at the second temperature for a period of time. In some embodiments, the method further comprises packaging the stem cells in a volume comprising about 10 x 102 living cells/mL to about 10 x 1010 living cells/mL and maintaining the stem cells at the second temperature prior to changing the first temperature to the second temperature and maintaining the stem cells at the second temperature for a period of time. In some embodiments, the method further comprises packaging the stem cells in a volume comprising about 10 x 106 viable cells/mL and maintaining the stem cells at the second temperature prior to changing the first temperature to the second temperature and maintaining the stem cells at the second temperature for a period of time. In some embodiments, the method further comprises packaging the stem cells in a volume comprising less than 10mL, 9mL, 8mL, 7mL, or 6mL prior to changing the first temperature to the second temperature and maintaining the stem cells at the second temperature for a period of time. In some embodiments, the method further comprises packaging the stem cells in a volume comprising 5mL prior to changing the first temperature to the second temperature and maintaining the stem cells at the second temperature for a period of time. In some embodiments, the stem cells are maintained at a first temperature in a medium comprising one or more amino acids. In some embodiments, the medium comprising one or more amino acids further comprises one or more vitamins. In some embodiments, the medium comprising one or more amino acids further comprises table 1. In some embodiments, the first period of time is a period of time in which the stem cells are not multiplied. In some embodiments, the first time period is a time period in which the stem cells undergo one or more population doublings. In some embodiments, the stem cells are maintained at less than 80% confluency relative to the cell culture storage medium during the first period of time or the period of time. In some embodiments, the stem cells are maintained in a wash medium at a second temperature, wherein the wash medium comprises an electrolyte formulation, human Serum Albumin (HSA), or both. In some embodiments, the stem cells comprise vertebrae adherent mesenchymal stem cells (vBA-MSC), vertebral bone marrow mesenchymal stem cells (vBM-MSC), or both. In some embodiments, the stem cells are derived from cadavers. In some embodiments, the stem cells comprise at least about 10,000 to about 300,000CFU-F/1 million viable cells. In some embodiments, the stem cells comprise from less than about 1% to about 10% cd45+ cells.
Another aspect of the present disclosure is a method for preparing a sample of stem cells for direct infusion, the method comprising: providing a cell culture comprising a sample of stem cells, wherein the cell culture is cryopreserved and passaged; thawing the cell culture; the cell culture was maintained at less than 40 ℃. In some embodiments, the cell culture is passaged at least once. In some embodiments, the method further comprises, after thawing the cell culture, not doubling the cell culture. In some embodiments, the method further comprises, after thawing the cell culture, maintaining the cell culture to undergo one or more population doublings. In some embodiments, the method further comprises, after thawing the cell culture, maintaining the cell culture at less than 80% confluency relative to the cell culture storage medium. In some embodiments, the cell culture is maintained in a medium comprising one or more amino acids. In some embodiments, the medium comprising one or more amino acids further comprises one or more vitamins. In some embodiments, the medium comprising one or more amino acids further comprises table 1. In some embodiments, the cell culture is passaged at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, or at least 10 times. In some embodiments, the cell culture is cryopreserved in a cryopreservation medium, wherein the cryopreservation medium comprises an electrolyte formulation, human Serum Albumin (HSA), dimethyl sulfoxide (DMSO), or any combination thereof. In some embodiments, the cryopreservation media comprises about 1% to about 5% HSA. In some embodiments, the cryopreservation media comprises about 2.5% HSA. In some embodiments, the cryopreservation media comprises about 1% to about 10% DMSO. In some embodiments, the cryopreservation medium comprises about 5% DMSO. In some embodiments, the electrolyte formulation is a boy pulse force A (Plasmalyte A). In some embodiments, thawing occurs at about 30 ℃ to about 40 ℃. In some embodiments, thawing occurs at about 37 ℃. In some embodiments, the method further comprises re-suspending the cell culture in a wash medium, wherein the wash medium comprises an electrolyte formulation, human Serum Albumin (HSA), or both; and maintaining the cell culture at below room temperature. In some embodiments, the method further comprises, prior to (c), re-suspending the cell culture in a wash medium, wherein the wash medium comprises an electrolyte formulation, human Serum Albumin (HSA), or both. In some embodiments, the rinse medium is fresh. In some embodiments, the wash medium comprises about 1% to about 5% HSA. In some embodiments, the wash medium comprises about 2.5% HSA. In some embodiments, the electrolyte formulation is boy vein a. In some embodiments, the cell culture is maintained at a temperature below room temperature for at least about 30 minutes. In some embodiments, the cell culture is maintained at room temperature for at least 12 hours. In some embodiments, the cell culture is maintained at below room temperature for at least 24 hours. In some embodiments, the cell culture is maintained at room temperature for at least 48 hours. In some embodiments, the cell culture is maintained at less than 37 ℃. In some embodiments, the cell culture is maintained at less than 35 ℃. In some embodiments, the cell culture is maintained at less than 30 ℃. In some embodiments, the method further comprises, after (b), maintaining the cell culture under conditions of low temperature. In some embodiments, the cell culture is maintained at less than 25 ℃. In some embodiments, the cell culture is maintained at less than 20 ℃. In some embodiments, the cell culture is maintained at about 2 ℃ to about 8 ℃. In some embodiments, the method further comprises culturing the cell culture at about 10,000 cells/cm 2 to about 50,000 cells/cm 2. In some embodiments, the method further comprises culturing the cell culture at about 32,000 cells/cm 2. In some embodiments, the method further comprises packaging the cell culture in a volume comprising about 10 x 102 living cells/mL to about 10 x 1010 living cells/mL. In some embodiments, the method further comprises packaging the cell culture in a volume comprising about 10 x 106 viable cells/mL. In some embodiments, the method further comprises packaging the cell culture in a volume comprising less than 10mL, 9mL, 8mL, 7mL, or 6 mL. In some embodiments, the method further comprises packaging the cell culture in a volume comprising 5 mL. In some embodiments, the sample of stem cells comprises at least about 70% viable cells. In some embodiments, the cell culture comprises vertebrae adherent mesenchymal stem cells (vBA-MSC), vertebral bone marrow mesenchymal stem cells (vBM-MSC), or both. In some embodiments, the cell culture is derived from cadavers. In some embodiments, the cell culture comprises at least about 10,000 to about 300,000CFU-F/1 million viable cells. In some embodiments, the cell culture comprises from less than about 1% to about 10% cd45+ cells.
Another aspect of the present disclosure is a method for preparing a sample of stem cells for direct infusion, the method comprising: providing a cell culture comprising a sample of stem cells, wherein the cell culture is cryopreserved and passaged; thawing the cell culture; the cell culture was maintained at above 0 ℃. In some embodiments, the sample of stem cells comprises at least about 70% viable cells after maintaining the cell culture above 0 ℃.
Another aspect of the present disclosure is a method for preparing a sample of stem cells for direct infusion, the method comprising: providing a cell culture comprising a sample of stem cells, wherein the cell culture is cryopreserved and passaged; thawing the cell culture; maintaining the cell culture at below room temperature; wherein the sample of stem cells comprises at least about 70% viable cells. In some embodiments, the sample of stem cells comprises at least about 70% viable cells after maintaining the cell culture above 0 ℃.
Another aspect of the present disclosure is a method for preparing a sample of stem cells for direct infusion, the method comprising: providing a cell culture comprising a sample of stem cells, wherein the cell culture is cryopreserved and passaged; thawing the cell culture; maintaining the cell culture at below room temperature; wherein the sample of stem cells comprises at least about 70% viable cells. In some embodiments, the sample of stem cells comprises at least about 70% viable cells after maintaining the cell culture above 0 ℃.
Another aspect of the present disclosure is a method for preparing a sample of stem cells for direct infusion, the method comprising: providing a cell culture comprising a sample of stem cells, wherein the cell culture is cryopreserved and passaged; thawing the cell culture; maintaining the cell culture at below room temperature; wherein the sample of stem cells comprises from less than about 1% to about 10% cd45+ cells. In some embodiments, the sample of stem cells comprises less than about 5% cd45+ cells. In some embodiments, the sample of stem cells comprises from less than about 1% to about 10% cd35+ cells. In some embodiments, the sample of stem cells comprises less than about 5% cd35+ cells.
Another aspect of the present disclosure is a method for preparing a sample of stem cells for direct infusion, the method comprising: providing a cell culture comprising a sample of stem cells, wherein the cell culture is cryopreserved and passaged; thawing the cell culture; maintaining the cell culture at below room temperature; wherein the sample of stem cells comprises at least about 90% to about 99% cd90+ cells. In some embodiments, the sample of stem cells comprises at least about 95% cd90+ cells.
Another aspect of the present disclosure is a method for preparing a sample of stem cells for direct infusion, the method comprising: providing a cell culture comprising a sample of stem cells, wherein the cell culture is cryopreserved and passaged; thawing the cell culture; maintaining the cell culture at below room temperature; wherein the sample of stem cells comprises at least about 90% to about 99% cd105+ cells. In some embodiments, the sample of stem cells comprises at least about 95% cd105+ cells. In some embodiments, the sample of stem cells comprises at least about 70% viable cells after maintaining the cell culture above 0 ℃.
Another aspect of the present disclosure is a method for preparing a sample of stem cells for direct infusion, the method comprising: providing a cell culture comprising a sample of stem cells, wherein the cell culture is cryopreserved and passaged; thawing the cell culture; maintaining the cell culture at below room temperature; wherein the sample of stem cells comprises at least about 10,000 to about 300,000CFU-F/1 million viable cells. In some embodiments, the sample of stem cells comprises at least about 10,000CFU-F/1 million viable cells. In some embodiments, the sample of stem cells comprises at least about 70% viable cells after maintaining the cell culture above 0 ℃. In some embodiments, the sample of stem cells comprises vertebrae adherent mesenchymal stem cells (vBA-MSC), vertebral bone marrow mesenchymal stem cells (vBM-MSC), or both. In some embodiments, the method further comprises, after thawing the cell culture, not doubling the cell culture. In some embodiments, the method further comprises, after thawing the cell culture, maintaining the cell culture to undergo one or more population doublings. In some embodiments, the method further comprises, after thawing the cell culture, maintaining the cell culture at less than 80% confluency relative to the cell culture storage medium. In some embodiments, the cell culture is maintained in a medium comprising one or more amino acids. In some embodiments, the medium comprising one or more amino acids further comprises one or more vitamins. In some embodiments, the medium comprising one or more amino acids further comprises table 1. In some embodiments, the cell culture is passaged at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, or at least 10 times. In some embodiments, the cell culture is cryopreserved in a cryopreservation medium, wherein the cryopreservation medium comprises an electrolyte formulation, human Serum Albumin (HSA), dimethyl sulfoxide (DMSO), or any combination thereof. In some embodiments, the cryopreservation media comprises about 1% to about 5% HSA. In some embodiments, the cryopreservation media comprises about 2.5% HSA. In some embodiments, the cryopreservation media comprises about 1% to about 10% DMSO. In some embodiments, the cryopreservation medium comprises about 5% DMSO. In some embodiments, the electrolyte formulation is boy vein a. In some embodiments, thawing occurs at about 30 ℃ to about 40 ℃. In some embodiments, thawing occurs at about 37 ℃. In some embodiments, the method further comprises re-suspending the cell culture in a wash medium, wherein the wash medium comprises an electrolyte formulation, human Serum Albumin (HSA), or both; and maintaining the cell culture at below room temperature. In some embodiments, the method further comprises, prior to (c), re-suspending the cell culture in a wash medium, wherein the wash medium comprises an electrolyte formulation, human Serum Albumin (HSA), or both. In some embodiments, the rinse medium is fresh. In some embodiments, the wash medium comprises about 1% to about 5% HSA. In some embodiments, the wash medium comprises about 2.5% HSA. In some embodiments, the electrolyte formulation is boy vein a. In some embodiments, the cell culture is maintained at a temperature below room temperature for at least about 30 minutes. In some embodiments, the cell culture is maintained at room temperature for at least 12 hours. In some embodiments, the cell culture is maintained at below room temperature for at least 24 hours. In some embodiments, the cell culture is maintained at room temperature for at least 48 hours. In some embodiments, the cell culture is maintained at less than 37 ℃. In some embodiments, the cell culture is maintained at less than 35 ℃. In some embodiments, the cell culture is maintained at less than 30 ℃. In some embodiments, the method further comprises, after thawing the cell culture, maintaining the cell culture at a low temperature. In some embodiments, the cell culture is maintained at less than 25 ℃. In some embodiments, the cell culture is maintained at less than 20 ℃. In some embodiments, the cell culture is maintained at about 2 ℃ to about 8 ℃. In some embodiments, the method further comprises culturing the cell culture at about 10,000 cells/cm 2 to about 50,000 cells/cm 2. In some embodiments, the method further comprises culturing the cell culture at about 32,000 cells/cm 2. In some embodiments, the method further comprises packaging the cell culture in a volume comprising about 10 x 102 living cells/mL to about 10 x 1010 living cells/mL. In some embodiments, the method further comprises packaging the cell culture in a volume comprising about 10 x 106 viable cells/mL. In some embodiments, the method further comprises packaging the cell culture in a volume comprising less than 10mL, 9mL, 8mL, 7mL, or 6 mL. In some embodiments, the method further comprises packaging the cell culture in a volume comprising 5 mL. In some embodiments, the sample of stem cells comprises at least about 70% viable cells. In some embodiments, the cell culture comprises vertebrae adherent mesenchymal stem cells (vBA-MSC), vertebral bone marrow mesenchymal stem cells (vBM-MSC), or both. In some embodiments, the cell culture is derived from cadavers.
In some embodiments, the first temperature is greater than 0 ℃. In some embodiments, the second temperature is less than 40 ℃. In some embodiments, the second temperature is low temperature.
In some embodiments, the period of time is less than one week. In some embodiments, the period of time is less than 5 days. In some embodiments, the period of time is less than 2 days. In some embodiments, the period of time is less than 1 day. In some embodiments, the period of time is less than 12 hours. In some embodiments, the period of time is less than 6 hours. In some embodiments, the period of time is less than 2 hours.
In some embodiments, the stem cells are passaged prior to (a). In some embodiments, the stem cells are passaged at least once prior to (a). In some embodiments, the stem cells are passaged at least 2 times prior to (a). In some embodiments, the stem cells are passaged at least 4 times prior to (a).
In some embodiments, the method further comprises, prior to (c), at the first temperature, at about 10,000 cells/cm 2 Up to about 50,000 cells/cm 2 Culturing the stem cells. In some embodiments, the method further comprises, prior to (c), at the first temperature, at about 32,000 cells/cm 2 Culturing the stem cells.
In some embodiments, the method further comprises, prior to (c), contacting the stem cells with a cell culture medium comprising about 10 x 10 cells 2 From about 10X 10 cells/mL 10 A volume of living cells/mL, and maintaining the stem cells at the second temperature. In some embodiments, the method further comprises, prior to (c), contacting the stem cells with a medium comprising about 10 x 10 6 A volume of living cells/mL, and maintaining the stem cells at the second temperature.
In some embodiments, the method further comprises, prior to (c), packaging the stem cells in a volume comprising less than 10mL, 9mL, 8mL, 7mL, or 6 mL. In some embodiments, the method further comprises, prior to (c), packaging the stem cells in a volume comprising 5 mL.
In another aspect, provided herein is a method for preparing a sample of stem cells for direct infusion, the method comprising: (a) Providing a cell culture comprising a sample of said stem cells, wherein said cell culture is cryopreserved and passaged; (b) thawing the cell culture; and (c) maintaining the cell culture at less than 40 ℃.
In some embodiments, the cell culture is passaged at least once. In some embodiments, after (b), the cell culture is not multiplied. In some embodiments, the cell culture is passaged at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, or at least 10 times.
In some embodiments, the cell culture is maintained at less than 37 ℃. The method of any one of the preceding claims, wherein the cell culture is maintained at less than 35 ℃. In some embodiments, the cell culture is maintained at less than 30 ℃. In some embodiments, after (b), the cell culture is maintained under conditions of low temperature. In some embodiments, the cell culture is maintained at less than 25 ℃. In some embodiments, the cell culture is maintained at less than 20 ℃. In some embodiments, the cell culture is maintained at about 2 ℃ to about 8 ℃.
In some embodiments, the cell culture is cryopreserved in a cryopreservation medium, wherein the cryopreservation medium comprises an electrolyte formulation, human Serum Albumin (HSA), dimethyl sulfoxide (DMSO), or any combination thereof. In some embodiments, the cryopreservation media comprises about 1% to about 5% HSA. In some embodiments, the cryopreservation media comprises about 2.5% HSA. In some embodiments, the cryopreservation media comprises about 1% to about 10% DMSO. In some embodiments, the cryopreservation media comprises about 5% DMSO. In some embodiments, the electrolyte formulation is boy vein a.
In some embodiments, the thawing occurs at about 30 ℃ to about 40 ℃. In some embodiments, the thawing occurs at about 37 ℃.
In some embodiments, the cell culture is maintained at below room temperature for at least about 30 minutes. In some embodiments, the cell culture is maintained at room temperature for at least 12 hours. In some embodiments, the cell culture is maintained at below room temperature for at least 24 hours. In some embodiments, the cell culture is maintained at room temperature for at least 48 hours.
In some embodiments, the method further comprises, prior to (c), re-suspending the cell culture in a wash medium, wherein the wash medium comprises an electrolyte formulation, human Serum Albumin (HSA), or both. In some embodiments, the method further comprises, prior to (c), re-suspending the cell culture in a wash medium, wherein the wash medium comprises an electrolyte formulation, human Serum Albumin (HSA), or both. In some embodiments, the rinse medium is fresh. In some embodiments, the wash medium comprises about 1% to about 5% HSA. In some embodiments, the wash medium comprises about 2.5% HSA. In some embodiments, the electrolyte formulation is boy vein a.
In some embodiments, the method further comprises, prior to (c), at about 10,000 cells/cm 2 Up to about 50,000 cells/cm 2 Culturing the cell culture. In some embodiments, the method further comprises, prior to (c), at about 32,000 cells/cm 2 Culturing the cell culture.
In some embodiments, the method further comprises, prior to (c), culturing the cells to comprise about 10 x 10 2 From about 10X 10 cells/mL 10 A volume of viable cells/mL. In some embodiments, the method further comprises, prior to (c), culturing the cells to comprise about 10 x 10 6 A volume of viable cells/mL.
In some embodiments, the method further comprises, prior to (c), packaging the cell culture in a volume comprising less than 10mL, 9mL, 8mL, 7mL, or 6 mL. In some embodiments, the method further comprises, prior to (c), packaging the cell culture in a volume comprising 5 mL.
In some embodiments, after (c), the sample of stem cells comprises at least about 70% viable cells.
In another aspect, provided herein is a method for preparing a sample of stem cells for direct infusion, the method comprising: (a) Providing a cell culture comprising a sample of said stem cells, wherein said cell culture is cryopreserved and passaged; (b) thawing the cell culture; and (c) maintaining the cell culture above 0 ℃.
In some embodiments, after (c), the sample of stem cells comprises at least about 70% viable cells.
In another aspect, provided herein is a method for preparing a sample of stem cells for direct infusion, the method comprising: (a) Providing a cell culture comprising a sample of said stem cells, wherein said cell culture is cryopreserved and passaged; (b) thawing the cell culture; and (c) maintaining the cell culture at below room temperature;
wherein the sample of stem cells comprises at least about 70% viable cells.
In some embodiments, after (c), the sample of stem cells comprises at least about 70% viable cells.
In another aspect, provided herein is a method for preparing a sample of stem cells for direct infusion, the method comprising: (a) Providing a cell culture comprising a sample of said stem cells, wherein said cell culture is cryopreserved and passaged; (b) thawing the cell culture; and (c) maintaining the cell culture at below room temperature;
wherein the sample of stem cells comprises from less than about 1% to about 10% cd45+ cells.
In some embodiments, the sample of stem cells comprises less than about 5% cd45+ cells.
In some embodiments, the sample of stem cells comprises from less than about 1% to about 10% cd34+ cells. In some embodiments, the sample of stem cells comprises less than about 5% cd34+ cells.
In another aspect, provided herein is a method for preparing a sample of stem cells for direct infusion, the method comprising: (a) Providing a cell culture comprising a sample of said stem cells, wherein said cell culture is cryopreserved and passaged; (b) thawing the cell culture; and (c) maintaining the cell culture at below room temperature;
wherein the sample of stem cells comprises at least about 90% to about 99% cd90+ cells.
In some embodiments, the sample of stem cells comprises at least about 95% cd90+ cells.
In another aspect, provided herein is a method for preparing a sample of stem cells for direct infusion, the method comprising: (a) Providing a cell culture comprising a sample of said stem cells, wherein said cell culture is cryopreserved and passaged; (b) thawing the cell culture; and (c) maintaining the cell culture at below room temperature;
Wherein the sample of stem cells comprises at least about 90% to about 99% cd105+ cells.
In some embodiments, the sample of stem cells comprises at least about 95% cd105+ cells.
In some embodiments, after (c), the sample of stem cells comprises at least about 70% viable cells.
In another aspect, provided herein is a method for preparing a sample of stem cells for direct infusion, the method comprising: (a) Providing a cell culture comprising a sample of said stem cells, wherein said cell culture is cryopreserved and passaged; (b) thawing the cell culture; and (c) maintaining the cell culture at below room temperature; wherein the sample of stem cells comprises at least about 10,000 to about 300,000CFU-F/1 million viable cells.
In some embodiments, the sample of stem cells comprises at least about 10,000CFU-F/1 million viable cells.
In some embodiments, after (c), the sample of stem cells comprises at least about 70% viable cells.
In some embodiments, the sample of stem cells comprises vertebrae adherent mesenchymal stem cells (vBA-MSC), vertebral bone marrow mesenchymal stem cells (vBM-MSC), or both.
In another aspect, provided herein is a method for preparing a sample of vertebral adherent mesenchymal stem cells (vBA-MSCs) for direct infusion, the method comprising: (a) Providing a cell culture comprising a sample of said stem cells, wherein said cell culture is cryopreserved and passaged; (b) thawing the cell culture; and (c) maintaining the cell culture at below room temperature.
Incorporation by reference
All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.
Drawings
The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:
FIG. 1 shows the number of fibroblast colony forming units (CFU-F) in 100 ten thousand cells per plating at 24 hours and 48 hours after plating.
Detailed Description
In general, patients undergoing life-saving procedures for Stem Cell Transplantation (SCT), solid Organ Transplantation (SOT), and Vascularized Composite Allograft (VCA) have a long-felt need for immunosuppressive drugs that prevent the patient's body from rejecting organ or tissue grafts. Unfortunately, immunosuppressive drugs have been found to be associated with serious medical problems such as cancer, renal failure, and infection. Thus, new medical procedures that reduce or overcome the need for immunosuppressive drugs are highly desirable. Previously deceased donor Vertebral Body (VB) Bone Marrow (BM) SCT from matched diseased organ donors has been found to induce immune tolerance after SOT and VCA. This suggests that immunosuppression induction from transplanted VB BM STC is dependent on the establishment of mixed chimeras by SCT. MSCs have also been shown to promote engraftment of stem cells in BM and induce foxp3+ TREG cell expansion, as well as have additional synergistic immunomodulatory properties. Furthermore, MSCs have been demonstrated to have potent immune system modulating activity, thereby facilitating extensive testing in clinical trials of various diseases associated with immune system dysfunction. Unfortunately, MSCs are very difficult to obtain and must then be grown in the laboratory to produce the sufficient number required to treat a typical adult patient. To address this shortcoming, the systems and methods disclosed herein provide a desirable supplement to existing MSC and BM sources and extraction methods that can produce sufficient quantities of MSCs and BMs for the applications described above.
The resulting processing steps mean that further preparation of MSCs is required prior to direct infusion into a subject. In practice, the site where the direct infusion of MSCs is performed has no personnel trained in the preparation of MSC samples. Thus, there is a need for methods and systems for sample preparation of MSCs for direct infusion, wherein MSCs do not require further processing steps upon arrival at the site of direct infusion.
The methods and systems disclosed herein enable the cryopreservation of large numbers of MSCs from human vertebrae, known as (vBA-MSCs) and centrum marrow mesenchymal stem cells (vBM-MSCs), to allow their distribution for clinical use.
The methods and systems disclosed herein are further capable of enabling sample preparation of MSCs for direct infusion. In some embodiments, the methods and systems for sample preparation described herein do not require further processing steps at the site of infusion. In some embodiments, the methods and systems described herein provide a sample of MSCs, wherein the MSCs are present in the sample comprising a quality suitable for direct infusion into a subject. Some of these qualities suitable for direct infusion into a subject include, but are not limited to: dilutions and/or concentrations suitable for direct infusion; a survival rate of at least 70%; wherein the MSC present in the sample results in at least 20,000 colony forming units (per 1X 10 6 Individual cells); which is a kind ofThe cells present in the sample comprise at least 90% cd105+ cells.
The methods and systems disclosed herein are further capable of effecting sample preparation of MSCs, thereby providing at least some or all of the above qualities after cryopreservation of MSCs. The methods and systems disclosed herein are further capable of effecting sample preparation of MSCs, thereby providing at least some or all of the above qualities after thawing cryopreserved MSCs and culturing MSCs for a period of time. Finally, the methods and systems disclosed herein further enable sample preparation of MSCs to provide at least some or all of the above qualities after thawing cryopreserved MSCs, culturing the MSCs for a period of time, and then maintaining the MSCs at a temperature different from the temperature at which the MSCs were cultured after thawing. In some embodiments, this temperature change is cooling. In some embodiments, the MSCs are maintained at a low temperature after thawing until direct infusion into a subject.
Recovery of MSC from processed bone
In another feature of the systems and methods disclosed herein, a method for preparing a composition of cadaveric human MSCs from bone is provided. In some embodiments, preparing may include providing bone from a deceased donor, grinding the bone into one or more ground bone segments, filtering the one or more ground bone segments, and extracting cadaveric human MSCs from the one or more ground bone segments. In some embodiments, MSCs may be recovered from thawed or cryopreserved VB bone fragments. In some embodiments, the extracted cadaveric human MSCs may be adherent centrum mesenchymal stem cells (vBA-MSCs). In some embodiments, the extracted cadaveric human MSCs are derived from bone or fragments thereof that have been processed to remove bone marrow or derivatives thereof (e.g., bone marrow-derived cells, hematopoietic stem cells) associated with the bone or fragments thereof. In some embodiments, the extracted cadaveric human MSCs are derived from bone or fragments thereof that have been processed as described herein for use with bone marrow and/or bone marrow-derived cells (e.g., hematopoietic stem cells). In some embodiments, after filtering and/or extracting and/or isolating bone marrow and/or bone marrow-derived cells as described herein, extracted cadaveric human MSCs are obtained from bone fragments and/or bone fragments as described herein. Processing and extraction of viable vBA-MSCs from bone and/or derivatives thereof (e.g., bone fragments described herein) results in significant improvements in cell yield, particularly relative to the total cell yield per unit weight of bone derived from the donor (vBA-MSCs and hematopoietic stem cells), as well as relative to cell viability of the prior art. In some embodiments, vBA-MSCs described herein may be combined with bone marrow-derived MSCs isolated from bone marrow isolated and processed as described herein.
In some embodiments, extracting cadaveric human MSCs may include contacting bone or derivatives thereof with a digestive solution. In some embodiments, the digestion solution may comprise one or more different enzymes. In some embodiments, the one or more different enzymes may include one or more collagenases and a neutral protease. In some embodiments, the digestion solution may be present in a ratio of volume to weight of the one or more ground bone fragments and enzyme solution of about 1:1 to about 15:1. In some embodiments, the ratio may be 1:1, 2.5:1, 5:1, 7.5:1, 10:1, and 15:1 (volume: weight). In some embodiments, a combination of one or more collagenases and a neutral protease is used to obtain the highest possible yield of vBA-MSCs.
In some embodiments, the collagenase may include clostridium histolyticum (Clostridium histolyticum), which further includes two active isoforms C1 and C2. In some embodiments, one or more collagenases including the C1 and C2 isoforms may be present in the digestion solution at a ratio comprising more collagenase isoform C1 than collagenase isoform C2. In some embodiments, the ratio of collagenase isoform C1 to collagenase isoform C2 can be about 30 to about 70:about 10 to about 29. In some embodiments, the ratio of collagenase isoform C1 to collagenase C2 can be 35:15. In some embodiments, the mass ratio of C1 to C2 may be 70:30, 54:46, 37:63, 82:18, 54:46, and 90:10 for each concentration.
In some embodiments, the neutral protease may be a paenibacillus polymyxa (Paneibacillus polymyxa) neutral protease. In some embodiments, the neutral protease concentration may be about 2U/ml to about 21U/ml. In some embodiments, the neutral protease concentration may be about 2U/ml to about 7U/ml, about 2U/ml to about 12U/ml, about 2U/ml to about 17U/ml, about 2U/ml to about 21U/ml, about 7U/ml to about 12U/ml, about 7U/ml to about 17U/ml, about 7U/ml to about 21U/ml, about 12U/ml to about 17U/ml, about 12U/ml to about 21U/ml, or about 17U/ml to about 21U/ml. In some embodiments, the neutral protease concentration may be about 2U/ml, about 7U/ml, about 12U/ml, about 17U/ml, or about 21U/ml. In some embodiments, the neutral protease concentration may be at least about 2U/ml, about 7U/ml, about 12U/ml, or about 17U/ml. In some embodiments, the neutral protease concentration may be up to about 7U/ml, about 12U/ml, about 17U/ml, or about 21U/ml. In some embodiments, the digestion solution may contain neutral protease with an activity of about 19.6U/ml.
In some embodiments, the collagenase concentration is from about 0.05U/ml to about 1.6U/ml. In some embodiments of the present invention, in some embodiments, about 0.05U/ml to about 0.4U/ml, about 0.05U/ml to about 0.15U/ml, about 0.05U/ml to about 0.2U/ml, about 0.05U/ml to about 0.25U/ml, about 0.05U/ml to about 0.3U/ml, about 0.05U/ml to about 0.35U/ml, about 0.05U/ml to about 0.4U/ml, about 0.05U/ml to about 0.8U/ml, about 0.05U/ml to about 1.2U/ml, about 0.05U/ml to about 1.6U/ml, about 0.1U/ml to about 0.15U/ml, about 0.1U/ml to about 0.2U/ml, about 0.1U/ml to about 0.25U/ml, about 0.1U/3U/ml to about 0.3U/ml, about 0.0.0.0.05U to about 0.3U/ml, about 0.0.0.0.0.5U/ml to about 0.3U/ml, about 0.0.0.5U to about 0.0.5U/ml, about 0.0.0.15U/ml to about 0.15U/ml, about 0.15U/ml to about 0.0.0.0.0.0.0U/ml, about 0.05U to about 0.0.0.0.0.0.0.5U/ml, about 0.0.0.0.0.5U/ml to about 0.0.5U/ml, about 0.15U/ml to about 0.0.0.0.0.0.5U/ml, about 0.0.0.0.0.5U/ml, about 0.25U/ml to about 0.4U/ml, about 0.25U/ml to about 0.8U/ml, about 0.25U/ml to about 1.2U/ml, about 0.25U/ml to about 1.6U/ml, about 0.3U/ml to about 0.35U/ml, about 0.3U/ml to about 0.4U/ml, about 0.3U/ml to about 0.8U/ml, about 0.3U/ml to about 1.2U/ml, about 0.3U/ml to about 1.6U/ml, about 0.35U/ml to about 0.4U/ml, about 0.35U/ml to about 0.8U/ml, about 0.35U/ml to about 1.2U/ml, about 0.35U/ml to about 1.6U/ml, about 0.4U/ml to about 0.8U/ml, about 0.4U/ml to about 1.2U/ml, about 1.2U/ml to about 1.6U/ml, about 0.3U/ml to about 1.6U/ml, about 0.8U/ml to about 1.8U/ml. In some embodiments, the collagenase concentration is about 0.05U/ml, about 0.1U/ml, about 0.15U/ml, about 0.2U/ml, about 0.25U/ml, about 0.3U/ml, about 0.35U/ml, about 0.4U/ml, about 0.8U/ml, about 1.2U/ml, or about 1.6U/ml. In some embodiments, the collagenase concentration is at least about 0.05U/ml, about 0.1U/ml, about 0.15U/ml, about 0.2U/ml, about 0.25U/ml, about 0.3U/ml, about 0.35U/ml, about 0.4U/ml, about 0.8U/ml, or about 1.2U/ml. In some embodiments, the collagenase concentration is up to about 0.1U/ml, about 0.15U/ml, about 0.2U/ml, about 0.25U/ml, about 0.3U/ml, about 0.35U/ml, about 0.4U/ml, about 0.8U/ml, about 1.2U/ml, or about 1.6U/ml.
According to one aspect of the present disclosure, neutral protease concentration and collagenase concentration (C1 and C2 collagenase) and the ratio of solution volume (ml) to bone fragment weight (mg) are determined.
In some embodiments, the total collagenase concentration (C1 and C2 collagenase) is from about 25 μg/ml to about 100 μg/ml. In some embodiments of the present invention, in some embodiments, the total collagenase concentration is from about 25 to about 32.5 μg/ml, from about 25 to about 47.5 μg/ml, from about 25 to about 42.5 μg/ml, from about 25 to about 50 μg/ml, from about 25 to about 65 μg/ml, from about 25 to about 77.5 μg/ml, from about 25 to about 85 μg/ml, from about 25 to about 100 μg/ml, from about 32.5 to about 47.5 μg/ml, from about 32.5 to about 42.5 μg/ml, from about 32.5 to about 50 μg/ml, from about 32.5 to about 65 μg/ml, from about 32.5 to about 77.5 μg/ml, from about 32.5 to about 85 μg/ml about 32.5 to about 100 μg/ml, about 47.5 to about 42.5 μg/ml, about 47.5 to about 50 μg/ml, about 47.5 to about 65 μg/ml, about 47.5 to about 77.5 μg/ml, about 47.5 to about 85 μg/ml, about 47.5 to about 100 μg/ml, about 42.5 to about 50 μg/ml, about 42.5 to about 65 μg/ml, about 42.5 to about 77.5 μg/ml, about 42.5 to about 5.5 μg/ml, about 42.5 to about 85 μg/ml, about 42.5 to about 100 μg/ml, about 50 to about 65 μg/ml, about 50 to about 77.5 μg/ml, about 50 μg/ml to about 85 μg/ml, about 50 μg/ml to about 100 μg/ml, about 65 μg/ml to about 77.5 μg/ml, about 65 μg/ml to about 85 μg/ml, about 65 μg/ml to about 100 μg/ml, about 77.5 μg/ml to about 85 μg/ml, about 77.5 μg/ml to about 100 μg/ml, or about 85 μg/ml to about 100 μg/ml. In some embodiments, the total collagenase concentration is about 25 μg/ml, about 32.5 μg/ml, about 47.5 μg/ml, about 42.5 μg/ml, about 50 μg/ml, about 65 μg/ml, about 77.5 μg/ml, about 85 μg/ml, or about 100 μg/ml. In some embodiments, the total collagenase concentration is at least about 25 μg/ml, about 32.5 μg/ml, about 47.5 μg/ml, about 42.5 μg/ml, about 50 μg/ml, about 65 μg/ml, about 77.5 μg/ml, or about 85 μg/ml. In some embodiments, the total collagenase concentration is up to about 32.5 μg/ml, about 47.5 μg/ml, about 42.5 μg/ml, about 50 μg/ml, about 65 μg/ml, about 77.5 μg/ml, about 85 μg/ml, or about 100 μg/ml.
In some embodiments, the mass ratio of C1 to C2 is 70:30, 54:46, 37:63, 82:18, and 90:10, respectively, for each concentration.
According to the method, fragments of VB bone are placed in a composition comprising PLASMA-LYTE TM In a cryoprotectant solution of 2.5% human serum albumin and 10% dimethyl sulfoxide (DMSO), and incubated at 4 ℃ for 1 hour. In some embodiments, the incubation period is from about 1 hour to about 3 hours. In some embodiments, the incubation period is from about 1 hour to about 1.5 hours, from about 1 hour to about 2 hours, from about 1 hour to about 2.5 hours, from about 1 hour to about 3 hours, from about 1.5 hours to about 2 hours, from about 1.5 hours to about 2.5 hours, from about 1.5 hours to about 3 hours, from about 2 hours to about 2.5 hours, from about 2 hours to about 3 hours, or from about 2.5 hours to about 3 hours. In some embodiments, the incubation period is about 1 hour, about 1.5 hours, about 2 hours, about 2.5 hours, or about 3 hours. In some embodiments, the incubation period is at least about 1 hour, about 1.5 hours, about 2 hours, or about 2.5 hours. In some embodiments, the incubation period is up to about 1.5 hours, about 2 hours, about 2.5 hours, or about 3 hours. The solution was removed and the bone fragments were cooled to-86℃at a rate of about 1℃per minute and then poured into the liquid Nitrogen. After 24-48 hours in liquid nitrogen, the bone fragments were quickly thawed in a water bath set to 37 ℃ and then washed in saline and digested using the collagenase/protease solution described above.
In some embodiments, the volume to weight ratio is 5:1 at an incubation time of 2.5 hours. In some embodiments, the protease produces 19.6U/ml of neutral protease activity.
A mixture of cells released by digestion of VB bone fragments was cultured on tissue coated plastic in the presence of Mesencult medium to select for proliferated vBA-MSCs. Freshly digested preparations (preparations) of VB-MSC can be characterized in vitro by flow cytometry, fibroblast colony forming unit (CFU-F) potential, population Doubling Time (PDT) and three-line (adipogenic, chondrogenic and osteogenic) differentiation.
In some embodiments, the methods of cadaveric human MSC extraction disclosed herein are capable of extracting amounts of about 1000 tens of thousands to about 100 billion. In some embodiments, cadaveric human MSCs may be administered in an amount of about 1000 tens of thousands to about 1 million, about 1000 tens of thousands to about 10 million, about 1000 tens of thousands to about 100 million, about 1 million to about 10 million, about 1 million to about 100 million, or about 10 million to about 100 million. In some embodiments, cadaveric human MSCs may be administered in an amount of about 1000 tens of thousands, about 1 million, about 10 million, or about 100 million. In some embodiments, cadaveric human MSCs may be administered in an amount of at least about 1000 tens of thousands, about 1 million, or about 10 million. In some embodiments, cadaveric human MSCs may be administered in an amount up to about 1 million, about 10 million, or about 100 million.
MSC composition
MSCs have been shown to be potentially useful adjuvants for Stem Cell Transplantation (SCT) for promoting mixed chimeras and for promoting complementary peripheral immunomodulatory functions. MSCs have demonstrated therapeutic potential for inducing surgical tolerance of SOT and VCA, providing a rationale demonstration for clinical testing in a transplantation setting. The effect of MSC infusion (including in humans and non-human primates) is compared to T EM Cells bias T cell populations towards T REG And (3) cells. It is evident that mixed chimeras are essential for central tolerance of SOT and VCA, which is resisted by peripheral homologsPrimordial specific regulatory T cells (T REGS ) And expansion of donor-derived tolerogenic dendritic cells (toldcs).
In some embodiments, cadaveric MSCs may be administered prophylactically, perioperatively, or post-operatively via SOT or other VCA procedures. In some embodiments, the cadaveric MSCs administered may include bone marrow derived cadaveric human MSCs, adherent vertebral MSCs (vBA-MSCs), or both.
In some embodiments, compositions providing cadaveric human MSCs for suppressing immune responses may be administered in amounts of about 1000 tens of thousands to about 100 billion. In some embodiments, cadaveric human MSCs may be administered in an amount of about 1000 tens of thousands to about 1 million, about 1000 tens of thousands to about 10 million, about 1000 tens of thousands to about 100 million, about 1 million to about 10 million, about 1 million to about 100 million, or about 10 million to about 100 million. In some embodiments, cadaveric human MSCs may be administered in an amount of about 1000 tens of thousands, about 1 million, about 10 million, or about 100 million. In some embodiments, cadaveric human MSCs may be administered in an amount of at least about 1000 tens of thousands, about 1 million, or about 10 million. In some embodiments, cadaveric human MSCs may be administered in an amount up to about 1 million, about 10 million, or about 100 million.
In some embodiments, a composition of cadaveric MSCs (e.g., vBA-MSCs) may comprise less than 5% cd45+. In some embodiments, the composition of cadaveric MSCs may comprise from less than about 0.5% cd45+ to about 10% cd45+. In some embodiments of the present invention, in some embodiments, the composition of cadaveric MSCs may comprise less than about 10% cd45+ to about 9% cd45+, about 10% cd45+ to about 8% cd45+, about 10% cd45+ to about 6% cd45+, about 10% cd45+ to about 4% cd45+, about 10% cd45+ to about 2% cd45+, about 10% cd45+ to about 1% cd45+, about 10% cd45+ to about 0.5% cd45+, about 9% cd45+ to about 8% cd45+, about 9% cd45+ to about 7% cd45+, about 9% cd45+ to about 6% cd45+, about 9% cd45+ to about 5% cd45% cd45+, about 9% cd45+ to about 3% cd45+, about 9% cd45+ to about 9% cd45% cd4+, about 9% cd4+ to about 9% cd45% cd4+ and about 9% cd45+ to about 9% cd4+ and about 5% cd4+ and about 9+ and about 45+ to about 3% cd4+ and about 5%. About 9% cd45+ to about 0.5% cd45+, about 8% cd45+ to about 7% cd45+, about 8% cd45+ to about 6% cd45+, about 8% cd45+ to about 8% cd45+, about 8% cd45+ to about 3% cd45+, about 8% cd45+ to about 2% cd45+, about 8% cd45+ to about 1% cd45+, about 8% cd45+ to about 0.5% cd45+, about 7% cd45+ to about 6% cd45+, about 7% cd45+ to about 4% cd4+, about 7% cd45+ to about 3% cd45+, about 7% cd45+ to about 2% cd45+, about 7% cd45+ to about 1% cd45+, about 7% cd45+ to about 5% cd45+, about 5% cd45+ to about 45+, about 7% cd45+ to about 45+ + to about 4% cd45% About 6% cd45+ to about 3% cd45+, about 6% cd45+ to about 2% cd45+, about 6% cd45+ to about 1% cd45+ to about 0.5% cd45+ to about 4% cd45+, about 5% cd45+ to about 3% cd45+, about 5% cd45+ to about 1% cd45+, about 5% cd45+ to about 0.5% cd45+, about 4% cd45+ to about 3% cd45+, about 4% cd45+ to about 4% cd45+, about 4% cd45+ to about 2% cd45+, about 4% cd45+ to about 1% cd45+ to about 0.5% cd45+ to about 2% cd45+, about 3% cd45+ to about 0.5% cd4+ to about 1% cd45+. In some embodiments, the composition of cadaveric MSCs may comprise less than about 10% cd45+, about 9% cd45+, about 8% cd45+, about 7% cd45+, about 5% cd45+, about 3% cd45+, about 2% cd45+, about 1% cd45+, or about 0.5% cd45+. In some embodiments, the composition of cadaveric MSCs may comprise less than at least about 10% cd45+, about 9% cd45+, about 8% cd45+, about 7% cd45+, about 5% cd4+, about 3% cd4+, about 2% cd45+, or about 1% cd45+. In some embodiments, the composition of cadaveric MSCs may comprise less than up to about 9% cd45+, about 8% cd45+, about 7% cd45+, about 6% cd45+, about 4% cd45+, about 3% cd45+, about 2% cd45+, about 1% cd45+, or about 0.5% cd45+.
In some embodiments, a composition of cadaveric MSCs (e.g., vBA-MSCs) can comprise more than 1% cd45+ cells. In some embodiments, a composition of cadaveric MSCs may comprise more than 1.1% cd45+ cells. In some embodiments, the composition of cadaveric MSCs may comprise more than 1.2% cd45+ cells. In some embodiments, the composition of cadaveric MSCs may comprise more than 1.3% cd45+ cells. In some embodiments, the composition of cadaveric MSCs may comprise more than 1.4% cd45+ cells. In some embodiments, the composition of cadaveric MSCs may comprise more than 1.5% cd45+ cells. In some embodiments, the composition of cadaveric MSCs may comprise more than 1.6% cd45+ cells. In some embodiments, the composition of cadaveric MSCs may comprise more than 1.7% cd45+ cells. In some embodiments, the composition of cadaveric MSCs may comprise more than 1.8% cd45+ cells. In some embodiments, the composition of cadaveric MSCs may comprise more than 1.9% cd45+ cells. In some embodiments, the composition of cadaveric MSCs may comprise more than 2% cd45+ cells.
In some embodiments, a composition of cadaveric MSCs may comprise at least 90% cd105+ cells. In some embodiments, a composition of cadaveric MSCs may comprise at least about 70% cd105+ cells to about 100% cd105+ cells. In some embodiments of the present invention, in some embodiments, the composition of cadaveric MSCs may comprise at least about 100% cd105+ cells to about 95% cd105+ cells, about 100% cd105+ cells to about 94% cd105+ cells, about 100% cd105+ cells to about 93% cd105+ cells, about 100% cd105+ cells to about 92% cd105+ cells, about 100% cd105+ cells to about 91% cd105+ cells, about 100% cd105+ cells to about 90% cd105+ cells, about 100% cd105+ cells to about 85% cd105+ cells, about 100% cd105+ cells to about 80% cd105+ cells, about 100% cd105+ cells to about 75% cd105+ cells, about 100% cd105+ cells to about 70% cd105+ cells, about 95% cd105+ cells to about 94% cd105+ cells, about 95% cd105+ cells to about 93% cd105+ cells, about 95% cd105+ cells to about 95% cd105+ cells, about 95% cd105+ cells to about 91% cd105+ cells. About 95% cd105+ cells to about 90% cd105+ cells, about 95% cd105+ cells to about 85% cd105+ cells, about 95% cd105+ cells to about 80% cd105+ cells, about 95% cd105+ cells to about 75% cd105+ cells, about 95% cd105+ cells to about 70% cd105+ cells, about 94% cd105+ cells to about 93% cd105+ cells, about 94% cd105+ cells to about 92% cd105+ cells, about 94% cd105+ cells to about 91% cd105+ cells, about 94% cd105+ cells to about 90% cd105+ cells, about 94% cd105+ cells to about 85% cd105+ cells, about 94% cd105+ cells to about 80% cd105+ cells, about 94% cd105+ cells to about 75% cd105+ cells, about 94% cd105+ cells to about 70% cd105+ cells, about 92% cd105+ cells, about 93% cd105+ cells About 93% cd105+ cells to about 91% cd105+ cells, about 93% cd105+ cells to about 90% cd105+ cells, about 93% cd105+ cells to about 85% cd105+ cells, about 93% cd105+ cells to about 80% cd105+ cells, about 93% cd105+ cells to about 75% cd105+ cells, about 93% cd105+ cells to about 70% cd105+ cells, about 92% cd105+ cells to about 91% cd105+ cells, about 92% cd105+ cells to about 90% cd105+ cells, about 92% cd105+ cells to about 85% cd105+ cells, about 92% cd105+ cells to about 80% cd105+ cells, about 92% cd105+ cells to about 75% cd105+ cells, about 92% cd105+ cells to about 70% cd105+ cells, about 91% cd105+ cells to about 90% cd105+ cells, about 91% cd105+ cells to about 85% cd105+ cells, about 92% cd105+ cells. About 91% cd105+ cells to about 80% cd105+ cells, about 91% cd105+ cells to about 75% cd105+ cells, about 91% cd105+ cells to about 70% cd105+ cells, about 90% cd105+ cells to about 85% cd105+ cells, about 90% cd105+ cells to about 80% cd105+ cells, about 90% cd105+ cells to about 75% cd105+ cells, about 90% cd105+ cells to about 70% cd105+ cells, about 85% cd105+ cells to about 80% cd105+ cells, about 85% cd105+ cells to about 75% cd105+ cells, about 85% cd105+ cells to about 70% cd105+ cells, about 80% cd105+ cells to about 75% cd105+ cells, about 80% cd105+ cells to about 70% cd105+ cells, or about 75% cd105+ cells to about 70% cd105+ cells. In some embodiments, the composition of cadaveric MSCs may comprise at least about 100% cd105+ cells, about 95% cd105+ cells, about 94% cd105+ cells, about 93% cd105+ cells, about 92% cd105+ cells, about 91% cd105+ cells, about 90% cd105+ cells, about 85% cd105+ cells, about 80% cd105+ cells, about 75% cd105+ cells, or about 70% cd105+ cells. In some embodiments, the composition of cadaveric MSCs may comprise at least about 100% cd105+ cells, about 95% cd105+ cells, about 94% cd105+ cells, about 93% cd105+ cells, about 92% cd105+ cells, about 91% cd105+ cells, about 90% cd105+ cells, about 85% cd105+ cells, about 80% cd105+ cells, or about 75% cd105+ cells. In some embodiments, the composition of cadaveric MSCs may comprise at least up to about 95% cd105+ cells, about 94% cd105+ cells, about 93% cd105+ cells, about 92% cd105+ cells, about 91% cd105+ cells, about 90% cd105+ cells, about 85% cd105+ cells, about 80% cd105+ cells, about 75% cd105+ cells, or about 70% cd105+ cells.
In some embodiments, a composition of cadaveric MSCs may comprise at least 90% cd166+ cells. In some embodiments, a composition of cadaveric MSCs may comprise at least about 70% cd166+ cells to about 100% cd166+ cells. In some embodiments of the present invention, in some embodiments, the composition of cadaveric MSCs may comprise at least about 100% cd166+ cells to about 95% cd166+ cells, about 100% cd166+ cells to about 94% cd166+ cells, about 100% cd166+ cells to about 93% cd166+ cells, about 100% cd166+ cells to about 92% cd166+ cells, about 100% cd166+ cells to about 91% cd166+ cells, about 100% cd166+ cells to about 90% cd166+ cells, about 100% cd166+ cells to about 85% cd166+ cells, about 100% cd166+ cells to about 80% cd166+ cells, about 100% cd166+ cells to about 75% cd166+ cells, about 100% cd166+ cells to about 70% cd166+ cells, about 95% cd166+ cells to about 94% cd166+ cells, about 95% cd166+ cells to about 93% cd166+ cells, about 95% cd166+ cells to about 95% cd166+ cells, about 91% cd166+ cells. About 95% cd166+ to about 90% cd166+ cells, about 95% cd166+ to about 85% cd166+ cells, about 95% cd166+ to about 80% cd166+ cells, about 95% cd166+ to about 75% cd166+ cells, about 95% cd166+ to about 70% cd166+ cells, about 94% cd166+ to about 93% cd166+ cells, about 94% cd166+ to about 92% cd166+ cells, about 94% cd166+ to about 91% cd166+ cells, about 94% cd166+ to about 90% cd166+ cells, about 94% cd166+ to about 85% cd166+ cells, about 94% cd166+ to about 80% cd166+ cells, about 94% cd166+ to about 75% cd166+ cells, about 94% cd166+ to about 70% cd166+ cells, about 93% cd166+ to about 93% cd166+ cells About 93% cd166+ to about 91% cd166+ cells, about 93% cd166+ to about 90% cd166+ cells, about 93% cd166+ to about 85% cd166+ cells, about 93% cd166+ to about 80% cd166+ cells, about 93% cd166+ to about 75% cd166+ cells, about 93% cd166+ to about 70% cd166+ cells, about 92% cd166+ to about 91% cd166+ cells, about 92% cd166+ to about 90% cd166+ cells, about 92% cd166+ to about 85% cd166+ cells, about 92% cd166+ to about 80% cd166+ cells, about 92% cd166+ to about 75% cd166+ cells, about 92% cd166+ to about 70% cd166+ cells, about 91% cd166+ to about 90% cd166+ cells, about 91% cd166+ to about 85% cd166+ cells. About 91% cd166+ cells to about 80% cd166+ cells, about 91% cd166+ cells to about 75% cd166+ cells, about 91% cd166+ cells to about 70% cd166+ cells, about 90% cd166+ cells to about 85% cd166+ cells, about 90% cd166+ cells to about 80% cd166+ cells, about 90% cd166+ cells to about 75% cd166+ cells, about 90% cd166+ cells to about 70% cd166+ cells, about 85% cd166+ cells to about 80% cd166+ cells, about 85% cd166+ cells to about 75% cd166+ cells, about 85% cd166+ cells to about 70% cd166+ cells, about 80% cd166+ cells to about 75% cd166+ cells, about 80% cd166+ cells to about 70% cd166+ cells, or about 75% cd166+ cells to about 70% cd166+ cells. In some embodiments, the composition of cadaveric MSCs may comprise at least about 100% cd166+ cells, about 95% cd166+ cells, about 94% cd166+ cells, about 93% cd166+ cells, about 92% cd166+ cells, about 91% cd166+ cells, about 90% cd166+ cells, about 85% cd166+ cells, about 80% cd166+ cells, about 75% cd166+ cells, or about 70% cd166+ cells. In some embodiments, the composition of cadaveric MSCs may comprise at least about 100% cd166+ cells, about 95% cd166+ cells, about 94% cd166+ cells, about 93% cd166+ cells, about 92% cd166+ cells, about 91% cd166+ cells, about 90% cd166+ cells, about 85% cd166+ cells, about 80% cd166+ cells, or about 75% cd166+ cells. In some embodiments, the composition of cadaveric MSCs may comprise at least up to about 95% cd166+ cells, about 94% cd166+ cells, about 93% cd166+ cells, about 92% cd166+ cells, about 91% cd166+ cells, about 90% cd166+ cells, about 85% cd166+ cells, about 80% cd166+ cells, about 75% cd166+ cells, or about 70% cd166+ cells.
Culture of MSC
In one aspect of the disclosure, extracted MSCs (e.g., vBA-MSCs) can be cultured and passaged to achieve a clinically-sized MSC formulation with a desired number of MSCs having the antigen profile (profile) taught herein. In some embodiments, a clinically-sized formulation may be obtained by serial passage amplification, wherein each passage includes the step of dividing the previous culture into multiple cultures at a given ratio. Each passaging step increased the number of parallel cultures in the formulation. In some embodiments, clinical scale formulations with the formulation profiles of the present invention, e.g., antigen profile, TNFRI profile, cryopreservation profile, differentiation profile, and/or sterility (relative to pathogen) were successfully produced.
In some embodiments, the extracted MSCs are cultured in a medium, wherein the medium is configured to produce MSCs having a formulation profile of the invention, e.g., an antigen profile, TNFRI profile, cryopreservation profile, differentiation profile, and/or sterility (relative to pathogen). In some embodiments, the medium comprises Minimal Essential Medium (MEM). In some embodiments, the culture medium comprises αmem. In some embodiments, the culture medium comprises human platelet lysate (hPL). In some embodiments, the medium comprises a carrier-free Fibroblast Growth Factor (FGF). In some embodiments, the medium comprises a carrier-free Epidermal Growth Factor (EGF). In some embodiments, the culture medium comprises α MEM, hPL, FGF, EGF or any combination thereof. In some embodiments, the culture medium comprises α MEM, hPL, FGF and EGF. In some embodiments, the medium does not further require heparin.
In some embodiments, hPL is present in the culture medium from about 1% to about 21%. In some embodiments of the present invention, in some embodiments, hPL may be about 1% to about 3%, about 1% to about 5%, about 1% to about 7%, about 1% to about 9%, about 1% to about 10%, about 1% to about 11%, about 1% to about 13%, about 1% to about 15%, about 1% to about 17%, about 1% to about 19%, about 1% to about 21%, about 3% to about 5%, about 3% to about 7%, about 3% to about 9%, about 3% to about 10%, about 3% to about 11%, about 3% to about 13%, about 3% to about 15%, about 3% to about 17%, about 3% to about 19%, about 3% to about 21%, about 5% to about 7%, about 5% to about 9%, about 5% to about 10%, about 5% to about 11%, about 5% to about 13%, about 5% to about 15%, about 5% to about 17%, about 5% to about 19%, about 5% to about 21%, about 7% to about 9%, about 7% to about 10%, about 10% to about 10%, about about 7% to about 11%, about 7% to about 13%, about 7% to about 15%, about 7% to about 17%, about 7% to about 19%, about 7% to about 21%, about 9% to about 10%, about 9% to about 11%, about 9% to about 13%, about 9% to about 15%, about 9% to about 17%, about 9% to about 19%, about 9% to about 21%, about 10% to about 11%, about 10% to about 13%, about 10% to about 15%, about about 10% to about 17%, about 10% to about 19%, about 10% to about 21%, about 11% to about 13%, about 11% to about 15%, about 11% to about 17%, about 11% to about 19%, about 11% to about 21%, about 13% to about 15%, about 13% to about 17%, about 13% to about 19%, about 13% to about 21%, about 15% to about 17%, about 15% to about 19%, about 15% to about 21%, about, about 17% to about 19%, about 17% to about 21%, or about 19% to about 21% are present in the medium. In some embodiments, hPL is present in the culture medium at about 1%, about 3%, about 5%, about 7%, about 9%, about 10%, about 11%, about 13%, about 15%, about 17%, about 19%, or about 21%. In some embodiments, hPL is present in the culture medium at least about 1%, about 3%, about 5%, about 7%, about 9%, about 10%, about 11%, about 13%, about 15%, about 17%, or about 19%. In some embodiments, hPL is present in the culture medium at up to about 3%, about 5%, about 7%, about 9%, about 10%, about 11%, about 13%, about 15%, about 17%, about 19%, or about 21%. In some embodiments, FGF is present in the medium at about 0.5ng/mL to about 5 ng/mL. In some embodiments, the FGF is provided in an amount of about 0.5ng to about 1ng/mL, about 0.5ng to about 1.5ng/mL, about 0.5ng to about 2ng/mL, about 1ng to about 2.5ng/mL, about 0.5ng to about 3ng/mL, about 0.5ng to about 3.5ng/mL, about 0.5ng to about 4.5ng/mL, about 0.5ng to about 5ng/mL, about 1ng to about 1.5ng/mL, about 1ng to about 3ng/mL, about 1ng to about 3.5ng to about 4ng/mL, about 1.5ng to about 4ng to about 4.5ng, about 1.5ng to about 4.5ng to about 3.5ng to about 2.5ng/mL, about 2.5ng to about 2.5ng/mL, about 1.5ng to about 2.5ng/mL, about 1ng to about 3ng/mL, about 3ng/mL to about 4ng/mL, about 3ng/mL to about 4.5ng/mL, about 3ng/mL to about 5ng/mL, about 3.5ng/mL to about 4ng/mL, about 3.5ng/mL to about 4.5ng/mL, about 3.5ng/mL to about 5ng/mL, about 4ng/mL to about 4.5ng/mL, about 4ng/mL to about 5ng/mL, or about 4.5ng/mL to about 5ng/mL is present in the medium. In some embodiments, FGF is present in the medium at about 0.5ng/mL, about 1ng/mL, about 1.5ng/mL, about 2ng/mL, about 2.5ng/mL, about 3ng/mL, about 3.5ng/mL, about 4ng/mL, about 4.5ng/mL, or about 5 ng/mL. In some embodiments, FGF is present in the medium at least about 0.5ng/mL, about 1ng/mL, about 1.5ng/mL, about 2ng/mL, about 2.5ng/mL, about 3ng/mL, about 3.5ng/mL, about 4ng/mL, or about 4.5 ng/mL. In some embodiments, FGF is present in the medium up to about 1ng/mL, about 1.5ng/mL, about 2ng/mL, about 2.5ng/mL, about 3ng/mL, about 3.5ng/mL, about 4ng/mL, about 4.5ng/mL, or about 5 ng/mL.
In some embodiments, EGF is present in the medium from about 0.5ng/mL to about 5 ng/mL. In some embodiments, EGF is present in an amount of about 0.5ng to about 1ng/mL, about 0.5ng to about 1.5ng/mL, about 0.5ng to about 2ng/mL, about 1ng to about 2.5ng/mL, about 0.5ng to about 3ng/mL, about 0.5ng to about 3.5ng/mL, about 0.5ng to about 4.5ng/mL, about 0.5ng to about 5ng/mL, about 1ng to about 1.5ng/mL, about 1ng to about 3ng/mL, about 1ng to about 3.5ng to about 4ng/mL, about 1.5ng to about 4ng to about 4.5ng, about 1.5ng to about 3.5ng to about 2.5ng/mL, about 2.5ng to about 2.5ng/mL, about 1ng to about 3ng/mL, about 3ng/mL to about 4ng/mL, about 3ng/mL to about 4.5ng/mL, about 3ng/mL to about 5ng/mL, about 3.5ng/mL to about 4ng/mL, about 3.5ng/mL to about 4.5ng/mL, about 3.5ng/mL to about 5ng/mL, about 4ng/mL to about 4.5ng/mL, about 4ng/mL to about 5ng/mL, or about 4.5ng/mL to about 5ng/mL is present in the medium. In some embodiments, EGF is present in the medium at about 0.5ng/mL, about 1ng/mL, about 1.5ng/mL, about 2ng/mL, about 2.5ng/mL, about 3ng/mL, about 3.5ng/mL, about 4ng/mL, about 4.5ng/mL, or about 5 ng/mL. In some embodiments, EGF is present in the medium at least about 0.5ng/mL, about 1ng/mL, about 1.5ng/mL, about 2ng/mL, about 2.5ng/mL, about 3ng/mL, about 3.5ng/mL, about 4ng/mL, or about 4.5 ng/mL. In some embodiments, EGF is present in the medium up to about 1ng/mL, about 1.5ng/mL, about 2ng/mL, about 2.5ng/mL, about 3ng/mL, about 3.5ng/mL, about 4ng/mL, about 4.5ng/mL, or about 5 ng/mL.
In some embodiments, the culture medium comprises modified αmem. In some embodiments, the modified αmem comprises one or more inorganic salts, one or more amino acids, one or more vitamins, glucose, lipoic acid, sodium bicarbonate, sodium pyruvate, or any combination thereof.
In some embodiments, the one or more inorganic salts include calcium chloride (dihydrate), magnesium sulfate (heptahydrate), potassium chloride, sodium dihydrogen phosphate (dehydrated), or any combination thereof. In some embodiments, each inorganic salt present in the medium is present at about 100 mg/liter to about 800 mg/liter. In some embodiments, each inorganic salt present in the medium is present in about 100 mg/liter to about 200 mg/liter, about 100 mg/liter to about 300 mg/liter, about 100 mg/liter to about 400 mg/liter, about 100 mg/liter to about 500 mg/liter, about 100 mg/liter to about 600 mg/liter, about 100 mg/liter to about 700 mg/liter, about 100 mg/liter to about 800 mg/liter, about 200 mg/liter to about 300 mg/liter, about 200 mg/liter to about 400 mg/liter, about 200 mg/liter to about 500 mg/liter, about 200 mg/liter to about 600 mg/liter, about 200 mg/liter to about 700 mg/liter, about 200 mg/liter to about 800 mg/liter, about 300 mg/liter to about 400 mg/liter, about 300 mg/liter to about 500 mg/liter, about 300 mg/liter to about 600 mg/liter, about 300 mg/liter to about 700 mg/liter, about 300 mg/liter to about 800 mg/liter, about 400mg to about 400 mg/liter, about 400mg to about 700 mg/liter, about 500mg to about 600 mg/liter, about 500mg to about 500 mg/liter. In some embodiments, each inorganic salt present in the medium is present at about 100 mg/liter, about 200 mg/liter, about 300 mg/liter, about 400 mg/liter, about 500 mg/liter, about 600 mg/liter, about 700 mg/liter, or about 800 mg/liter. In some embodiments, each inorganic salt present in the medium is present at least about 100 mg/liter, about 200 mg/liter, about 300 mg/liter, about 400 mg/liter, about 500 mg/liter, about 600 mg/liter, or about 700 mg/liter. In some embodiments, each inorganic salt present in the medium is present at up to about 200 mg/liter, about 300 mg/liter, about 400 mg/liter, about 500 mg/liter, about 600 mg/liter, about 700 mg/liter, or about 800 mg/liter.
In some embodiments, the one or more amino acids include glycine, alanine, alanyl-glutamine, arginine (HCl), asparagine (monohydrate), aspartic acid, cysteine (HCl) (monohydrate), cystine, glutamic acid, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, or any combination thereof. In some embodiments, one or more amino acids are present in the L isoform. In some embodiments, one or more amino acids are present as the D isoform. In some embodiments, one or more amino acids are present in both isoforms. In some embodiments, each amino acid present in the medium is present at about 10 mg/liter to about 100 mg/liter. In some embodiments of the present invention, in some embodiments, each amino acid present in the medium is present in an amount of about 10 mg/liter to about 20 mg/liter, about 10 mg/liter to about 30 mg/liter, about 10 mg/liter to about 40 mg/liter, about 10 mg/liter to about 50 mg/liter, about 10 mg/liter to about 60 mg/liter, about 10 mg/liter to about 70 mg/liter, about 10 mg/liter to about 80 mg/liter, about 10 mg/liter to about 90 mg/liter, about 10 mg/liter to about 100 mg/liter, about 20 mg/liter to about 30 mg/liter, about 20 mg/liter to about 40 mg/liter, about 20 mg/liter to about 50 mg/liter, about 20 mg/liter to about 60 mg/liter, about 20 mg/liter to about 70 mg/liter, about 20 mg/liter to about 80 mg/liter, about 30 mg/liter to about 40 mg/liter, about 30 mg/liter to about 90 mg/liter, about 50 mg/liter, about 60mg to about 40 mg/liter, about 50mg to about 50 mg/liter, about 60mg to about 60 mg/liter, about 50 mg/liter to about 60 mg/liter, about 50mg to about 60 mg/liter, about 60mg to about 60 mg/liter, about 50mg to about 60 mg/liter, about 90 mg/liter, about 50mg to about 60 mg/liter, about 50 mg/liter to about 80 mg/liter, about 60mg to about 90 mg/liter, about 90mg to about 60 mg/liter, about 60 mg/liter to about 60 mg/liter About 60 mg/liter to about 80 mg/liter, about 60 mg/liter to about 90 mg/liter, about 60 mg/liter to about 100 mg/liter, about 70 mg/liter to about 80 mg/liter, about 70 mg/liter to about 90 mg/liter, about 70 mg/liter to about 100 mg/liter, about 80 mg/liter to about 90 mg/liter, about 80 mg/liter to about 100 mg/liter, or about 90 mg/liter to about 100 mg/liter. In some embodiments, each amino acid present in the medium is present at about 10 mg/liter, about 20 mg/liter, about 30 mg/liter, about 40 mg/liter, about 50 mg/liter, about 60 mg/liter, about 70 mg/liter, about 80 mg/liter, about 90 mg/liter, or about 100 mg/liter. In some embodiments, each amino acid present in the medium is present at least about 10 mg/liter, about 20 mg/liter, about 30 mg/liter, about 40 mg/liter, about 50 mg/liter, about 60 mg/liter, about 70 mg/liter, about 80 mg/liter, or about 90 mg/liter. In some embodiments, each amino acid present in the medium is present at up to about 20 mg/liter, about 30 mg/liter, about 40 mg/liter, about 50 mg/liter, about 60 mg/liter, about 70 mg/liter, about 80 mg/liter, about 90 mg/liter, or about 100 mg/liter. In some embodiments, each amino acid present in the medium is present at about 100 mg/liter to about 500 mg/liter. In some embodiments, each amino acid present in the medium is present at about 100 mg/liter to about 200 mg/liter, about 100 mg/liter to about 300 mg/liter, about 100 mg/liter to about 400 mg/liter, about 100 mg/liter to about 500 mg/liter, about 200 mg/liter to about 300 mg/liter, about 200 mg/liter to about 400 mg/liter, about 200 mg/liter to about 500 mg/liter, about 300 mg/liter to about 400 mg/liter, about 300 mg/liter to about 500 mg/liter, or about 400 mg/liter to about 500 mg/liter. In some embodiments, each amino acid present in the medium is present at about 100 mg/liter, about 200 mg/liter, about 300 mg/liter, about 400 mg/liter, or about 500 mg/liter. In some embodiments, each amino acid present in the medium is present at least about 100 mg/liter, about 200 mg/liter, about 300 mg/liter, or about 400 mg/liter. In some embodiments, each amino acid present in the medium is present at up to about 200 mg/liter, about 300 mg/liter, about 400 mg/liter, or about 500 mg/liter.
In some embodiments, the one or more vitamins include ascorbic acid, biotin, choline chloride, calcium pantothenate, folic acid, inositol, nicotinamide, pyridoxal (HCl), pyruvic acid (sodium salt), riboflavin, thiamine (HCl), vitamin B12, or any combination thereof. In some embodiments, one or more vitamins are present in the L isoform. In some embodiments, one or more vitamins are present as the D isoform. In some embodiments, one or more vitamins are present in both isoforms. In some embodiments, each vitamin present in the medium is present at about 0.1 mg/liter to about 2 mg/liter. In some embodiments of the present invention, in some embodiments, each vitamin present in the medium is present in an amount of about 0.1 mg/liter to about 0.3 mg/liter, about 0.1 mg/liter to about 0.5 mg/liter, about 0.1 mg/liter to about 0.7 mg/liter, about 0.1 mg/liter to about 0.9 mg/liter, about 0.1 mg/liter to about 1.1 mg/liter, about 0.1 mg/liter to about 1.3 mg/liter, about 0.1 mg/liter to about 1.5 mg/liter, about 0.1 mg/liter to about 1.7 mg/liter, about 0.1 mg/liter to about 1.9 mg/liter, about 0.1 mg/liter to about 2 mg/liter, about 0.3 mg/liter to about 0.5 mg/liter, about 0.3 mg/liter to about 0.7 mg/liter, about 0.3 mg/liter to about 0.9 mg/liter, about 0.3 mg/liter to about 1.1.1 mg/liter, about 0.1 mg/liter to about 1.7 mg/liter, about 0.1.1 mg/liter, about 0.1 mg/liter to about 1.9 mg/liter, about 0.1 mg/liter to about 2.1.1 mg/liter, about 0.1 mg/liter, about 1.1 mg/liter, about 0.1 mg/liter to about 2.1 mg/liter, 1.1 mg/liter, about 3.1 mg/liter to about 0.5 mg/liter about 0.3 mg/liter to about 1.9 mg/liter, about 0.3 mg/liter to about 2 mg/liter, about 0.5 mg/liter to about 0.7 mg/liter, about 0.5 mg/liter to about 0.9 mg/liter, about 0.5 mg/liter to about 1.1 mg/liter, about 0.5 mg/liter to about 1.3 mg/liter, about 0.5 mg/liter to about 1.5 mg/liter, about 0.5 mg/liter to about 1.7 mg/liter, about 0.5 mg/liter to about 1.9 mg/liter, about 0.5 mg/liter to about 2 mg/liter, about 0.7 mg/liter to about 0.9 mg/liter, about 0.7 mg/liter to about 1.1 mg/liter, about 0.7 mg/liter to about 1.3 mg/liter, about 0.7 mg/liter to about 1.5 mg/liter, about 0.7 mg/liter to about 1.7 mg/liter, about 0.7 mg/liter to about 1.9 mg/liter, about 0.5 mg/liter to about 2 mg/liter, about 0.7 mg/liter to about 0.7 mg/liter, about 0.9 mg/liter to about 1.5 mg/liter, about 0.9 mg/liter to about 1.7 mg/liter, about 0.9 mg/liter to about 1.9 mg/liter, about 0.9 mg/liter to about 2 mg/liter, about 1.1 mg/liter to about 1.3 mg/liter, about 1.1 mg/liter to about 1.5 mg/liter, about 1.1 mg/liter to about 1.7 mg/liter, about 1.1 mg/liter to about 1.9 mg/liter, about 1.1 mg/liter to about 2 mg/liter, about 1.3 mg/liter to about 1.5 mg/liter, about 1.3 mg/liter to about 1.7 mg/liter, about 1.3 mg/liter to about 1.9 mg/liter, about 1.3 mg/liter to about 2 mg/liter, about 1.5 mg/liter to about 1.7 mg/liter, about 1.5 mg/liter to about 1.9 mg/liter, about 1.5 mg/liter to about 2 mg/liter, about 1.7 mg/liter, about 1.3mg to about 2 mg/liter, or about 1.5mg to about 2 mg/liter. In some embodiments, each vitamin present in the medium is present at about 0.1 mg/liter, about 0.3 mg/liter, about 0.5 mg/liter, about 0.7 mg/liter, about 0.9 mg/liter, about 1.1 mg/liter, about 1.3 mg/liter, about 1.5 mg/liter, about 1.7 mg/liter, about 1.9 mg/liter, or about 2 mg/liter. In some embodiments, each vitamin present in the medium is present at least about 0.1 mg/liter, about 0.3 mg/liter, about 0.5 mg/liter, about 0.7 mg/liter, about 0.9 mg/liter, about 1.1 mg/liter, about 1.3 mg/liter, about 1.5 mg/liter, about 1.7 mg/liter, or about 1.9 mg/liter. In some embodiments, each vitamin present in the medium is present at up to about 0.3 mg/liter, about 0.5 mg/liter, about 0.7 mg/liter, about 0.9 mg/liter, about 1.1 mg/liter, about 1.3 mg/liter, about 1.5 mg/liter, about 1.7 mg/liter, about 1.9 mg/liter, or about 2 mg/liter. In some embodiments, each vitamin present in the medium is present at about 10 mg/liter to about 120 mg/liter. In some embodiments, each vitamin present in the medium is present in an amount of about 10 mg/liter to about 20 mg/liter, about 10 mg/liter to about 30 mg/liter, about 10 mg/liter to about 40 mg/liter, about 10 mg/liter to about 50 mg/liter, about 10 mg/liter to about 60 mg/liter, about 10 mg/liter to about 70 mg/liter, about 10 mg/liter to about 80 mg/liter, about 10 mg/liter to about 90 mg/liter, about 10 mg/liter to about 100 mg/liter, about 10 mg/liter to about 110 mg/liter, about 10 mg/liter to about 120 mg/liter, about 20 mg/liter to about 30 mg/liter, about 20 mg/liter to about 40 mg/liter, about 20 mg/liter to about 50 mg/liter, about 20 mg/liter to about 60 mg/liter, about 20 mg/liter to about 70 mg/liter, about 20 mg/liter to about 80 mg/liter, about 20 mg/liter to about 90 mg/liter, about 20mg to about 90 mg/liter, about 40mg to about 40 mg/liter, about 40mg to about 30 mg/liter, about 40mg to about 40 mg/liter, about 20mg to about 30 mg/liter, about 20mg to about 60 mg/liter, about 60mg to about 60 mg/liter, about 20mg to about 60 mg/liter, about 70mg to about 70 mg/liter, about 20mg to about 50 mg/liter, about 20mg to about 60 mg/liter, about 60mg to about 70 mg/liter, about 70mg to about 70 mg/liter About 40 mg/liter to about 110 mg/liter, about 40 mg/liter to about 120 mg/liter, about 50 mg/liter to about 60 mg/liter, about 50 mg/liter to about 70 mg/liter, about 50 mg/liter to about 80 mg/liter, about 50 mg/liter to about 90 mg/liter, about 50 mg/liter to about 100 mg/liter, about 50 mg/liter to about 110 mg/liter, about 50 mg/liter to about 120 mg/liter, about 60 mg/liter to about 70 mg/liter, about 60 mg/liter to about 80 mg/liter, about 60 mg/liter to about 90 mg/liter, about 60 mg/liter to about 100 mg/liter, about 60 mg/liter to about 110 mg/liter, about 60 mg/liter to about 120 mg/liter, about 70 mg/liter to about 80 mg/liter, about 70 mg/liter to about 90 mg/liter, about 70 mg/liter to about 100 mg/liter, about 70 mg/liter to about 110 mg/liter, about 70 mg/liter to about 120 mg/liter, about 80mg to about 90 mg/liter, about 80mg to about 120 mg/liter, about 60mg to about 120 mg/liter. In some embodiments, each vitamin present in the medium is present at about 10 mg/liter, about 20 mg/liter, about 30 mg/liter, about 40 mg/liter, about 50 mg/liter, about 60 mg/liter, about 70 mg/liter, about 80 mg/liter, about 90 mg/liter, about 100 mg/liter, about 110 mg/liter, or about 120 mg/liter. In some embodiments, each vitamin present in the medium is present at least about 10 mg/liter, about 20 mg/liter, about 30 mg/liter, about 40 mg/liter, about 50 mg/liter, about 60 mg/liter, about 70 mg/liter, about 80 mg/liter, about 90 mg/liter, about 100 mg/liter, or about 110 mg/liter. In some embodiments, each vitamin present in the medium is present at up to about 20 mg/liter, about 30 mg/liter, about 40 mg/liter, about 50 mg/liter, about 60 mg/liter, about 70 mg/liter, about 80 mg/liter, about 90 mg/liter, about 100 mg/liter, about 110 mg/liter, or about 120 mg/liter.
In some embodiments, the glucose contained in the medium is anhydrous. In some embodiments, the glucose is present as an L isoform. In some embodiments, the glucose is present as the D isoform. In some embodiments, glucose exists in both isoforms. In some embodiments, the glucose present in the medium is present at about 500 mg/liter to about 1,600 mg/liter. In some embodiments of the present invention, in some embodiments, glucose is present in the medium in an amount of about 500 mg/liter to about 600 mg/liter, about 500 mg/liter to about 700 mg/liter, about 500 mg/liter to about 800 mg/liter, about 500 mg/liter to about 900 mg/liter, about 500 mg/liter to about 1,000 mg/liter, about 500 mg/liter to about 1,100 mg/liter, about 500 mg/liter to about 1,200 mg/liter, about 500 mg/liter to about 1,400 mg/liter, about 500 mg/liter to about 1,500 mg/liter, about 500 mg/liter to about 1,600 mg/liter, about 600 mg/liter to about 700 mg/liter, about 600 mg/liter to about 800 mg/liter, about 600 mg/liter to about 900 mg/liter, about 600 mg/liter to about 1,000 mg/liter, about 600 mg/liter to about 1,100 mg/liter, about 600 mg/liter to about 1,200 mg/liter, about 600 mg/liter to about 1,500 mg/liter, about 600 mg/liter, about 300 mg/liter, about 600 mg/liter, about 1,600 mg/liter, 300 mg/liter, about 600 mg/liter, about 1,600 mg/liter, 600 mg/liter, about about 600 mg/liter to about 1,400 mg/liter, about 600 mg/liter to about 1,500 mg/liter, about 600 mg/liter to about 1,600 mg/liter, about 700 mg/liter to about 800 mg/liter, about 700 mg/liter to about 900 mg/liter, about 700 mg/liter to about 1,000 mg/liter, about 700 mg/liter to about 1,100 mg/liter, about 700 mg/liter to about 1,200 mg/liter, about 700 mg/liter to about 1,300 mg/liter, about 700 mg/liter to about 1,400 mg/liter, about 700 mg/liter to about 1,500 mg/liter, about 700 mg/liter to about 1,600 mg/liter, about 800 mg/liter to about 900 mg/liter, about 800 mg/liter to about 1,000 mg/liter, about 800 mg/liter to about 1,100 mg/liter, about 800 mg/liter to about 1,200 mg/liter, about 800 mg/liter to about 1,300 mg/liter, about 400 mg/liter, about 800 mg/liter, 400 mg/liter, about 1,500 mg/liter, about 800 mg/liter, about 000 mg/liter, about 800 mg/liter to about 1,500 mg/liter, about 800 mg/liter to about 1,600 mg/liter, about 900 mg/liter to about 1,000 mg/liter, about 900 mg/liter to about 1,100 mg/liter, about 900 mg/liter to about 1,200 mg/liter, about 900 mg/liter to about 1,300 mg/liter, about 900 mg/liter to about 1,400 mg/liter, about 900 mg/liter to about 1,500 mg/liter, about 1,000 mg/liter to about 1,100 mg/liter, about 1,000 mg/liter to about 1,200 mg/liter, about 1,000 mg/liter to about 1,300 mg/liter, about 1,000 mg/liter to about 1,400 mg/liter, about 1,000 mg/liter to about 1,000 mg/liter, about 1,000 mg/liter to about 1,400 mg/liter, about 1,100 mg/liter to about 1,400 mg/liter, about 1,400 mg/liter to about 1,100 mg/liter, about 400 mg/liter to about 1,400 mg/liter, about 1,100 mg/liter to about 1,400 mg/liter, about 1,000 mg/liter to about 1,100 mg/liter, about 1,100 mg/liter to about 1,1,100 mg/liter to about 1,1 mg/liter to about 1,1,1 mg/liter to about 1,1 mg/liter to about 1 mg/liter. In some embodiments, the glucose present in the medium is present at about 500 mg/liter, about 600 mg/liter, about 700 mg/liter, about 800 mg/liter, about 900 mg/liter, about 1,000 mg/liter, about 1,100 mg/liter, about 1,200 mg/liter, about 1,300 mg/liter, about 1,400 mg/liter, about 1,500 mg/liter, or about 1,600 mg/liter. In some embodiments, the glucose present in the medium is present at least about 500 mg/liter, about 600 mg/liter, about 700 mg/liter, about 800 mg/liter, about 900 mg/liter, about 1,000 mg/liter, about 1,100 mg/liter, about 1,200 mg/liter, about 1,300 mg/liter, about 1,400 mg/liter, or about 1,500 mg/liter. In some embodiments, the glucose present in the medium is present at up to about 600 mg/liter, about 700 mg/liter, about 800 mg/liter, about 900 mg/liter, about 1,000 mg/liter, about 1,100 mg/liter, about 1,200 mg/liter, about 1,300 mg/liter, about 1,400 mg/liter, about 1,500 mg/liter, or about 1,600 mg/liter.
In some embodiments, lipoic acid is present in the medium from about 0.05 mg/liter to about 0.5 mg/liter. In some embodiments, the lipoic acid is present in the medium as DL-lipoic acid (thio acid). In some embodiments, lipoic acid is present in the medium at about 0.05 mg/liter to about 0.1 mg/liter, about 0.05 mg/liter to about 0.15 mg/liter, about 0.05 mg/liter to about 0.2 mg/liter, about 0.05 mg/liter to about 0.25 mg/liter, about 0.05 mg/liter to about 0.3 mg/liter, about 0.05 mg/liter to about 0.35 mg/liter, about 0.05 mg/liter to about 0.4 mg/liter, about 0.05 mg/liter to about 0.45 mg/liter, about 0.05 mg/liter to about 0.5 mg/liter, about 0.1 mg/liter to about 0.15 mg/liter, about 0.1 mg/liter to about 0.2 mg/liter, about 0.1 mg/liter to about 0.25 mg/liter, about 0.3 mg/liter to about 0.5 mg/liter, about 0.5 mg/liter to about 0.15 mg/liter, about 0.5 mg/liter to about 0.5 mg/liter, about 0.5mg to about 0.15 mg/liter, about 0.5 mg/liter to about 0.5 mg/liter, about 0.15mg to about 0.5 mg/liter, mg to about 0.15 mg/liter, about 0.3 mg/liter to about 0.4 mg/liter, about 0.3 mg/liter to about 0.45 mg/liter, about 0.3 mg/liter to about 0.5 mg/liter, about 0.35 mg/liter to about 0.4 mg/liter, about 0.35 mg/liter to about 0.45 mg/liter, about 0.35 mg/liter to about 0.5 mg/liter, about 0.4 mg/liter to about 0.45 mg/liter, about 0.4 mg/liter to about 0.5 mg/liter, or about 0.45 mg/liter to about 0.5 mg/liter. In some embodiments, lipoic acid is present in the medium at about 0.05 mg/liter, about 0.1 mg/liter, about 0.15 mg/liter, about 0.2 mg/liter, about 0.25 mg/liter, about 0.3 mg/liter, about 0.35 mg/liter, about 0.4 mg/liter, about 0.45 mg/liter, or about 0.5 mg/liter. In some embodiments, lipoic acid is present in the medium at least about 0.05 mg/liter, about 0.1 mg/liter, about 0.15 mg/liter, about 0.2 mg/liter, about 0.25 mg/liter, about 0.3 mg/liter, about 0.35 mg/liter, about 0.4 mg/liter, or about 0.45 mg/liter. In some embodiments, lipoic acid is present in the medium at up to about 0.1 mg/liter, about 0.15 mg/liter, about 0.2 mg/liter, about 0.25 mg/liter, about 0.3 mg/liter, about 0.35 mg/liter, about 0.4 mg/liter, about 0.45 mg/liter, or about 0.5 mg/liter.
In some embodiments, sodium bicarbonate is present in the medium at about 250 mg/liter to about 2,000 mg/liter. In some embodiments, sodium bicarbonate is present in the medium at about 250 mg/liter to about 500 mg/liter, about 250 mg/liter to about 750 mg/liter, about 250 mg/liter to about 1,000 mg/liter, about 250 mg/liter to about 1,750 mg/liter, about 250 mg/liter to about 2,000 mg/liter, about 500 mg/liter to about 750 mg/liter, about 500 mg/liter to about 1,000 mg/liter, about 500 mg/liter to about 1,250 mg/liter, about 500 mg/liter to about 1,750 mg/liter, about 500 mg/liter to about 1,000 mg/liter, about 750 mg/liter to about 2,000 mg/liter, about 750 mg/liter to about 1,750 mg/liter, about 750,750 mg/liter to about 1,000 mg/liter, about 1,750 mg/liter to about 1,750 mg/liter, about 1,750 mg/liter to about 1,000 mg/liter, about 750 mg/liter to about 1,750 mg/liter, about 1,000mg to about 1,000 mg/liter, about 750 mg/liter to about 1,000 mg/liter, about 1,000mg to about 1,000 mg/liter, about 750 mg/liter, about 1,000mg to about 1,000 mg/liter. In some embodiments, sodium bicarbonate is present in the medium at about 250 mg/liter, about 500 mg/liter, about 750 mg/liter, about 1,000 mg/liter, about 1,250 mg/liter, about 1,500 mg/liter, about 1,750 mg/liter, or about 2,000 mg/liter. In some embodiments, sodium bicarbonate is present in the medium at least about 250 mg/liter, about 500 mg/liter, about 750 mg/liter, about 1,000 mg/liter, about 1,250 mg/liter, about 1,500 mg/liter, or about 1,750 mg/liter. In some embodiments, sodium bicarbonate is present in the medium at up to about 500 mg/liter, about 750 mg/liter, about 1,000 mg/liter, about 1,250 mg/liter, about 1,500 mg/liter, about 1,750 mg/liter, or about 2,000 mg/liter.
In some embodiments, sodium pyruvate present in the medium is present at about 50 mg/liter to about 160 mg/liter. In some embodiments, sodium pyruvate is present in the medium at about 50 mg/liter to about 60 mg/liter, about 50 mg/liter to about 70 mg/liter, about 50 mg/liter to about 80 mg/liter, about 50 mg/liter to about 90 mg/liter, about 50 mg/liter to about 100 mg/liter, about 50 mg/liter to about 110 mg/liter, about 50 mg/liter to about 120 mg/liter, about 50 mg/liter to about 130 mg/liter, about 50 mg/liter to about 140 mg/liter, about 50 mg/liter to about 150 mg/liter, about 50 mg/liter to about 160 mg/liter, about 60 mg/liter to about 70 mg/liter, about 60 mg/liter to about 80 mg/liter, about 60 mg/liter to about 90 mg/liter, about 60 mg/liter to about 100 mg/liter, about 60 mg/liter to about 110 mg/liter, about 60mg to about 120 mg/liter, about 60 mg/liter to about 130 mg/liter, about 60mg to about 60 mg/liter, about 60mg to about 130 mg/liter, about 70mg to about 70 mg/liter, about 70mg to about 80 mg/liter, about 70mg to about 70 mg/liter, about 80mg to about 70 mg/liter, about 60mg to about 70mg to about 80 mg/liter, about 60mg to about 60 mg/liter, about 60mg to about 90 mg/liter, about 60mg to about 60 mg/liter, about 60mg to about 90 mg/liter, about 60mg to about 60 mg/liter, about 60mg to about 100 mg/liter, about 60mg to about 60 mg/liter About 80 mg/liter to about 150 mg/liter, about 80 mg/liter to about 160 mg/liter, about 90 mg/liter to about 100 mg/liter, about 90 mg/liter to about 110 mg/liter, about 90 mg/liter to about 120 mg/liter, about 90 mg/liter to about 130 mg/liter, about 90 mg/liter to about 140 mg/liter, about 90 mg/liter to about 150 mg/liter, about 90 mg/liter to about 160 mg/liter, about 100 mg/liter to about 110 mg/liter, about 100 mg/liter to about 120 mg/liter, about 100 mg/liter to about 130 mg/liter, about 100 mg/liter to about 140 mg/liter, about 100 mg/liter to about 150 mg/liter, about 100 mg/liter to about 160 mg/liter, about 110 mg/liter to about 120 mg/liter, about 110 mg/liter to about 130 mg/liter, about 110 mg/liter to about 140 mg/liter, about 110 mg/liter to about 150 mg/liter, about 160mg to about 160 mg/liter, about 160mg to about 130 mg/liter, about 160 mg/liter, about 150mg to about 120 mg/liter, about 120mg to about 130 mg/liter. In some embodiments, sodium pyruvate present in the culture medium is present at about 50 mg/liter, about 60 mg/liter, about 70 mg/liter, about 80 mg/liter, about 90 mg/liter, about 100 mg/liter, about 110 mg/liter, about 120 mg/liter, about 130 mg/liter, about 140 mg/liter, about 150 mg/liter, or about 160 mg/liter. In some embodiments, sodium pyruvate present in the culture medium is present at least about 50 mg/liter, about 60 mg/liter, about 70 mg/liter, about 80 mg/liter, about 90 mg/liter, about 100 mg/liter, about 110 mg/liter, about 120 mg/liter, about 130 mg/liter, about 140 mg/liter, or about 150 mg/liter. In some embodiments, sodium pyruvate present in the medium is present at up to about 60 mg/liter, about 70 mg/liter, about 80 mg/liter, about 90 mg/liter, about 100 mg/liter, about 110 mg/liter, about 120 mg/liter, about 130 mg/liter, about 140 mg/liter, about 150 mg/liter, or about 160 mg/liter.
In some embodiments, the αmem has a pH between 7.0 and 7.4.
In some embodiments, αmem comprises the ingredients shown in table 1.
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In some embodiments, vBA-MSCs are cultured in a medium comprising αmem as described in table 1, 10% stemlate hPL (heparin is not required), 2ng/mL recombinant carrier free FGF, and 2ng/mL recombinant carrier free EGF.
In some embodiments, the cells are cultured by culturing about 1,000 to about 100 ten thousand nucleated cells/cm in a culture dish 2 Is of the order of 5,900 cells/cm (for example 2 Plus and minus about 1,200), and then cultured for an additional number of days (e.g., about 14±about 2 days), the primary MSCs can be further passaged to non-primary cells (e.g., removed from the culture surface and expanded into additional areas). In suitable embodiments, the primary cells may be grown to confluence, and in some cases, the secondary culture of non-primary cells may be passaged to the primary cells by seeding the primary cells from the confluent primary cell culture in an amount below confluence in the secondary culture surface, and growing the non-primary culture to confluence. This method can be repeated for additional passages.
In some embodiments, MSCs in a therapeutic composition may be derived from a contiguous number of generations (i.e., they are within about 1 or about 2 or about 3 or about 4 cell doublings of each other). Optionally, the average number of cell doublings in a therapeutic composition of the compositions of the invention may be about 20 to about 25 doublings. Optionally, the average number of cell doublings in a therapeutic composition of the invention can be about 9 to about 13 (e.g., about 11 or about 11.2) doublings from primary culture, plus about 1, about 2, about 3, or about 4 doublings/passaging (e.g., about 2.5 doublings/passaging). Exemplary average cell doublings in the formulations of the invention, when produced by about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 passages, can be about 13.5, about 16, about 18.5, about 21, about 23.5, about 26, about 28.5, about 31, about 33.5, or about 36, respectively.
In some embodiments, MSCs in the therapeutic composition (e.g., vBA-MSCs) may be derived from MSCs that are subcultured by about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 times despite one or more population doublings.
In some embodiments, the formulations and compositions of the present disclosure may comprise at least 1 million vBA-MSCs, the vBA-MSCs having an antigen profile of greater than about 1.75% cd45+ cells, at least about 95% cd105+ cells, and at least about 95% cd166+ cells, and the cells may expand ex vivo from passage 2 through passage 4 while maintaining population uniformity based on the antigen profile (i.e., greater than about 1.75% cd45+ cells, at least about 95% cd105+ cells, and at least about 95% cd166+ cells).
In some embodiments, the formulations and compositions of the present disclosure may comprise vBA-MSCs having an antigen profile with reduced expression of one or more senescent cell markers compared to bone marrow derived MSCs prepared according to known MSC culture techniques. In some embodiments, the one or more senescent cell markers comprises MIC-A, MIC-B, ULBP2 or any combination thereof. NK cell mediated immune responses are stimulated by MIC-A, MIC-B and/or ULBP 2.
In some embodiments, the vBA-MSC formulations and compositions described herein comprise about 1% less cells than bone marrow derived MSCs to about 100% less cells than bone marrow derived MSCs that express one or more markers of senescent cells. In some embodiments of the present invention, in some embodiments, the vBA-MSC formulations and compositions described herein comprise an amount of cells expressing one or more markers of senescent cells that is about 100% to about 90% less than, about 100% to about 80% less than, about 100% to about 70% less than, about 100% to about 60% less than, about 100% to about 50% less than, about 100% to about 40% less than, about 100% to about 30% less than, about 100% to about 20% less than, about 100% to about 10% less than bone marrow-derived MSC about 100% to about 5% less than bone marrow derived MSCs, about 100% to about 1% less than bone marrow derived MSCs, about 90% to about 80% less than bone marrow derived MSCs, about 90% to about 70% less than bone marrow derived MSCs, about 90% to about 60% less than bone marrow derived MSCs, about 90% to about 50% less than bone marrow derived MSCs, about 90% to about 40% less than bone marrow derived MSCs, about 90% to about 30% less than bone marrow derived MSCs, about 90% to about 20% less than bone marrow derived MSCs, about 90% to about 10% less than bone marrow derived MSCs, about 90% to about 5% less than bone marrow derived MSCs, about 5% less than bone marrow derived MSCs About 90% to about 1% less than bone marrow derived MSCs, about 80% to about 70% less than bone marrow derived MSCs, about 80% to about 60% less than bone marrow derived MSCs, about 80% to about 50% less than bone marrow derived MSCs, about 80% to about 40% less than bone marrow derived MSCs, about 80% to about 30% less than bone marrow derived MSCs, about 80% to about 20% less than bone marrow derived MSCs, about 80% to about 10% less than bone marrow derived MSCs, about 80% to about 5% less than bone marrow derived MSCs, about 80% to about 1% less than bone marrow derived MSCs, about 70% to about 60% less than bone marrow derived MSCs, about 0% to about 60% less than bone marrow derived MSCs. About 70% to about 50% less than bone marrow derived MSCs, about 70% to about 40% less than bone marrow derived MSCs, about 70% to about 30% less than bone marrow derived MSCs, about 70% to about 20% less than bone marrow derived MSCs, about 70% to about 10% less than bone marrow derived MSCs, about 70% to about 5% less than bone marrow derived MSCs, about 70% to about 1% less than bone marrow derived MSCs, about 60% to about 50% less than bone marrow derived MSCs, about 60% to about 40% less than bone marrow derived MSCs, about 60% to about 30% less than bone marrow derived MSCs, about 60% to about 20% less than bone marrow derived MSCs, about 5% less than bone marrow derived MSCs, about 70% to about 1% less than bone marrow derived MSCs, about 60% to about 50% less than bone marrow derived MSCs, about 60% to about 40% less than bone marrow derived MSCs About 60% to about 10% less than bone marrow derived MSCs, about 60% to about 5% less than bone marrow derived MSCs, about 60% to about 1% less than bone marrow derived MSCs, about 50% to about 40% less than bone marrow derived MSCs, about 50% to about 30% less than bone marrow derived MSCs, about 50% to about 20% less than bone marrow derived MSCs, about 50% to about 10% less than bone marrow derived MSCs, about 50% to about 5% less than bone marrow derived MSCs, about 50% to about 1% less than bone marrow derived MSCs, about 40% to about 30% less than bone marrow derived MSCs, about 40% to about 20% less than bone marrow derived MSCs, about 5% less than bone marrow derived MSCs, about 40% to about 20% less than bone marrow derived MSCs. About 40% to about 10% less than bone marrow derived MSCs, about 40% to about 5% less than bone marrow derived MSCs, about 40% to about 1% less than bone marrow derived MSCs, about 30% to about 20% less than bone marrow derived MSCs, about 30% to about 10% less than bone marrow derived MSCs, about 30% to about 5% less than bone marrow derived MSCs, about 30% to about 1% less than bone marrow derived MSCs, about 20% to about 10% less than bone marrow derived MSCs, about 20% to about 5% less than bone marrow derived MSCs, about 20% to about 20% less than bone marrow derived MSCs, about 20% to about 1% less than bone marrow derived MSCs, about 10% to about 5% less than bone marrow derived MSCs, about 5% less than bone marrow derived MSCs About 10% less than bone marrow derived MSCs to about 1% less than bone marrow derived MSCs, about 5% less than bone marrow derived MSCs to about 1% less than bone marrow derived MSCs. In some embodiments, the vBA-MSC formulations and compositions described herein comprise about 100% less cells than bone marrow derived MSCs, about 90% less bone marrow derived MSCs, about 80% less bone marrow derived MSCs, about 70% less bone marrow derived MSCs, about 60% less bone marrow derived MSCs, about 50% less bone marrow derived MSCs, about 40% less bone marrow derived MSCs, about 30% less bone marrow derived MSCs, about 20% less bone marrow derived MSCs, about 10% less bone marrow derived MSCs, about 5% less bone marrow derived MSCs, or about 1% less bone marrow derived MSCs than the amount of cells expressing one or more markers of senescent cells. In some embodiments, the vBA-MSC formulations and compositions described herein comprise at least about 100% less cells than bone marrow derived MSCs, about 90% less bone marrow derived MSCs, about 80% less bone marrow derived MSCs, about 70% less bone marrow derived MSCs, about 60% less bone marrow derived MSCs, about 50% less bone marrow derived MSCs, about 40% less bone marrow derived MSCs, about 30% less bone marrow derived MSCs, about 20% less bone marrow derived MSCs, about 10% less bone marrow derived MSCs, or about 5% less bone marrow derived MSCs than bone marrow derived MSCs in amounts of cells expressing one or more markers of senescent cells. In some embodiments, the vBA-MSC formulations and compositions described herein comprise an amount of cells expressing one or more markers of senescent cells up to about 90% less than, about 80% less than, about 70% less than, about 60% less than, about 50% less than, about 40% less than, about 30% less than, about 20% less than, about 10% less than, about 5% or about 1% less than bone marrow-derived MSCs.
Thawing and storing
In some embodiments, provided herein is a method of warming a cryopreserved stem cell population to a first temperature and storing the stem cells at a second temperature that is less than about 40 ℃. In some embodiments, the cryopreserved stem cell population is warmed to a first temperature above about 0 ℃. In some embodiments, the first temperature is greater than about 20 ℃. In some embodiments, the second temperature at which the stem cells are stored is a cryogenic temperature.
In one aspect, provided herein is a method for preparing stem cells for infusion, the method comprising: (a) Providing a cryopreserved population of cells comprising the stem cells; (b) Warming the stem cells to a first temperature and maintaining the stem cells at the first temperature for a first period of time; and (c) changing the first temperature to a second temperature and maintaining the stem cells at the second temperature for a second period of time.
In some embodiments, the first temperature is greater than about 0 ℃, about 1 ℃, about 2 ℃, about 3 ℃, about 4 ℃, about 5 ℃, about 6 ℃, about 7 ℃, about 8 ℃, about 9 ℃, about 10 ℃, about 11 ℃, about 12 ℃, about 13 ℃, about 14 ℃, about 15 ℃, about 16 ℃, about 17 ℃, about 18 ℃, about 19 ℃, about 20 ℃, about 21 ℃, about 22 ℃, about 23 ℃, about 24 ℃, about 25 ℃, about 26 ℃, about 27 ℃, about 28 ℃, about 29 ℃, about 30 ℃, about 31 ℃, about 32 ℃, about 33 ℃, about 34 ℃, about 35 ℃, about 36 ℃, about 37 ℃, about 38 ℃, about 39 ℃, about 40 ℃. In some embodiments, the first temperature is about 0 ℃, about 1 ℃, about 2 ℃, about 3 ℃, about 4 ℃, about 5 ℃, about 6 ℃, about 7 ℃, about 8 ℃, about 9 ℃, about 10 ℃, about 11 ℃, about 12 ℃, about 13 ℃, about 14 ℃, about 15 ℃, about 16 ℃, about 17 ℃, about 18 ℃, about 19 ℃, about 20 ℃, about 21 ℃, about 22 ℃, about 23 ℃, about 24 ℃, about 25 ℃, about 26 ℃, about 27 ℃, about 28 ℃, about 29 ℃, about 30 ℃, about 31 ℃, about 32 ℃, about 33 ℃, about 34 ℃, about 35 ℃, about 36 ℃, about 37 ℃, about 38 ℃, about 39 ℃, about 40 ℃. In some embodiments, the first temperature is greater than 0 ℃.
In some embodiments, the second temperature is less than about 0 ℃, about 1 ℃, about 2 ℃, about 3 ℃, about 4 ℃, about 5 ℃, about 6 ℃, about 7 ℃, about 8 ℃, about 9 ℃, about 10 ℃, about 11 ℃, about 12 ℃, about 13 ℃, about 14 ℃, about 15 ℃, about 16 ℃, about 17 ℃, about 18 ℃, about 19 ℃, about 20 ℃, about 21 ℃, about 22 ℃, about 23 ℃, about 24 ℃, about 25 ℃, about 26 ℃, about 27 ℃, about 28 ℃, about 29 ℃, about 30 ℃, about 31 ℃, about 32 ℃, about 33 ℃, about 34 ℃, about 35 ℃, about 36 ℃, about 37 ℃, about 38 ℃, about 39 ℃, about 40 ℃. In some embodiments, the second temperature is about 0 ℃, about 1 ℃, about 2 ℃, about 3 ℃, about 4 ℃, about 5 ℃, about 6 ℃, about 7 ℃, about 8 ℃, about 9 ℃, about 10 ℃, about 11 ℃, about 12 ℃, about 13 ℃, about 14 ℃, about 15 ℃, about 16 ℃, about 17 ℃, about 18 ℃, about 19 ℃, about 20 ℃, about 21 ℃, about 22 ℃, about 23 ℃, about 24 ℃, about 25 ℃, about 26 ℃, about 27 ℃, about 28 ℃, about 29 ℃, about 30 ℃, about 31 ℃, about 32 ℃, about 33 ℃, about 34 ℃, about 35 ℃, about 36 ℃, about 37 ℃, about 38 ℃, about 39 ℃, about 40 ℃. In some embodiments, the second temperature is low temperature. In some embodiments, the second temperature is less than 40 ℃.
In some embodiments, the first period of time is less than about one week. In some embodiments, the period of time is less than about 7 days, about 6 days, about 5 days, about 4 days, about 3 days, about 2 days, or about 1 day. In some embodiments, the period of time is less than about 5 days. In some embodiments, the period of time is less than about 2 days. In some embodiments, the period of time is less than about 1 day. In some embodiments, the time period is less than about 24 hours, about 23 hours, about 22 hours, about 21 hours, about 20 hours, about 19 hours, about 18 hours, about 17 hours, about 16 hours, about 15 hours, about 14 hours, about 13 hours, about 12 hours, about 11 hours, about 10 hours, about 9 hours, about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, or about 1 hour. In some embodiments, the period of time is less than about 12 hours. In some embodiments, the period of time is less than about 6 hours. In some embodiments, the period of time is less than about 2 hours. In some embodiments, the time period is less than about 60 minutes, about 59 minutes, about 58 minutes, about 57 minutes, about 56 minutes, about 55 minutes, about 54 minutes, about 53 minutes, about 52 minutes, about 51 minutes, about 50 minutes, about 49 minutes, about 48 minutes, about 47 minutes, about 46 minutes, about 45 minutes, about 44 minutes, about 43 minutes, about 42 minutes, about 41 minutes, about 40 minutes, about 39 minutes, about 38 minutes, about 37 minutes, about 36 minutes, about 35 minutes, about 34 minutes, about 33 minutes, about 32 minutes, about 31 minutes, about 30 minutes, about 29 minutes, about 28 minutes, about 27 minutes, about 26 minutes, about 25 minutes, about 24 minutes, about 23 minutes, about 22 minutes, about 21 minutes, about 20 minutes, about 19 minutes, about 18 minutes, about 17 minutes, about 16 minutes, about 15 minutes, about 14 minutes, about 13 minutes, about 12 minutes, about 11 minutes, about 10 minutes, about 9 minutes, about 8 minutes, about 7 minutes, about 6 minutes, about 5 minutes, about 4 minutes, about 3 minutes, about 2 minutes, or about 1 minute.
In some embodiments, the stem cells are cultured after they are warmed/thawed. In some embodiments, the culturing is not calculated in the first time period described herein. In some embodiments, the culturing is calculated in a first time period. In some embodiments, the culturing follows the MSC culturing methods described herein. In some embodiments, the post-thawing culture method comprises not allowing for MSC population doubling. In some embodiments, MSCs are recovered and/or packaged prior to doubling. In some embodiments, the post-thawing culture method comprises culturing the cells at the first temperature at about 10,000 to about 50,000, about 10,000 to about 40,000, about 10,000 to about 30,000, about 10,000 to about 20,000, about 20,000 to about 50,000, about 20,000 to about 40,000, about 20,000 to about 30,000, about 30,000 to about 50,000, about 30,000 to about 40,000, or about 40,000 to about 50,000 cells/cm 2 Culturing the stem cells. In some embodiments, for the methods provided herein, the method further comprises, prior to (c), at the first temperature, at about 10,000 cells/cm 2 Up to about 50,000 cells/cm 2 Culturing the stem cells. In some embodiments, for the methods provided herein, the method further comprises, prior to (c), at the first temperature, at about 10,000, about 20,000, about 30,000, about 40,000, or about 50,000 cells/cm 2 Culturing the stem cells. In some embodiments, for the methods provided herein, the method further comprises, prior to (c), at the first temperature, at about 32,000 cells/cm 2 Culturing the stem cells. In some embodiments, post-thawing culturing is performed in a T25 flask. In some embodiments, post-thawing culturing is performed in a T75 flask.In some embodiments, post-thawing culturing is performed in flasks of other sizes.
In some embodiments, the stem cells in Plasma-Lyte A+2.5% HSA (rinse medium) at 10×10 6 A 5mL volume package of living cells/mL. In some embodiments, stem cells are present at about 1×10 6 From about 12X 10 cells/mL 6 A volume of viable cells/mL. In some embodiments, stem cells are used at a rate of about 1X 10≡6 viable cells/mL to about 2X 10 6 Living cells/mL, about 1X 10 6 From about 3X 10 cells/mL 6 Living cells/mL, about 1X 10 6 From about 4X 10 cells/mL 6 Living cells/mL, about 1X 10 6 From about 5X 10 cells/mL 6 Living cells/mL, about 1X 10 6 From about 6X 10 cells/mL 6 Living cells/mL, about 1X 10 6 From about 7X 10 cells/mL 6 Living cells/mL, about 1X 10 6 From about 8X 10 cells/mL 6 Living cells/mL, about 1X 10 6 From about 9X 10 cells/mL 6 Living cells/mL, about 1X 10 6 From about 10X 10 cells/mL 6 Living cells/mL, about 1X 10 6 From about 11X 10 cells/mL 6 Living cells/mL, about 1X 10 6 From about 12X 10 cells/mL 6 Living cells/mL, about 2X 10 6 From about 3X 10 cells/mL 6 Living cells/mL, about 2X 10 6 From about 4X 10 cells/mL 6 Living cells/mL, about 2X 10 6 From about 5X 10 cells/mL 6 Living cells/mL, about 2X 10 6 From about 6X 10 cells/mL 6 Living cells/mL, about 2X 10 6 From about 7X 10 cells/mL 6 Living cells/mL, about 2X 10 6 From about 8X 10 cells/mL 6 Living cells/mL, about 2X 10 6 From about 9X 10 cells/mL 6 Living cells/mL, about 2X 10 6 From about 10X 10 cells/mL 6 Living cells/mL, about 2X 10 6 From about 11X 10 cells/mL 6 Living cells/mL, about 2X 10 6 From about 12X 10 cells/mL 6 Living cells/mL, about 3X 10 6 From about 4X 10 cells/mL 6 Living cells/mL, about 3X 10 6 From about 5X 10 cells/mL 6 Living cells/mL, about 3X 10 6 From about 6X 10 cells/mL 6 Living cells/mL, about 3X 10 6 From about 7X 10 cells/mL 6 Living cells/mL, about 3X 10 6 From about 8X 10 cells/mL 6 Living cells/mL, about 3X 10 6 From about 9X 10 cells/mL 6 Living cells/mL, about 3X 10 6 From about 10X 10 cells/mL 6 Living cells/mL, about 3X 10 6 From about 11X 10 cells/mL 6 Living cells/mL, about 3X 10 6 From about 12X 10 cells/mL 6 Individual living cells/mL, about 4X 10 6 From about 5X 10 cells/mL 6 Individual living cells/mL, about 4X 10 6 From about 6X 10 cells/mL 6 Individual living cells/mL, about 4X 10 6 From about 7X 10 cells/mL 6 Individual living cells/mL, about 4X 10 6 From about 8X 10 cells/mL 6 Individual living cells/mL, about 4X 10 6 From about 9X 10 cells/mL 6 Individual living cells/mL, about 4X 10 6 From about 10X 10 cells/mL 6 Individual living cells/mL, about 4X 10 6 From about 11X 10 cells/mL 6 Individual living cells/mL, about 4X 10 6 From about 12X 10 cells/mL 6 About 5X 10 cells/mL 6 From about 6X 10 cells/mL 6 About 5X 10 cells/mL 6 From about 7X 10 cells/mL 6 About 5X 10 cells/mL 6 From about 8X 10 cells/mL 6 About 5X 10 cells/mL 6 From about 9X 10 cells/mL 6 About 5X 10 cells/mL 6 From about 10X 10 cells/mL 6 About 5X 10 cells/mL 6 From about 11X 10 cells/mL 6 About 5X 10 cells/mL 6 From about 12X 10 cells/mL 6 About 6X 10 cells/mL 6 From about 7X 10 cells/mL 6 About 6X 10 cells/mL 6 From about 8X 10 cells/mL 6 About 6X 10 cells/mL 6 From about 9X 10 cells/mL 6 About 6X 10 cells/mL 6 From about 10X 10 cells/mL 6 About 6X 10 cells/mL 6 From about 11X 10 cells/mL 6 About 6X 10 cells/mL 6 From about 12X 10 cells/mL 6 Individual living cells/mL, about 7X 10 6 From about 8X 10 cells/mL 6 Individual living cells/mL, about 7X 10 6 From about 9X 10 cells/mL 6 Individual living cells/mL, about 7X 10 6 From about 10X 10 cells/mL 6 Individual living cells/mL, about 7X 10 6 From about 11X 10 cells/mL 6 Individual living cells/mL, about 7X 10 6 From about 12X 10 cells/mL 6 Individual living cells/mL, about 8X 10 6 From about 9X 10 cells/mL 6 Individual living cells/mL, about 8X 10 6 From about 10X 10 cells/mL 6 Individual living cells/mL, about 8X 10 6 From about 11X 10 cells/mL 6 Individual living cells/mL, about 8X 10 6 From about 12X 10 cells/mL 6 Individual living cells/mL, about 9X 10 6 From about 10X 10 cells/mL 6 Individual living cells/mL, about 9X 10 6 From about 11X 10 cells/mL 6 Individual living cells/mL, about 9X 10 6 From about 12X 10 cells/mL 6 Individual living cells/mL, about 10X 10 6 From about 11X 10 cells/mL 6 Individual living cells/mL, about 10X 10 6 From about 12X 10 cells/mL 6 Individual living cells/mL or about 11X 10 6 From about 12X 10 cells/mL 6 A volume of viable cells/mL. In some embodiments, stem cells are present at about 1×10 6 Living cells/mL, about 2X 10 6 Living cells/mL, about 3X 10 6 Individual living cells/mL, about 4X 10 6 About 5X 10 cells/mL 6 About 6X 10 cells/mL 6 Individual living cells/mL, about 7X 10 6 Individual living cells/mL, about 8X 10 6 Individual living cells/mL, about 9X 10 6 Individual living cells/mL, about 10X 10 6 About 11X 10 cells/mL 6 Individual living cells/mL or about 12X 10 6 A volume of viable cells/mL. In some embodiments, the stem cells are present in at least about 1×10 6 Living cells/mL, about 2X 10 6 Living cells/mL, about 3X 10 6 Individual living cells/mL, about 4X 10 6 About 5X 10 cells/mL 6 About 6X 10 cells/mL 6 Individual living cells/mL, about 7X 10 6 Individual living cells/mL, about 8X 10 6 Individual living cells/mL, about 9X 10 6 Individual living cells/mL, about 10X 10 6 Individual living cells/mL or about 11X 10 6 A volume of viable cells/mL. In some embodiments, the stem cells are present in an amount up to about 2X 10 6 Living cells/mL, about 3X 10 6 Individual living cells/mL, about 4X 10 6 About 5X 10 cells/mL 6 About 6X 10 cells/mL 6 Individual living cells/mL, about 7X 10 6 Individual living cells/mL, about 8X 10 6 Individual living cells/mL, about 9X 10 6 Individual living cells/mL, about 10X 10 6 About 11X 10 cells/mL 6 Individual living cells/mL or about 12X 10 6 A volume of viable cells/mL.
In some embodiments, the living stem cells are packaged in a vial containing about 1mL to about 12 mL. In some embodiments of the present invention, in some embodiments, packaging the living stem cells in a container comprising about 1mL to about 2mL, about 1mL to about 3mL, about 1mL to about 4mL, about 1mL to about 5mL, about 1mL to about 6mL, about 1mL to about 7mL, about 1mL to about 8mL, about 1mL to about 9mL, about 1mL to about 10mL, about 1mL to about 11mL, about 1mL to about 12mL, about 2mL to about 3mL, about 2mL to about 4mL, about 2mL to about 5mL, about 2mL to about 6mL, about 2mL to about 7mL, about 2mL to about 8mL, about 2mL to about 9mL, about 2mL to about 10mL, about 2mL to about 11mL, about 2mL to about 12mL, about 3mL to about 4mL, about 3mL to about 5mL, about 3mL to about 6mL, about 3mL to about 7mL, about 3mL to about 8mL, about 3mL to about 9mL, about 3mL to about 10mL, about 3mL to about 11mL, about 3mL to about 12mL, about 4mL to about 5mL, about 4mL to about 6mL, about 4mL to about 7mL, from about 4mL to about 8mL, from about 4mL to about 9mL, from about 4mL to about 10mL, from about 4mL to about 11mL, from about 4mL to about 12mL, from about 5mL to about 6mL, from about 5mL to about 7mL, from about 5mL to about 8mL, from about 5mL to about 9mL, from about 5mL to about 10mL, from about 5mL to about 11mL, from about 5mL to about 12mL, from about 6mL to about 7mL, from about 6mL to about 8mL, from about 6mL to about 9mL, from about 6mL to about 10mL, from about 6mL to about 11mL, from about 6mL to about 12mL, from about 7mL to about 8mL, from about 7mL to about 9mL, from about 7mL to about 11mL, from about 7mL to about 12mL, from about 8mL to about 9mL, from about 8mL to about 11mL, from about 8mL to about 12mL, from about 9mL to about 10mL, from about 9mL to about 11mL, from about 9mL to about 12mL, from about 10mL to about 10mL, from about 7mL to about 11mL, from about 7mL to about 12mL, from about 11mL, from about 8mL to about 11mL, from about 11 mL. In some embodiments, the living stem cells are packaged in a vial containing about 1mL, about 2mL, about 3mL, about 4mL, about 5mL, about 6mL, about 7mL, about 8mL, about 9mL, about 10mL, about 11mL, or about 12 mL. In some embodiments, the living stem cells are packaged in a vial containing at least about 1mL, about 2mL, about 3mL, about 4mL, about 5mL, about 6mL, about 7mL, about 8mL, about 9mL, about 10mL, or about 11 mL. In some embodiments, the living stem cells are packaged in a vial containing up to about 2mL, about 3mL, about 4mL, about 5mL, about 6mL, about 7mL, about 8mL, about 9mL, about 10mL, about 11mL, or about 12 mL.
Once the stem cells are packaged, the stem cells are placed at a second temperature for a second period of time. In some embodiments, the second temperature is less than about 40 ℃, about 39 ℃, about 38 ℃, about 37 ℃, about 36 ℃, about 35 ℃, about 34 ℃, about 33 ℃, about 32 ℃, about 31 ℃, about 30 ℃, about 29 ℃, about 28 ℃, about 27 ℃, about 26 ℃, about 25 ℃, about 24 ℃, about 23 ℃, about 22 ℃, about 21 ℃, about 20 ℃, about 19 ℃, about 18 ℃, about 17 ℃, about 16 ℃, about 15 ℃, about 14 ℃, about 13 ℃, about 12 ℃, about 11 ℃, about 10 ℃, about 9 ℃, about 8 ℃, about 7 ℃, about 6 ℃, about 5 ℃, about 4 ℃, about 3 ℃, about 2 ℃, or about 1 ℃. In some embodiments, the cell culture is maintained at about 40 ℃, about 39 ℃, about 38 ℃, about 37 ℃, about 36 ℃, about 35 ℃, about 34 ℃, about 33 ℃, about 32 ℃, about 31 ℃, about 30 ℃, about 29 ℃, about 28 ℃, about 27 ℃, about 26 ℃, about 25 ℃, about 24 ℃, about 23 ℃, about 22 ℃, about 21 ℃, about 20 ℃, about 19 ℃, about 18 ℃, about 17 ℃, about 16 ℃, about 15 ℃, about 14 ℃, about 13 ℃, about 12 ℃, about 11 ℃, about 10 ℃, about 9 ℃, about 8 ℃, about 7 ℃, about 6 ℃, about 5 ℃, about 4 ℃, about 3 ℃, about 2 ℃, or about 1 ℃. In some embodiments, the second temperature is less than 37 ℃. In some embodiments, the second temperature is less than 35 ℃. In some embodiments, the second temperature is less than 30 ℃. In some embodiments, the second temperature is less than 25 ℃. In some embodiments, the second temperature is less than 20 ℃. In some embodiments, the second temperature is about 2 ℃ to about 8 ℃.
In some embodiments, the second period of time is less than about one week. In some embodiments, the period of time is less than about 7 days, about 6 days, about 5 days, about 4 days, about 3 days, about 2 days, or about 1 day. In some embodiments, the period of time is less than about 5 days. In some embodiments, the period of time is less than about 2 days. In some embodiments, the period of time is less than about 1 day. In some embodiments, the time period is less than about 24 hours, about 23 hours, about 22 hours, about 21 hours, about 20 hours, about 19 hours, about 18 hours, about 17 hours, about 16 hours, about 15 hours, about 14 hours, about 13 hours, about 12 hours, about 11 hours, about 10 hours, about 9 hours, about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, or about 1 hour. In some embodiments, the period of time is less than about 12 hours. In some embodiments, the period of time is less than about 6 hours. In some embodiments, the period of time is less than about 2 hours. In some embodiments, the time period is less than about 60 minutes, about 59 minutes, about 58 minutes, about 57 minutes, about 56 minutes, about 55 minutes, about 54 minutes, about 53 minutes, about 52 minutes, about 51 minutes, about 50 minutes, about 49 minutes, about 48 minutes, about 47 minutes, about 46 minutes, about 45 minutes, about 44 minutes, about 43 minutes, about 42 minutes, about 41 minutes, about 40 minutes, about 39 minutes, about 38 minutes, about 37 minutes, about 36 minutes, about 35 minutes, about 34 minutes, about 33 minutes, about 32 minutes, about 31 minutes, about 30 minutes, about 29 minutes, about 28 minutes, about 27 minutes, about 26 minutes, about 25 minutes, about 24 minutes, about 23 minutes, about 22 minutes, about 21 minutes, about 20 minutes, about 19 minutes, about 18 minutes, about 17 minutes, about 16 minutes, about 15 minutes, about 14 minutes, about 13 minutes, about 12 minutes, about 11 minutes, about 10 minutes, about 9 minutes, about 8 minutes, about 7 minutes, about 6 minutes, about 5 minutes, about 4 minutes, about 3 minutes, about 2 minutes, or about 1 minute.
In another aspect, provided herein is a method for preparing a sample of stem cells for direct infusion, the method comprising:
a. providing a cell culture comprising a sample of said stem cells, wherein said cell culture is cryopreserved and passaged;
b. thawing the cell culture; and
c. the cell culture was maintained at less than 40 ℃.
In some embodiments, after (b), the cell culture is not multiplied.
In some embodiments, the cell culture is maintained under conditions of low temperature. In some embodiments, the cell culture is maintained at less than about 40 ℃, about 39 ℃, about 38 ℃, about 37 ℃, about 36 ℃, about 35 ℃, about 34 ℃, about 33 ℃, about 32 ℃, about 31 ℃, about 30 ℃, about 29 ℃, about 28 ℃, about 27 ℃, about 26 ℃, about 25 ℃, about 24 ℃, about 23 ℃, about 22 ℃, about 21 ℃, about 20 ℃, about 19 ℃, about 18 ℃, about 17 ℃, about 16 ℃, about 15 ℃, about 14 ℃, about 13 ℃, about 12 ℃, about 11 ℃, about 10 ℃, about 9 ℃, about 8 ℃, about 7 ℃, about 6 ℃, about 5 ℃, about 4 ℃, about 3 ℃, about 2 ℃, or about 1 ℃. In some embodiments, the cell culture is maintained at about 40 ℃, about 39 ℃, about 38 ℃, about 37 ℃, about 36 ℃, about 35 ℃, about 34 ℃, about 33 ℃, about 32 ℃, about 31 ℃, about 30 ℃, about 29 ℃, about 28 ℃, about 27 ℃, about 26 ℃, about 25 ℃, about 24 ℃, about 23 ℃, about 22 ℃, about 21 ℃, about 20 ℃, about 19 ℃, about 18 ℃, about 17 ℃, about 16 ℃, about 15 ℃, about 14 ℃, about 13 ℃, about 12 ℃, about 11 ℃, about 10 ℃, about 9 ℃, about 8 ℃, about 7 ℃, about 6 ℃, about 5 ℃, about 4 ℃, about 3 ℃, about 2 ℃, or about 1 ℃. In some embodiments, the cell culture is maintained at less than 37 ℃. In some embodiments, the cell culture is maintained at less than 35 ℃. In some embodiments, the cell culture is maintained at less than 30 ℃. In some embodiments, the cell culture is maintained at less than 25 ℃. In some embodiments, the cell culture is maintained at less than 20 ℃. In some embodiments, the cell culture is maintained at about 2 ℃ to about 8 ℃.
In some embodiments of the present invention, in some embodiments, the thawing occurs at about 10 to about 100 ℃, about 10 to about 90 ℃, about 10 to about 80 ℃, about 10 to about 70 ℃, about 10 to about 60 ℃, about 10 to about 50 ℃, about 10 to about 40 ℃, about 10 to about 30 ℃, about 10 to about 20 ℃, about 20 to about 100 ℃, about 20 to about 90 ℃, about 20 to about 80 ℃, about 20 to about 70 ℃, about 20 to about 60 ℃, about 20 to about 50 ℃, about 20 to about 40 ℃, about 20 to about 30 ℃, about 30 to about 100 ℃, about 30 to about 90 ℃, about 30 to about 80 ℃, about 30 to about 70 ℃, about 30 to about 60 ℃, about about 30 ℃ to about 50 ℃, about 30 ℃ to about 40 ℃, about 40 ℃ to about 100 ℃, about 40 ℃ to about 90 ℃, about 40 ℃ to about 80 ℃, about 40 ℃ to about 70 ℃, about 40 ℃ to about 60 ℃, about 40 ℃ to about 50 ℃, about 50 ℃ to about 100 ℃, about 50 ℃ to about 90 ℃, about 50 ℃ to about 80 ℃, about 50 ℃ to about 70 ℃, about 50 ℃ to about 60 ℃, about 60 ℃ to about 100 ℃, about 60 ℃ to about 90 ℃, about 60 ℃ to about 80 ℃, about 60 ℃ to about 70 ℃, about 70 ℃ to about 100 ℃, about 70 ℃ to about 90 ℃, about 70 ℃ to about 80 ℃, about 80 ℃ to about 100 ℃, about 80 ℃ to about 90 ℃, or about 90 ℃ to about 100 ℃. In some embodiments, the thawing occurs at about 30 ℃ to about 40 ℃. In some embodiments, the thawing occurs at about 30 ℃. In some embodiments, the thawing occurs at about 31 ℃. In some embodiments, the thawing occurs at about 32 ℃. In some embodiments, the thawing occurs at about 33 ℃. In some embodiments, the thawing occurs at about 34 ℃. In some embodiments, the thawing occurs at about 35 ℃. In some embodiments, the thawing occurs at about 36 ℃. In some embodiments, the thawing occurs at about 37 ℃. In some embodiments, the thawing occurs at about 38 ℃. In some embodiments, the thawing occurs at about 39 ℃. In some embodiments, the thawing occurs at about 40 ℃.
In some embodiments, the cell culture is maintained at less than room temperature for at least about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 11 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 16 minutes, about 17 minutes, about 18 minutes, about 19 minutes, about 20 minutes, about 21 minutes, about 22 minutes, about 23 minutes, about 24 minutes, about 25 minutes, about 26 minutes, about 27 minutes, about 28 minutes, about 29 minutes, about 30 minutes, about 31 minutes, about 32 minutes, about 33 minutes, about 34 minutes, about 35 minutes, about 36 minutes, about 37 minutes, about 38 minutes, about 39 minutes, about 40 minutes, about 41 minutes, about 42 minutes, about 43 minutes, about 44 minutes, about 45 minutes, about 46 minutes, about 47 minutes, about 48 minutes, about 49 minutes, about 50 minutes, about 51 minutes, about 52 minutes, about 53 minutes, about 54 minutes, about 55 minutes, about 57, or about 57 minutes. In some embodiments, the cell culture is maintained at below room temperature for at least about 30 minutes. In some embodiments, the cell culture is maintained at less than room temperature for at least about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 31 hours, about 32 hours, about 33 hours, about 34 hours, about 35 hours, about 36 hours, about 37 hours, about 38 hours, about 39 hours, about 40 hours, about 41 hours, about 42 hours, about 43 hours, about 44 hours, about 45 hours, about 46 hours, about 48 hours, or about 46 hours. In some embodiments, the cell culture is maintained at room temperature for at least 12 hours. In some embodiments, the cell culture is maintained at below room temperature for at least 24 hours. In some embodiments, the cell culture is maintained at room temperature for at least 48 hours.
In another aspect, provided herein is a method for preparing a sample of stem cells for direct infusion, the method comprising:
a. providing a cell culture comprising a sample of said stem cells, wherein said cell culture is cryopreserved and passaged;
b. thawing the cell culture; and
c. the cell culture is maintained at above 0 ℃.
In some embodiments, the cell culture is maintained at a temperature above about 0 ℃, about 1 ℃, about 2 ℃, about 3 ℃, about 4 ℃, about 5 ℃, about 6 ℃, about 7 ℃, about 8 ℃, about 9 ℃, about 10 ℃, about 11 ℃, about 12 ℃, about 13 ℃, about 14 ℃, about 15 ℃, about 16 ℃, about 17 ℃, about 18 ℃, about 19 ℃, about 20 ℃, about 21 ℃, about 22 ℃, about 23 ℃, about 24 ℃, about 25 ℃, about 26 ℃, about 27 ℃, about 28 ℃, about 29 ℃, about 30 ℃, about 31 ℃, about 32 ℃, about 33 ℃, about 34 ℃, about 35 ℃, about 36 ℃, about 37 ℃, about 38 ℃, about 39 ℃, or about 40 ℃. In some embodiments, the cell culture is maintained at about 0 ℃, about 1 ℃, about 2 ℃, about 3 ℃, about 4 ℃, about 5 ℃, about 6 ℃, about 7 ℃, about 8 ℃, about 9 ℃, about 10 ℃, about 11 ℃, about 12 ℃, about 13 ℃, about 14 ℃, about 15 ℃, about 16 ℃, about 17 ℃, about 18 ℃, about 19 ℃, about 20 ℃, about 21 ℃, about 22 ℃, about 23 ℃, about 24 ℃, about 25 ℃, about 26 ℃, about 27 ℃, about 28 ℃, about 29 ℃, about 30 ℃, about 31 ℃, about 32 ℃, about 33 ℃, about 34 ℃, about 35 ℃, about 36 ℃, about 37 ℃, about 38 ℃, about 39 ℃, or about 40 ℃. In some embodiments, the cell culture is maintained under conditions of low temperature. In some embodiments, the cell culture is maintained at greater than about 0 ℃. In some embodiments, the cell culture is maintained at about 2 ℃ to about 8 ℃.
Cell passage
In one aspect, provided herein is a method for preparing stem cells for infusion, the method comprising:
a. providing a cryopreserved population of cells comprising the stem cells;
b. warming the stem cells to a first temperature and maintaining the stem cells at the first temperature for a first period of time; and
c. changing the first temperature to a second temperature and maintaining the stem cells at the second temperature for a period of time.
In some embodiments, for the methods provided herein, the stem cells are passaged prior to (a), e.g., during the culture methods described herein. In some embodiments, for the methods provided herein, the stem cells are passaged at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times prior to (a). In some embodiments, for the methods provided herein, the stem cells are passaged at least once prior to (a). In some embodiments, for the methods provided herein, the stem cells are passaged at least 2 times prior to (a). In some embodiments, for the methods provided herein, the stem cells are passaged at least 4 times prior to (a). In some embodiments, for the methods provided herein, the stem cells are passaged 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times prior to (a). In some embodiments, for the methods provided herein, the stem cells are passaged more than 10 times prior to (a).
In another aspect, provided herein is a method for preparing a sample of stem cells for direct infusion, the method comprising:
a. providing a cell culture comprising a sample of said stem cells, wherein said cell culture is cryopreserved and passaged;
b. thawing the cell culture; and
c. the cell culture was maintained at less than 40 ℃.
In some embodiments, the cell culture is passaged. In some embodiments, the cell culture is passaged at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times. In some embodiments, the cell culture is passaged at least once. In some embodiments, the cell culture is passaged at least 2 times. In some embodiments, the cell culture is passaged at least 4 times. In some embodiments, the cell culture is passaged 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times.
Cell culture and packaging
In some embodiments, for the methods provided herein, the method further comprises, prior to (c), at the first temperature, at about 10,000 to about 50,000, about 10,000 to about 40,000, about 10,000 to about 30,000, about 10,000 to about 20,000, about 20,000 to about 50,000, about 20,000 to about 40,000, about 20,000 to about 30,000, about 30,000 to about 50,000, about 30,000 to about 40,000, or about 40,000 to about 50,000 cells/cm 2 Culturing the stem cells. In some embodiments, for the methods provided herein, the method further comprises, prior to (c), at the first temperature, at about 10,000 cells/cm 2 Up to about 50,000 cells/cm 2 Culturing the stem cells. In some embodiments, for the methods provided herein, the method further comprises, prior to (c), at the first temperature, at about 10,000, about 20,000, about 30,000, about 40,000, or about 50,000 cells/cm 2 Culturing the stem cells. In some embodiments, for the methods provided herein, the method further comprises, prior to (c), at the first temperature, at about 32,000 cells/cm 2 Culturing the stem cells.
In some embodiments, for the methods provided herein, the method further comprises, prior to (c), contacting the stem cells with a cell culture medium comprising about 10 x 10 2 Up to about 10X 10 10 About 10×10 2 Up to about 10X 10 9 About 10×10 2 Up to about 10X 10 8 About 10×10 2 Up to about 10X 10 7 About 10×10 2 Up to about 10X 10 6 About 10×10 2 Up to about 10X 10 5 About 10×10 2 Up to about 10X 10 4 About 10×10 2 Up to about 10X 10 3 About 10×10 3 Up to about 10X 10 10 About 10×10 3 Up to about 10X 10 9 About 10×10 3 Up to about 10X 10 8 About 10×10 3 Up to about 10X 10 7 About 10×10 3 Up to about 10X 10 6 About 10×10 3 Up to about 10X 10 5 About 10×10 3 Up to about 10X 10 4 About 10×10 4 Up to about 10X 10 10 About 10×10 4 Up to about 10X 10 9 About 10×10 4 Up to about 10X 10 8 About 10×10 4 Up to about 10X 10 7 About 10×10 4 Up to about 10X 10 6 About 10×10 4 Up to about 10X 10 5 About 10×10 5 Up to about 10X 10 10 About 10×10 5 Up to about 10X 10 9 About 10×10 5 Up to about 10X 10 8 About 10×10 5 Up to about 10X 10 7 About 10×10 5 Up to about 10X 10 6 About 10×10 6 Up to about 10X 10 10 About 10×10 6 Up to about 10X 10 9 About 10×10 6 Up to about 10X 10 8 About 10×10 6 Up to about 10X 10 7 About 10×10 7 Up to about 10X 10 10 About 10×10 7 Up to about 10X 10 9 About 10×10 7 Up to about 10X 10 8 About 10×10 8 Up to about 10X 10 10 About 10×10 8 Up to about 10X 10 9 Or about 10X 10 9 Up to about 10X 10 10 A volume of living cells/mL, and maintaining the stem cells at the second temperature. In some embodiments, for the methods provided herein, the method further comprises, prior to (c), contacting the stem cells with a cell culture medium comprising about 10 x 10 2 From about 10X 10 cells/mL 10 A volume of living cells/mL, and maintaining the stem cells at the second temperature. In some embodiments, for the methods provided herein, the method further comprises, prior to (c), contacting the stem cells with a cell culture medium comprising about 10 x 10 6 A volume of living cells/mL, and maintaining the stem cells at the second temperature.
In some embodiments, for the methods provided herein, the method further comprises, prior to (c), packaging the stem cells in a volume comprising less than 10mL, 9mL, 8mL, 7mL, or 6 mL. In some embodiments, for the methods provided herein, the method further comprises, prior to (c), packaging the stem cells in a volume comprising 10mL, 9mL, 8mL, 7mL, 6mL, 5mL, 4mL, 3mL, 2mL, or 1 mL. In some embodiments, for the methods provided herein, the method further comprises, prior to (c), packaging the stem cells in a volume comprising 5 mL.
Cell viability
In some embodiments, for the methods provided herein, after (c), the sample of stem cells comprises at least about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% viable cells. In some embodiments, for the methods provided herein, after (c), the sample of stem cells comprises about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% viable cells. In some embodiments, for the methods provided herein, after (c), the sample of stem cells comprises at least about 70% viable cells. In some embodiments, for the methods provided herein, after (c), the sample of stem cells comprises about 70% viable cells.
In another aspect, provided herein is a method for preparing a sample of stem cells for direct infusion, the method comprising:
a. providing a cell culture comprising a sample of said stem cells, wherein said cell culture is cryopreserved and passaged;
b. thawing the cell culture; and
c. maintaining the cell culture at below room temperature;
wherein the sample of stem cells comprises at least about 70% viable cells.
In some embodiments, for the methods provided herein, after (c), the sample of stem cells comprises at least about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% viable cells. In some embodiments, for the methods provided herein, after (c), the sample of stem cells comprises about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% viable cells. In some embodiments, for the methods provided herein, after (c), the sample of stem cells comprises at least about 70% viable cells. In some embodiments, for the methods provided herein, after (c), the sample of stem cells comprises about 70% viable cells.
Cryopreservation and rinsing Medium
In some embodiments, for the methods provided herein, the cell culture is cryopreserved in a cryopreservation medium, wherein the cryopreservation medium comprises an electrolyte formulation, human Serum Albumin (HSA), dimethyl sulfoxide (DMSO), or any combination thereof.
In some embodiments, the cryopreservation media comprises about 1% to about 10%, about 1% to about 9%, about 1% to about 8%, about 1% to about 7%, about 1% to about 6%, about 1% to about 5%, about 1% to about 4%, about 1% to about 3%, about 1% to about 2%, about 2% to about 10%, about 2% to about 9%, about 2% to about 8%, about 2% to about 7%, about 2% to about 6%, about 2% to about 5%, about 2% to about 4%, about 2% to about 3%, about 3% to about 10%, about 3% to about 9%, about 3% to about 8%, about 3% to about 7%, about 3% to about 5%, about 3% to about 4%, about 4% to about 10%, about 4% to about 9%, about 4% to about 8%, about 4% to about 6%, about 4% to about 5%, about 5% to about 10%, about 5% to about 9%, about 5% to about 7%, about 7% to about 6%, about 10% to about 10%, about 10% to about 7%, about 10% to about 8%, about 10% to about 9%, about 10% to about 8%. In some embodiments, the cryopreservation media comprises about 1% to about 5% HSA. In some embodiments, the cryopreservation media comprises about 2.5% HSA.
In some embodiments of the present invention, in some embodiments, the cryopreservation media comprises about 1% to about 10%, about 1% to about 9%, about 1% to about 8%, about 1% to about 7%, about 1% to about 6%, about 1% to about 5%, about 1% to about 4%, about 1% to about 3%, about 1% to about 2%, about 2% to about 10%, about 2% to about 9%, about 2% to about 8%, about 2% to about 7%, about 2% to about 6%, about 2% to about 5%, about 2% to about 4%, about 2% to about 3%, about 3% to about 10%, about 3% to about 9%, about 3% to about 8%, about 3% to about 7%, about 3% to about 6%, about about 3% to about 5%, about 3% to about 4%, about 4% to about 10%, about 4% to about 9%, about 4% to about 8%, about 4% to about 7%, about 4% to about 6%, about 4% to about 5%, about 5% to about 10%, about 5% to about 9%, about 5% to about 8%, about 5% to about 7%, about 5% to about 6%, about 6% to about 10%, about 6% to about 9%, about 6% to about 8%, about 6% to about 7%, about 7% to about 10%, about 7% to about 9%, about 7% to about 8%, about 8% to about 10%, about 8% to about 9%, or about 9% to about 10% DMSO. In some embodiments, the cryopreservation media comprises about 1% to about 10% DMSO. In some embodiments, the cryopreservation media comprises about 5% DMSO.
In some embodiments, the electrolyte formulation is boy vein a.
In some embodiments, for the methods provided herein, the method further comprises, prior to (c), re-suspending the cell culture in a wash medium, wherein the wash medium comprises an electrolyte formulation, human Serum Albumin (HSA), or both. In some embodiments, for the methods provided herein, the method further comprises, prior to (c), re-suspending the cell culture in a wash medium, wherein the wash medium comprises an electrolyte formulation, human Serum Albumin (HSA), or both.
In some embodiments, the rinse medium is fresh.
In some embodiments, the wash medium comprises about 1% to about 10%, about 1% to about 9%, about 1% to about 8%, about 1% to about 7%, about 1% to about 6%, about 1% to about 5%, about 1% to about 4%, about 1% to about 3%, about 1% to about 2%, about 2% to about 10%, about 2% to about 9%, about 2% to about 8%, about 2% to about 7%, about 2% to about 6%, about 2% to about 5%, about 2% to about 4%, about 2% to about 3%, about 3% to about 10%, about 3% to about 9%, about 3% to about 8%, about 3% to about 7%, about 3% to about 5%, about 3% to about 4%, about 4% to about 10%, about 4% to about 9%, about 4% to about 7%, about 4% to about 6%, about 4% to about 5%, about 5% to about 10%, about 5% to about 9%, about 7% to about 7%, about 10% to about 8%, about 10% to about 10%, about 10% to about 8%, about 10% to about 9%, about 10% to about 8%, or about 10%. In some embodiments, the wash medium comprises about 1% to about 5% HSA. In some embodiments, the wash medium comprises about 2.5% HSA.
In some embodiments, the electrolyte formulation is boy vein a.
Antigens of low temperature stored mesenchymal stem cells
In another aspect, provided herein is a method for preparing a sample of stem cells for direct infusion, the method comprising:
a. providing a cell culture comprising a sample of said stem cells, wherein said cell culture is cryopreserved and passaged;
b. thawing the cell culture; and
c. maintaining the cell culture at below room temperature;
wherein the sample of stem cells comprises from less than about 1% to about 10% cd45+ cells.
In some embodiments of the present invention, in some embodiments, the sample of stem cells comprises less than about 1% to about 10%, about 1% to about 9%, about 1% to about 8%, about 1% to about 7%, about 1% to about 6%, about 1% to about 5%, about 1% to about 4%, about 1% to about 3%, about 1% to about 2%, about 2% to about 10%, about 2% to about 9%, about 2% to about 8%, about 2% to about 7%, about 2% to about 6%, about 2% to about 5%, about 2% to about 4%, about 2% to about 3%, about 3% to about 10%, about 3% to about 9%, about 3% to about 8%, about 3% to about 7%, about 3% to about 6%, about about 3% to about 5%, about 3% to about 4%, about 4% to about 10%, about 4% to about 9%, about 4% to about 8%, about 4% to about 7%, about 4% to about 6%, about 4% to about 5%, about 5% to about 10%, about 5% to about 9%, about 5% to about 8%, about 5% to about 7%, about 5% to about 6%, about 6% to about 10%, about 6% to about 9%, about 6% to about 8%, about 6% to about 7%, about 7% to about 10%, about 7% to about 9%, about 7% to about 8%, about 8% to about 10%, about 8% to about 9%, or about 9% to about 10% of cd45+ cells. In some embodiments, the sample of stem cells comprises from less than about 1% to about 10% cd45+ cells. In some embodiments, the sample of stem cells comprises less than about 5% cd45+ cells.
In some embodiments of the present invention, in some embodiments, the sample of stem cells comprises less than about 1% to about 10%, about 1% to about 9%, about 1% to about 8%, about 1% to about 7%, about 1% to about 6%, about 1% to about 5%, about 1% to about 4%, about 1% to about 3%, about 1% to about 2%, about 2% to about 10%, about 2% to about 9%, about 2% to about 8%, about 2% to about 7%, about 2% to about 6%, about 2% to about 5%, about 2% to about 4%, about 2% to about 3%, about 3% to about 10%, about 3% to about 9%, about 3% to about 8%, about 3% to about 7%, about 3% to about 6%, about 3% to about 5%, about 3% to about 4%, about 4% to about 10%, about 4% to about 9%, about 4% to about 7%, about 4% to about 5%, about 5% to about 10%, about 5% to about 9%, about 5% to about 8%, about 7% to about 7%, about 7% to about 8%, about 10% to about 10%, about 10% to about 8%, about 10% to about 10%, about 7% to about 8%, about 10% to about 10%. In some embodiments, the sample of stem cells comprises from less than about 1% to about 10% cd34+ cells. In some embodiments, the sample of stem cells comprises less than about 5% cd34+ cells.
In another aspect, provided herein is a method for preparing a sample of stem cells for direct infusion, the method comprising:
a. providing a cell culture comprising a sample of said stem cells, wherein said cell culture is cryopreserved and passaged;
b. thawing the cell culture; and
c. maintaining the cell culture at below room temperature;
wherein the sample of stem cells comprises at least about 90% to about 99% cd90+ cells.
In some embodiments of the present invention, in some embodiments, the sample of stem cells comprises at least about 90% to about 99%, about 90% to about 98%, about 90% to about 97%, about 90% to about 96%, about 90% to about 95%, about 90% to about 94%, about 90% to about 93%, about 90% to about 92%, about 90% to about 91%, about 91% to about 99%, about 91% to about 98%, about 91% to about 97%, about 91% to about 96%, about 91% to about 95%, about 91% to about 94%, about 91% to about 93%, about 91% to about 92%, about 92% to about 99%, about 92% to about 98%, about 92% to about 97%, about 92% to about 96%, about 92% to about 95%, about about 92% to about 94%, about 92% to about 93%, about 93% to about 99%, about 93% to about 98%, about 93% to about 97%, about 93% to about 96%, about 93% to about 95%, about 93% to about 94%, about 94% to about 99%, about 94% to about 98%, about 94% to about 97%, about 94% to about 96%, about 94% to about 95%, about 95% to about 99%, about 95% to about 98%, about 95% to about 97%, about 95% to about 96%, about 96% to about 99%, about 96% to about 98%, about 96% to about 97%, about 97% to about 99%, about 97% to about 98%, or about 98% to about 99% of cd90+ cells. In some embodiments, the sample of stem cells comprises at least about 90% to about 99% cd90+ cells. In some embodiments, the sample of stem cells comprises at least about 95% cd90+ cells.
In another aspect, provided herein is a method for preparing a sample of stem cells for direct infusion, the method comprising:
a. providing a cell culture comprising a sample of said stem cells, wherein said cell culture is cryopreserved and passaged;
b. thawing the cell culture; and
c. maintaining the cell culture at below room temperature;
wherein the sample of stem cells comprises at least about 90% to about 99% cd105+ cells.
In some embodiments of the present invention, in some embodiments, the sample of stem cells comprises at least about 90% to about 99%, about 90% to about 98%, about 90% to about 97%, about 90% to about 96%, about 90% to about 95%, about 90% to about 94%, about 90% to about 93%, about 90% to about 92%, about 90% to about 91%, about 91% to about 99%, about 91% to about 98%, about 91% to about 97%, about 91% to about 96%, about 91% to about 95%, about 91% to about 94%, about 91% to about 93%, about 91% to about 92%, about 92% to about 99%, about 92% to about 98%, about 92% to about 97%, about 92% to about 96%, about 92% to about 95%, about about 92% to about 94%, about 92% to about 93%, about 93% to about 99%, about 93% to about 98%, about 93% to about 97%, about 93% to about 96%, about 93% to about 95%, about 93% to about 94%, about 94% to about 99%, about 94% to about 98%, about 94% to about 97%, about 94% to about 96%, about 94% to about 95%, about 95% to about 99%, about 95% to about 98%, about 95% to about 97%, about 95% to about 96%, about 96% to about 99%, about 96% to about 98%, about 96% to about 97%, about 97% to about 99%, about 97% to about 98%, or about 98% to about 99% of cd105+ cells. In some embodiments, the sample of stem cells comprises at least about 90% to about 99% cd105+ cells. In some embodiments, the sample of stem cells comprises at least about 95% cd105+ cells.
Fibroblast colony forming units (CFU-F) of low temperature stored mesenchymal stem cells
In another aspect, provided herein is a method for preparing a sample of stem cells for direct infusion, the method comprising:
a. providing a cell culture comprising a sample of said stem cells, wherein said cell culture is cryopreserved and passaged;
b. thawing the cell culture; and
c. maintaining the cell culture at below room temperature;
wherein the sample of stem cells comprises at least about 10,000 to about 300,000CFU-F/1 million viable cells.
In some embodiments of the present invention, in some embodiments, the sample of stem cells comprises at least about 10,000 to about 300,000, about 10,000 to about 250,000, about 10,000 to about 200,000, about 10,000 to about 150,000, about 10,000 to about 100,000, about 10,000 to about 50,000, about 10,000 to about 40,000, about 10,000 to about 30,000, about 10,000 to about 20,000, about 20,000 to about 300,000, about 20,000 to about 250,000, about 20,000 to about 200,000, about 20,000 to about 150,000, about 20,000 to about 100,000, about 20,000 to about 50,000, about 20,000 to about 40,000, about 20,000 to about 30,000, about 30,000 to about 300,000, about 30,000 to about 250,000, about 30,000 to about 150,000, about 30,000 to about 100,000, about 30,000 to about 30,000, about 30,000 to about 50,000, about 50,000 about 30,000 to about 40,000, about 40,000 to about 300,000, about 40,000 to about 250,000, about 40,000 to about 200,000, about 40,000 to about 150,000, about 40,000 to about 100,000, about 40,000 to about 50,000, about 50,000 to about 300,000, about 50,000 to about 250,000, about 50,000 to about 200,000, about 50,000 to about 150,000, about 50,000 to about 100,000, about 100,000 to about 300,000, about 100,000 to about 250,000, about 100,000 to about 200,000, about 100,000 to about 150,000, about 150,000 to about 300,000, about 150,000 to about 250,000, about 150,000 to about 200,000, about 200,000 to about 300,000, about 200,000 to about 250,000, or about 250,000 to about 300,000CFU-F/1 million viable cells. In some embodiments, the sample of stem cells comprises at least about 10,000 to about 300,000CFU-F/1 million viable cells. In some embodiments, the sample of stem cells comprises at least about 10,000CFU-F/1 million viable cells.
Type of stem cells
In some embodiments, for the methods provided herein, the sample of stem cells comprises vertebrae adherent mesenchymal stem cells (vBA-MSC), vertebral bone marrow mesenchymal stem cells (vBM-MSC), or both. In some embodiments, for the methods provided herein, the sample of stem cells comprises vertebrae adherent mesenchymal stem cells (vBA-MSCs). In some embodiments, for the methods provided herein, the sample of stem cells comprises a centrum bone marrow mesenchymal stem cell (vBM-MSC). In some embodiments, for the methods provided herein, the sample of stem cells comprises both vertebrae adherent mesenchymal stem cells (vBA-MSCs) and vertebral bone marrow mesenchymal stem cells (vBM-MSCs).
In another aspect, provided herein is a method for preparing a sample of vertebral adherent mesenchymal stem cells (vBA-MSCs) for direct infusion, the method comprising:
a. providing a cell culture comprising a sample of said stem cells, wherein said cell culture is cryopreserved and passaged;
b. thawing the cell culture; and
c. the cell culture is maintained at below room temperature.
In some embodiments, for the methods provided herein, the stem cells are derived from cadavers.
Definition of the definition
While preferred embodiments of the present disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Many variations, changes, and substitutions will now occur to those skilled in the art without departing from the disclosure. It should be understood that various alternatives to the embodiments of the disclosure described herein may be employed in practicing the disclosure. The following claims are intended to define the scope of the disclosure and their methods and structures within the scope of these claims and their equivalents are thereby covered.
The use of absolute or sequential terms, such as "to," "should," "must," "first," "initially," "next," "then," "before … …," "after … …," "last," and "final," is not meant to limit the scope of the embodiments of the invention disclosed herein, but is exemplary.
As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the term "includes," including, "" has, "" having, "" with, "or variants thereof are used in either the detailed description and/or the claims, such term is intended to be inclusive in a manner similar to the term" comprising.
As used herein, the phrases "at least one/at least one", "one or more/one or more", and "and/or" are open-ended expressions that are both conjunctive and disjunctive in operation. For example, each of the expressions "at least one of A, B and C", "at least one of A, B or C", "one or more of A, B and C", "one or more of A, B or C" and "A, B and/or C" means a alone, B alone, C, A and B together, a and C together, B and C together, or A, B and C together.
As used herein, "or" may refer to "and", "or", or "and/or", and may be used exclusively and in inclusion. For example, the term "a or B" may refer to "a or B", "a but not B", "B but not a", and "a and B". In some cases, the context may determine a particular meaning.
Any of the systems, methods, software, and platforms described herein are modular. Thus, terms such as "first" and "second" do not necessarily imply a priority, order of importance, or order of action.
When referring to a number or range of values, the term "about" means that the number or range of values referred to is an approximation within experimental variability (or within statistical experimental error), and that the number or range of values may vary, for example, from 1% to 15% of the number or range of values. In an example, the term "about" refers to ±10% of the number or value.
The terms "increase", "increase" or "increase" are generally used herein to mean an increase in a statistically significant amount. In some aspects, the term "increase" or "increase" means an increase of at least 10% compared to a reference level, e.g., an increase of at least about 10%, at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including 100% or any increase between 10-100% compared to a reference level, standard or control. Other examples of "increasing" include increasing by at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 1000-fold, or more as compared to a reference level.
The terms "reduced", "reduction" or "reduction" are generally meant herein to mean a reduction in a statistically significant amount. In some aspects, "reducing" or "reducing" means at least a 10% reduction from a reference level, such as at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% reduction (e.g., a lack of level or an undetectable level compared to a reference level), or any reduction between 10-100%. In the context of markers or symptoms, these terms mean a statistically significant reduction in such levels. The reduction may be, for example, at least 10%, at least 20%, at least 30%, at least 40% or more, and preferably to a level that is acceptable within normal ranges for individuals without the given disease.
As used herein, the term "passaging" refers to removing medium from a cell culture and transferring cells into freshly grown medium. Passaging allows further propagation of the cell line.
Examples
Example 1: recovery of Mesenchymal Stem Cells (MSC) after cryopreservation
According to standard protocols, generation 2 (P2) Mesenchymal Stem Cells (MSCs) from 3 donors were cryopreserved in 5% freezing medium (bomai a+2.5% human serum albumin+5% DMSO) in 2mL freezing vials. The vials were removed from the liquid nitrogen vapor storage and quickly thawed in a 37 ℃ water bath. The cells were diluted with 1 volume of wash medium (Boehmeria A+2.5% human serum albumin), centrifuged, resuspended, and concentrated at 32,000 cells/cm 2 Or 800,000 cells/T25 (which corresponds to a density of 100M cell end of production (EOP) in 5-chamber CellSTACK) were plated in T25 flasks. At 24 hours, cells were removed from the first set of flasks. At 48 hours, cells were removed from the second set of flasks. After removal, cells were counted and plated for fibroblast colony forming units (CFU-F). CFU-F was stained with crystal violet after 10 days and colonies containing 50 or more cells were counted.
24 hour data are listed in Table 2 and 48 hour data are listed in Table 3. The data included donor, viable cell/mL count, total cell/mL count,% recovery, and CFU-F results. FIG. 1 graphically illustrates CFU-F data.
Table 2:24 hour MSC culture recovery
Table 3:48 hours MSC culture recovery
These data indicate that logarithmic amplification after thawing begins between 24 and 48 hours.
The cells were then washed with 10X 10 in Plasma-Lyte A+2.5% HSA (washing medium) 6 5mL volumes of each live P4 vBM-MSC/mL were packed inIn 5mL frozen vials (Sexton Biotechnologies of indianapolis, indiana). The vials were placed on wet ice resulting in low temperature conditions (e.g., about 2 ℃ to 8 ℃). Packaging with wet ice, the cells are transported to the recipient. Transportation may take anywhere from 30 minutes to 2 days or more.
Example 2: recovery of vertebral adherent mesenchymal stem cells (vBA-MSC) after cryopreservation relative to vertebral bone marrow mesenchymal stem cells (vBM-MSC)
Vertebral adherent mesenchymal stem cells (vBA-MSCs) and vertebral bone marrow mesenchymal stem cells (vBM-MSCs) were cryopreserved in 5% freezing medium (bomai a+2.5% human serum albumin+5% DMSO) in 2mL freezing vials according to standard protocols. The vials were removed from the liquid nitrogen vapor storage and quickly thawed in a 37 ℃ water bath. The cells were diluted with 1 volume of wash medium (Boehmeria A+2.5% human serum albumin), centrifuged, resuspended, and concentrated at 32,000 cells/cm 2 Or 800,000 cells/T25 (which corresponds to a density of 100M cell end of production (EOP) in 5-chamber CellSTACK) were plated in T25 flasks. At 24 hours, cells were removed from the first set of flasks. At 48 hours, cells were removed from the second set of flasks. After removal, cells were counted and plated for fibroblast colony forming units (CFU-F). CFU-F was stained with crystal violet after 10 days and colonies containing 50 or more cells were counted.
The vertebrae adherent mesenchymal stem cells (vBA-MSC) are expected to produce a higher number of fibroblast colony forming units (CFU-F) than the vertebral bone marrow mesenchymal stem cells (vBM-MSC).
Example 3: low temperature storage and administration scheme for centrum mesenchymal stem cells
Frozen and recovered 4 th generation (P4) vertebral bone marrow mesenchymal stem cells (vBM-MSC) are prepared fromHuman cadaveric organs and tissue donors were generated at 10X 10 in Plasma-Lyte A+2.5% HSA (rinse Medium) 6 5mL volumes of each live P4 vBM-MSC/mL were packed inIn 5mL frozen vials (Sexton Biotechnologies of indianapolis, indiana). The product and packaging arrangement is designed for direct injection without further manipulation at the clinical site.
After receipt at the clinical site, the frozen recovered P4 vBM-MSC vials were stored at low temperature (2 ℃ to 8 ℃) in a cryoort C3 shipper (shipper) until ready for patient administration. Removing the cooler cover and phase change panel from the crystal C3 shipper, and removing the crystal C3 shipperA vial. Check if the vial is damaged and ensure that the label is clearly discernible and still adhered.
When the patient is ready for injection, a recovered P4 vBM-MSC vial is removed from cryogenic storage. The vials were gently inverted several times to mix thoroughly and placed upright in a biosafety cabinet with the tubing and ports at the top of the treatment rack. The foil was removed from the bottom of the withdrawal port, the withdrawal port septum was thoroughly wiped with a sterile alcohol pad, and allowed to air dry for up to one minute before entry. The 5mL syringe is preloaded with 2mL of air. The syringe is attached to the vial adapter via a luer lock connection. The preloaded air is slowly vented to ensure that the luer-activated valve is open.
Piercing with stable forceVial septum, ensuring vertical access of spike to septum. Cut open with clean scissors->A vent tube, care was taken to ensure that the fill tube was not sheared. Holding vial adapter master The body and syringe plunger are gradually withdrawn until the sample is retrieved. The vial adapter body is grasped and the syringe is disconnected. Treatment->Vials and vial adapters. The needle was attached to the syringe and the sample was administered according to the clinical protocol. The second vial was administered using the same procedure as the first vial. Two vials correspond to one dose.
Example 4: recovery, storage and transport of centrum mesenchymal stem cells
Vertebral adherent mesenchymal stem cells (vBA-MSCs) and vertebral bone marrow mesenchymal stem cells (vBM-MSCs) are recovered from one or more cadavers as described in PCT/US2021/055066 filed herein and on 10 months 14 of 2021, which is hereby incorporated by reference in its entirety.
After recovery, the MSC population was cryopreserved as described herein and in PCT/US 2021/055066. At the time of cryopreservation, MSCs are stored in one of a plurality of cell bank memory centers located at different geographical locations.
When MSC infusion/administration is required to a patient in need thereof, the attending physician and/or physician employer contacts the cell bank storage central network to identify a recommended population of MSCs based on the particular characteristics of the patient in need thereof, such as Human Leukocyte Antigen (HLA) profile.
After identifying the recommended population of MSCs, the sample comprising the recommended population of MSCs is maintained at a first temperature above 0 ℃ for thawing. After thawing, the recommended MSC population was resuspended in alpha-MEM medium as described in table 1. The recommended MSC population is then expanded to about 32,000 cells/cm 2 Is plated in alpha-MEM medium in a cell storage medium at about room temperature. The recommended MSC population is cultured for 24-48 hours under conditions that maintain less than about 70-90% cell storage medium matrix confluence and allowed to undergo 0 to no more than 3 population doublings (e.g., example 2 described herein).
Finally, the recommended MSC population, i.e. the recommended MSC population, is packaged and transported, e.g. according to example 3 described herein, is kept at a second temperature, which is a low temperature. The recommended MSC population is then administered to the patient in need thereof as described in example 3.
While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Many variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. The following claims are intended to define the scope of the invention and their methods and structures within the scope of these claims and their equivalents are thereby covered.
Claims (135)
1. A method for preparing stem cells for infusion, the method comprising:
a. providing a cryopreserved population of cells comprising the stem cells;
b. warming the stem cells to a first temperature and maintaining the stem cells at the first temperature for a first period of time; and
c. changing the first temperature to a second temperature and maintaining the stem cells at the second temperature for a period of time.
2. The method of any one of the preceding claims, wherein the first temperature is greater than 0 ℃.
3. The method of any one of the preceding claims, wherein the second temperature is less than 40 ℃.
4. The method of any one of the preceding claims, wherein the second temperature is cryogenic.
5. The method of any one of the preceding claims, wherein the first period of time or the period of time is less than one week.
6. The method of any one of the preceding claims, wherein the first period of time or the period of time is less than 5 days.
7. The method of any one of the preceding claims, wherein the first period of time or the period of time is less than 2 days.
8. The method of any one of the preceding claims, wherein the first period of time or the period of time is less than 1 day.
9. The method of any one of the preceding claims, wherein the first period of time or the period of time is less than 12 hours.
10. The method of any one of the preceding claims, wherein the first period of time or the period of time is less than 6 hours.
11. The method of any one of the preceding claims, wherein the first period of time or the period of time is less than 2 hours.
12. The method of any one of the preceding claims, wherein the stem cells are passaged prior to (a).
13. The method of any one of the preceding claims, wherein the stem cells are passaged at least once prior to (a).
14. The method of any one of the preceding claims, wherein the stem cells are passaged at least 2 times prior to (a).
15. The method of any one of the preceding claims, wherein the stem cells are passaged at least 4 times prior to (a).
16. The method of any one of the preceding claims, further comprising, prior to (c), at the first temperature, at about 10,000 cells/cm 2 Up to about 50,000 cells/cm 2 Culturing the stem cells.
17. The method of any one of the preceding claims, further comprising, prior to (c), at the first temperature, at about 32,000 cells/cm 2 Culturing the stem cells.
18. The method of any one of the preceding claims, further comprising, prior to (c), the stem cells to comprise about 10 x 10 2 From about 10X 10 cells/mL 10 A volume of living cells/mL, and maintaining the stem cells at the second temperature.
19. The method of any one of the preceding claims, further comprising, prior to (c), the stem cells to comprise about 10 x 10 6 A volume of living cells/mL, and maintaining the stem cells at the second temperature.
20. The method of any one of the preceding claims, further comprising, prior to (c), packaging the stem cells in a volume comprising less than 10mL, 9mL, 8mL, 7mL, or 6 mL.
21. The method of any one of the preceding claims, further comprising, prior to (c), packaging the stem cells in a volume comprising 5 mL.
22. The method of any one of the preceding claims, wherein the stem cells are maintained at the first temperature in a medium comprising one or more amino acids.
23. The method of any one of the preceding claims, wherein the medium comprising one or more amino acids further comprises one or more vitamins.
24. The method of any one of the preceding claims, wherein the medium comprising one or more amino acids further comprises table 1.
25. The method of any one of the preceding claims, wherein the first period of time is a period of time in which the stem cells are not multiplied.
26. The method of any one of the preceding claims, wherein the first period of time is a period of time in which the stem cells undergo one or more population doublings.
27. The method of any one of the preceding claims, wherein the stem cells are maintained at a confluency of less than 80% relative to a cell culture storage medium during the first period of time or the period of time.
28. The method of any one of the preceding claims, wherein the stem cells are maintained in a wash medium at the second temperature, wherein the wash medium comprises an electrolyte formulation, human Serum Albumin (HSA), or both.
29. The method of any one of the preceding claims, wherein the stem cells comprise vertebral adherent mesenchymal stem cells (vBA-MSC), vertebral bone marrow mesenchymal stem cells (vBM-MSC), or both.
30. The method of any one of the preceding claims, wherein the stem cells are derived from cadavers.
31. The method of any one of the preceding claims, wherein the stem cells comprise at least about 10,000 to about 300,000CFU-F/1 million viable cells.
32. The method of any one of the preceding claims, wherein the stem cells comprise less than about 1% to about 10% cd45+ cells.
33. A method for preparing a sample of stem cells for direct infusion, the method comprising:
a. providing a cell culture comprising a sample of said stem cells, wherein said cell culture is cryopreserved and passaged;
b. thawing the cell culture; and
c. the cell culture was maintained at less than 40 ℃.
34. The method of claim 33, wherein the cell culture is passaged at least once.
35. The method of any one of the preceding claims, wherein after (b), the cell culture is not multiplied.
36. The method of any one of the preceding claims, wherein after (b), the cell culture is maintained to undergo one or more population doublings.
37. The method of any one of the preceding claims, wherein after (b), the cell culture is maintained at a confluency of less than 80% relative to a cell culture storage medium.
38. The method of any one of the preceding claims, wherein the cell culture is maintained in a medium comprising one or more amino acids.
39. The method of any one of the preceding claims, wherein the medium comprising one or more amino acids further comprises one or more vitamins.
40. The method of any one of the preceding claims, wherein the medium comprising one or more amino acids further comprises table 1.
41. The method of any one of the preceding claims, wherein the cell culture is passaged at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, or at least 10 times.
42. The method of any one of the preceding claims, wherein the cell culture is cryopreserved in a cryopreservation medium, wherein the cryopreservation medium comprises an electrolyte formulation, human Serum Albumin (HSA), dimethyl sulfoxide (DMSO), or any combination thereof.
43. The method of any one of the preceding claims, wherein the cryopreservation media comprises about 1% to about 5% HSA.
44. The method of any one of the preceding claims, wherein the cryopreservation media comprises about 2.5% HSA.
45. The method of any one of the preceding claims, wherein the cryopreservation medium comprises about 1% to about 10% DMSO.
46. The method of any one of the preceding claims, wherein the cryopreservation medium comprises about 5% DMSO.
47. The method of any one of the preceding claims, wherein the electrolyte formulation is a boy pulse force a.
48. The method of any of the preceding claims, wherein the thawing occurs at about 30 ℃ to about 40 ℃.
49. The method of any of the preceding claims, wherein the thawing occurs at about 37 ℃.
50. The method of any one of the preceding claims, further comprising re-suspending the cell culture in a wash medium, wherein the wash medium comprises an electrolyte formulation, human Serum Albumin (HSA), or both; maintaining the cell culture at a temperature below room temperature
51. The method of any one of the preceding claims, further comprising, prior to (c), re-suspending the cell culture in a wash medium, wherein the wash medium comprises an electrolyte formulation, human Serum Albumin (HSA), or both.
52. The method of any one of the preceding claims, wherein the rinse medium is fresh.
53. The method of any one of the preceding claims, wherein the wash medium comprises about 1% to about 5% HSA.
54. The method of any one of the preceding claims, wherein the wash medium comprises about 2.5% HSA.
55. The method of any one of the preceding claims, wherein the electrolyte formulation is a boy pulse force a.
56. The method of any one of the preceding claims, wherein the cell culture is maintained at room temperature for at least about 30 minutes.
57. The method of any one of the preceding claims, wherein the cell culture is maintained at room temperature for at least 12 hours.
58. The method of any one of the preceding claims, wherein the cell culture is maintained at room temperature for at least 24 hours.
59. The method of any one of the preceding claims, wherein the cell culture is maintained at room temperature for at least 48 hours.
60. The method of any one of the preceding claims, wherein the cell culture is maintained at less than 37 ℃.
61. The method of any one of the preceding claims, wherein the cell culture is maintained at less than 35 ℃.
62. The method of any one of the preceding claims, wherein the cell culture is maintained at less than 30 ℃.
63. The method of any one of the preceding claims, wherein after (b), the cell culture is maintained under conditions of low temperature.
64. The method of any one of the preceding claims, wherein the cell culture is maintained at less than 25 ℃.
65. The method of any one of the preceding claims, wherein the cell culture is maintained at less than 20 ℃.
66. The method of any one of the preceding claims, wherein the cell culture is maintained at about 2 ℃ to about 8 ℃.
67. The method of any one of the preceding claims, further comprising at about 10,000 cells/cm 2 Up to about 50,000 cells/cm 2 Culturing the cell culture.
68. The method of any one of the preceding claims, further comprising at about 32,000 cells/cm 2 Culturing the cell culture.
69. The method of any one of the preceding claims, further comprising culturing the cells to comprise about 10 x 10 2 From about 10X 10 cells/mL 10 A volume of viable cells/mL.
70. The method of any one of the preceding claims, further comprising culturing the cells to comprise about 10 x 10 6 A volume of viable cells/mL.
71. The method of any one of the preceding claims, further comprising packaging the cell culture in a volume comprising less than 10mL, 9mL, 8mL, 7mL, or 6 mL.
72. The method of any one of the preceding claims, further comprising packaging the cell culture in a volume comprising 5 mL.
73. The method of any one of the preceding claims, wherein the sample of stem cells comprises at least about 70% viable cells.
74. The method of any one of the preceding claims, wherein the cell culture comprises vertebrae adherent mesenchymal stem cells (vBA-MSC), vertebral bone marrow mesenchymal stem cells (vBM-MSC), or both.
75. The method of any one of the preceding claims, wherein the cell culture is derived from cadavers.
76. The method of any one of the preceding claims, wherein the cell culture comprises at least about 10,000 to about 300,000CFU-F/1 million viable cells.
77. The method of any one of the preceding claims, wherein the cell culture comprises less than about 1% to about 10% cd45+ cells.
78. A method for preparing a sample of stem cells for direct infusion, the method comprising:
a. providing a cell culture comprising a sample of said stem cells, wherein said cell culture is cryopreserved and passaged;
b. thawing the cell culture; and
c. the cell culture is maintained at above 0 ℃.
79. The method of claim 78, wherein after (c), the sample of stem cells comprises at least about 70% viable cells.
80. A method for preparing a sample of stem cells for direct infusion, the method comprising:
a. providing a cell culture comprising a sample of said stem cells, wherein said cell culture is cryopreserved and passaged;
b. Thawing the cell culture; and
c. maintaining the cell culture at below room temperature;
wherein the sample of stem cells comprises at least about 70% viable cells.
81. The method of claim 80, wherein after (c), the sample of stem cells comprises at least about 70% viable cells.
82. A method for preparing a sample of stem cells for direct infusion, the method comprising:
a. providing a cell culture comprising a sample of said stem cells, wherein said cell culture is cryopreserved and passaged;
b. thawing the cell culture; and
c. maintaining the cell culture at below room temperature;
wherein the sample of stem cells comprises from less than about 1% to about 10% cd45+ cells.
83. The method of claim 82, wherein the sample of stem cells comprises less than about 5% cd45+ cells.
84. The method of any one of the preceding claims, wherein the sample of stem cells comprises less than about 1% to about 10% cd35+ cells.
85. The method of claim 84, wherein the sample of stem cells comprises less than about 5% cd35+ cells.
86. A method for preparing a sample of stem cells for direct infusion, the method comprising:
a. providing a cell culture comprising a sample of said stem cells, wherein said cell culture is cryopreserved and passaged;
b. thawing the cell culture; and
c. maintaining the cell culture at below room temperature;
wherein the sample of stem cells comprises at least about 90% to about 99% cd90+ cells.
87. The method of claim 86, wherein the sample of stem cells comprises at least about 95% cd90+ cells.
88. A method for preparing a sample of stem cells for direct infusion, the method comprising:
a. providing a cell culture comprising a sample of said stem cells, wherein said cell culture is cryopreserved and passaged;
b. thawing the cell culture; and
c. maintaining the cell culture at below room temperature;
wherein the sample of stem cells comprises at least about 90% to about 99% cd105+ cells.
89. The method of claim 88, wherein the sample of stem cells comprises at least about 95% cd105+ cells.
90. The method of any one of the preceding claims, wherein after (c), the sample of stem cells comprises at least about 70% viable cells.
91. A method for preparing a sample of stem cells for direct infusion, the method comprising:
a. providing a cell culture comprising a sample of said stem cells, wherein said cell culture is cryopreserved and passaged;
b. thawing the cell culture; and
c. maintaining the cell culture at below room temperature;
wherein the sample of stem cells comprises at least about 10,000 to about 300,000CFU-F/1 million viable cells.
92. The method of claim 91, wherein the sample of stem cells comprises at least about 10,000cfu-F/1 million viable cells.
93. The method of any one of the preceding claims, wherein after (c), the sample of stem cells comprises at least about 70% viable cells.
94. The method of any one of the preceding claims, wherein the sample of stem cells comprises vertebral adherent mesenchymal stem cells (vBA-MSC), vertebral bone marrow mesenchymal stem cells (vBM-MSC), or both.
95. The method of any one of the preceding claims, wherein after (b), the cell culture is not multiplied.
96. The method of any one of the preceding claims, wherein after (b), the cell culture is maintained to undergo one or more population doublings.
97. The method of any one of the preceding claims, wherein after (b), the cell culture is maintained at a confluency of less than 80% relative to a cell culture storage medium.
98. The method of any one of the preceding claims, wherein the cell culture is maintained in a medium comprising one or more amino acids.
99. The method of any one of the preceding claims, wherein the medium comprising one or more amino acids further comprises one or more vitamins.
100. The method of any one of the preceding claims, wherein the medium comprising one or more amino acids further comprises table 1.
101. The method of any one of the preceding claims, wherein the cell culture is passaged at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, or at least 10 times.
102. The method of any one of the preceding claims, wherein the cell culture is cryopreserved in a cryopreservation medium, wherein the cryopreservation medium comprises an electrolyte formulation, human Serum Albumin (HSA), dimethyl sulfoxide (DMSO), or any combination thereof.
103. The method of any one of the preceding claims, wherein the cryopreservation media comprises about 1% to about 5% HSA.
104. The method of any one of the preceding claims, wherein the cryopreservation media comprises about 2.5% HSA.
105. The method of any one of the preceding claims, wherein the cryopreservation medium comprises about 1% to about 10% DMSO.
106. The method of any one of the preceding claims, wherein the cryopreservation medium comprises about 5% DMSO.
107. The method of any one of the preceding claims, wherein the electrolyte formulation is a boy pulse force a.
108. The method of any of the preceding claims, wherein the thawing occurs at about 30 ℃ to about 40 ℃.
109. The method of any of the preceding claims, wherein the thawing occurs at about 37 ℃.
110. The method of any one of the preceding claims, further comprising re-suspending the cell culture in a wash medium, wherein the wash medium comprises an electrolyte formulation, human Serum Albumin (HSA), or both; and maintaining the cell culture at below room temperature.
111. The method of any one of the preceding claims, further comprising, prior to (c), re-suspending the cell culture in a wash medium, wherein the wash medium comprises an electrolyte formulation, human Serum Albumin (HSA), or both.
112. The method of any one of the preceding claims, wherein the rinse medium is fresh.
113. The method of any one of the preceding claims, wherein the wash medium comprises about 1% to about 5% HSA.
114. The method of any one of the preceding claims, wherein the wash medium comprises about 2.5% HSA.
115. The method of any one of the preceding claims, wherein the electrolyte formulation is a boy pulse force a.
116. The method of any one of the preceding claims, wherein the cell culture is maintained at room temperature for at least about 30 minutes.
117. The method of any one of the preceding claims, wherein the cell culture is maintained at room temperature for at least 12 hours.
118. The method of any one of the preceding claims, wherein the cell culture is maintained at room temperature for at least 24 hours.
119. The method of any one of the preceding claims, wherein the cell culture is maintained at room temperature for at least 48 hours.
120. The method of any one of the preceding claims, wherein the cell culture is maintained at less than 37 ℃.
121. The method of any one of the preceding claims, wherein the cell culture is maintained at less than 35 ℃.
122. The method of any one of the preceding claims, wherein the cell culture is maintained at less than 30 ℃.
123. The method of any one of the preceding claims, wherein after (b), the cell culture is maintained under conditions of low temperature.
124. The method of any one of the preceding claims, wherein the cell culture is maintained at less than 25 ℃.
125. The method of any one of the preceding claims, wherein the cell culture is maintained at less than 20 ℃.
126. The method of any one of the preceding claims, wherein the cell culture is maintained at about 2 ℃ to about 8 ℃.
127. The method of any one of the preceding claims, further comprising at about 10,000 cells/cm 2 Up to about 50,000 cells/cm 2 Culturing the cell culture.
128. The method of any one of the preceding claims, further comprising at about 32,000 cells/cm 2 Culturing the cell culture.
129. The method of any one of the preceding claims, further comprising culturing the cells to comprise about 10 x 10 2 From about 10X 10 cells/mL 10 A volume of viable cells/mL.
130. The method of any one of the preceding claims, further comprising culturing the cells to comprise about 10 x 10 6 Individual living cells/mLIs a volume package of (2).
131. The method of any one of the preceding claims, further comprising packaging the cell culture in a volume comprising less than 10mL, 9mL, 8mL, 7mL, or 6 mL.
132. The method of any one of the preceding claims, further comprising packaging the cell culture in a volume comprising 5 mL.
133. The method of any one of the preceding claims, wherein the sample of stem cells comprises at least about 70% viable cells.
134. The method of any one of the preceding claims, wherein the cell culture comprises vertebrae adherent mesenchymal stem cells (vBA-MSC), vertebral bone marrow mesenchymal stem cells (vBM-MSC), or both.
135. The method of any one of the preceding claims, wherein the cell culture is derived from cadavers.
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| US202063128780P | 2020-12-21 | 2020-12-21 | |
| US63/128,780 | 2020-12-21 | ||
| PCT/US2021/064433 WO2022140296A1 (en) | 2020-12-21 | 2021-12-20 | Methods for storage of stem cells |
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| CN117098542A true CN117098542A (en) | 2023-11-21 |
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| CN202180094235.XA Pending CN117098542A (en) | 2020-12-21 | 2021-12-20 | Method for storing stem cells |
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| US (1) | US20220195393A1 (en) |
| EP (1) | EP4262832A1 (en) |
| CN (1) | CN117098542A (en) |
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| WO2007117262A2 (en) * | 2005-07-29 | 2007-10-18 | Athersys, Inc. | Culture of non-embryonic cells at high cell density |
| WO2017156235A1 (en) * | 2016-03-09 | 2017-09-14 | Emory University | Methods of preserving mesenchymal stem cells |
| MY173966A (en) * | 2016-05-25 | 2020-02-28 | Cytopeutics Sdn Bhd | A cell-based composition and use thereof for treatment of acute stroke |
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- 2021-12-20 CN CN202180094235.XA patent/CN117098542A/en active Pending
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| US20220195393A1 (en) | 2022-06-23 |
| EP4262832A1 (en) | 2023-10-25 |
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