WO2016154206A1 - Milieu de bioconservation et ses utilisations pour la bioconservation de matières biologiques - Google Patents

Milieu de bioconservation et ses utilisations pour la bioconservation de matières biologiques Download PDF

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Publication number
WO2016154206A1
WO2016154206A1 PCT/US2016/023587 US2016023587W WO2016154206A1 WO 2016154206 A1 WO2016154206 A1 WO 2016154206A1 US 2016023587 W US2016023587 W US 2016023587W WO 2016154206 A1 WO2016154206 A1 WO 2016154206A1
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Prior art keywords
cells
keratinocytes
fibroblasts
medium
poloxamers
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PCT/US2016/023587
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English (en)
Inventor
Aleksa Jovanovic
Lei Shi
Yiling Lu
Vincent Ronfard
Jason Campbell
Jacquelyn COVARRUBIAS
Annette RUFF
Gladys MONTENEGRO
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Smith & Nephew, Inc.
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Publication of WO2016154206A1 publication Critical patent/WO2016154206A1/fr

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A01N1/0205—Chemical aspects
    • A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
    • A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A01N1/0205—Chemical aspects
    • A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids

Definitions

  • the invention generally relates to biopreservation of biological materials.
  • the invention relates to the cryopreservation, thawing, and hypothermic preservation (cold storage) of cells, cell preparations, biological tissues and organs.
  • Biopreservation involves the preservation of biological materials including cells, cell preparations, biological tissues, and organs at cryogenic temperatures and/or hypothermic temperatures (cold storage).
  • biological materials such as cells and cell preparations are preserved at cryogenic temperatures for long periods of time (e.g., up to 1 to 2 years), thawed, and then used immediately in uses such as tissue regeneration, wound healing, and cellular therapy.
  • the cell preparation DERMAGRAFT® must be stored continuously at minus 65°C to minus 85°C in order to ensure cell viability, according to "DERMAGRAFT Directions for Use", Organogenesis Inc., 2014, herein incorporated by reference.
  • DMSO dimethyl sulfoxide
  • Bovine serum is commonly used as a cryoprotectant, but must be removed before the cells or cell preparation is used on humans because DMSO is irritating to the skin and mucous membranes.
  • Bovine serum is also commonly used in cryopreservation media because it can improve the viability and stability of the cells during the freezing process, storage at cryogenic temperatures, and subsequent thawing.
  • hypothermic (cold storage) media solutions such as HYPOTHERMOSOL®
  • FRS available from BioLife Solutions, and PRIME-XVTM available from Irvine Scientific are commercially available for the transport and short term storage of biological materials; however, these solutions are not suitable for cryopreservation.
  • a manufacturer of a cell preparation were to thaw the cell preparation prior to shipping so that hypothermic temperatures could be maintained during shipping and storage of the preparation at the end user's facilities, the manufacturer would have to remove the cryoprotectant medium from the cell preparation and add the hypothermic medium solution to the cell preparation before shipping. This process would present the opportunity for contamination of the preparation and potential loss of cell viability, plus this would entail additional handling costs at the manufacturer.
  • the present invention provides a solution to the aforementioned limitations and deficiencies in the art relating to biopreservation of biological materials.
  • the present invention provides for a biopreservation medium, and methods of use thereof, such that the biopreservation medium can be used for each phase of biopreservation, i.e., cryopreservation, thawing, and hypothermic storage of biological materials such as cells, cell preparations, biological tissues and organs, carried out in succession without the need to change the medium between phases. This eliminates the need to change the medium after thawing, and thus facilitates the shipment and short term storage of biological materials.
  • the biological materials can remain under hypothermic conditions for at least 5 weeks which allows ample time for shipping and storing the biological materials at the end user's facility, such as a hospital, clinic, nursing facility, or doctor's office. It also eliminates the need for the end user to have a cryogenic freezer or liquid nitrogen on site. The end user only needs a refrigerator for storage of the thawed biological materials at refrigeration temperatures. Thus, in certain instances, the end user can even be a patient at home administering self-treatment, where the biological material could be stored in a home refrigerator. [0008] Additionally, the biopreservation medium of the invention does not need the inclusion of DMSO for cryopreservation.
  • the biopreservation medium of the invention has demonstrated a significant improvement of cell culture viability, quality and metabolic activity during cryopreservation and thawing over commercially available cryopreservation mediums such as SYNTH-A-FREEZE®, which contains 10% DMSO.
  • Another advantage of the present invention is that only one medium is needed for all the phases of biopreservation, i.e. cryopreservation, thawing, and hypothermic storage, which is more economical than using a different medium for each phase.
  • the biopreservation medium of the invention can also be animal origin free (AOF), since it does not need serum to promote cell viability during biopreservation and thawing.
  • OAF animal origin free
  • a method for cryopreservation, thawing, and subsequent hypothermic storage of a cell preparation or a plurality of cells comprising: a. combining the cell preparation or a plurality of cells with a biopreservation medium composition comprising a basal medium, one or more poloxamers, and one or more cryoprotectants, to form a mixture;
  • viability of the cells is at least 50% of the viable cells initially present in the thawed mixture after storage at hypothermic conditions for 5 weeks.
  • the viability of the cells is at least 85% of the viable cells initially present in the thawed mixture after storage at hypothermic conditions for 5 weeks.
  • the biopreservation medium composition is not changed between steps a and b. In further aspects, the biopreservation medium is not changed between steps c and d.
  • the one or more poloxamers includes poloxamer 407. [0013] In one embodiment, the one or more poloxamers is poloxamer 407.
  • the one or more poloxamers does not include poloxamer
  • the one or more poloxamers includes poloxamer 407 and does not include poloxamer 188.
  • the one or more cryoprotectants includes glycerin.
  • the one or more cryoprotectants is glycerin.
  • the one or more cryoprotectants does not include DMSO.
  • the one or more cryoprotectants includes glycerin and does not include DMSO.
  • the basal medium is animal origin free.
  • the biopreservation medium composition is animal origin free.
  • the plurality of cells comprises fibroblasts and/or keratinocytes. In another embodiment the plurality of cells comprises fibroblasts and/or keratinocytes, wherein the fibroblasts and/or keratinocytes are mitotically inactivated. In another embodiment, the plurality of cells comprises stem cells.
  • the cell preparation comprises a carrier, fibroblasts, and/or keratinocytes. In another embodiment, the cell preparation comprises a carrier, fibroblasts, and/or keratinocytes, wherein the fibroblasts and/or keratinocytes are mitotically inactivated. In another embodiment, the cell preparation comprises a carrier and stem cells. [0023] In one embodiment, the viability of the cells is at least 60% of the viable cells initially present in the thawed mixture after storage at hypothermic conditions for 2 weeks. In another embodiment, the viability of the cells is at least 90% of the viable cells initially present in the thawed mixture after storage at hypothermic conditions for 2 weeks.
  • biopreservation medium composition useful for the cryopreservation, thawing, and/or hypothermic preservation of a cell preparation or a plurality of cells, the biopreservation medium comprising a basal medium, one or more poloxamers, and one or more cryoprotectants.
  • the one or more poloxamers includes poloxamer 407.
  • the one or more poloxamers is poloxamer 407. [0027] In one embodiment, the one or more poloxamers does not include poloxamer 188.
  • the one or more poloxamers includes poloxamer 407 and does not include poloxamer 188.
  • the one or more cryoprotectants includes glycerin. [0030] In one embodiment, the one or more cryoprotectants is glycerin.
  • the one or more cryoprotectants does not include DMSO.
  • the one or more cryoprotectants includes glycerin and does not include DMSO.
  • the basal medium is animal origin free.
  • the biopreservation medium composition is animal origin free.
  • the plurality of cells comprises fibroblasts and/or keratinocytes. In another embodiment the plurality of cells comprises fibroblasts and/or keratinocytes, wherein the fibroblasts and/or keratinocytes are mitotically inactivated. In another embodiment, the plurality of cells comprises stem cells. [0035] In one embodiment, the cell preparation comprises a carrier, fibroblasts, and/or keratinocytes. In another embodiment, the cell preparation comprises a carrier, fibroblasts, and/or keratinocytes, wherein the fibroblasts and/or keratinocytes are mitotically inactivated. In another embodiment, the cell preparation comprises a carrier and stem cells.
  • a method for the cryopreservation of a cell preparation or a plurality of cells comprising: combining the cell preparation or a plurality of cells with a biopreservation medium composition comprising a basal medium, one or more poloxamers, and one or more cryoprotectants, to form a mixture and cryopreserving the mixture.
  • the method further comprises thawing the cryopreserved mixture.
  • the one or more poloxamers includes poloxamer 407.
  • the one or more poloxamers is poloxamer 407.
  • the one or more poloxamers does not include poloxamer 188.
  • the one or more poloxamers includes poloxamer 407 and does not include poloxamer 188.
  • the one or more cryoprotectants includes glycerin.
  • the one or more cryoprotectants is glycerin.
  • the one or more cryoprotectants does not include DMSO.
  • the one or more cryoprotectants includes glycerin and does not include DMSO.
  • the basal medium is animal origin free.
  • the biopreservation medium composition is animal origin free.
  • the plurality of cells comprises fibroblasts and/or keratinocytes. In another embodiment the plurality of cells comprises fibroblasts and/or keratinocytes, wherein the fibroblasts and/or keratinocytes are mitotically inactivated. In another embodiment, the plurality of cells comprises stem cells.
  • the cell preparation comprises a carrier, fibroblasts, and/or keratinocytes.
  • the cell preparation comprises a carrier, fibroblasts, and/or keratinocytes, wherein the fibroblasts and/or keratinocytes are mitotically inactivated.
  • the cell preparation comprises a carrier and stem cells.
  • a method for the hypothermic storage of a cell preparation or a plurality of cells comprising: combining the cell preparation or a plurality of cells with a biopreservation medium composition comprising a basal medium, one or more poloxamers, and one or more cryoprotectants, to form a mixture, and storing the thawed mixture at hypothermic conditions, wherein the viability of the cells is at least 50% of the viable cells initially present in the mixture after storage at hypothermic conditions for 5 weeks. In one embodiment, the viability of the cells is at least 85% of the viable cells initially present in the mixture after storage at hypothermic conditions for 5 weeks.
  • the viability of the cells is at least 60% of the viable cells initially present in the mixture after storage at hypothermic conditions for 2 weeks. In another embodiment, the viability of the cells is at least 90% of the viable cells initially present in the mixture after storage at hypothermic conditions for 2 weeks.
  • the one or more poloxamers includes poloxamer 407.
  • the one or more poloxamers is poloxamer 407.
  • the one or more poloxamers does not include poloxamer
  • the one or more poloxamers includes poloxamer 407 and does not include poloxamer 188.
  • the one or more cryoprotectants includes glycerin.
  • the one or more cryoprotectants is glycerin.
  • the one or more cryoprotectants does not include DMSO.
  • the one or more cryoprotectants includes glycerin and does not include DMSO.
  • the basal medium is animal origin free.
  • the biopreservation medium composition is animal origin free.
  • the plurality of cells comprises fibroblasts and/or keratinocytes. In another embodiment the plurality of cells comprises fibroblasts and/or keratinocytes, wherein the fibroblasts and/or keratinocytes are mitotically inactivated. In another embodiment, the plurality of cells comprises stem cells.
  • the cell preparation comprises a carrier, fibroblasts, and/or keratinocytes.
  • the cell preparation comprises a carrier, fibroblasts, and/or keratinocytes, wherein the fibroblasts and/or keratinocytes are mitotically inactivated.
  • the cell preparation comprises a carrier and stem cells.
  • cryopreservation temperatures mean temperatures at minus 20°C to minus 196°C.
  • “refrigeration temperatures”, “refrigerated conditions” or “cold storage” mean temperatures at O°C to 10 °C or 2°C to 8°C.
  • animal origin free with respect to the biopreservation mediums and basal mediums disclosed in the present invention means substantially free from animal derived components.
  • a number value with one or more decimal places can be rounded to the nearest whole number using standard rounding guidelines, i.e. round up if the number being rounded is 5, 6, 7, 8, or 9; and round down if the number being rounded is 0, 1, 2, 3, or 4. For example, 3.7 can be rounded to 4.
  • compositions and methods for their use can "comprise,” “consist essentially of,” or “consist of any of the ingredients or steps disclosed throughout the specification.
  • FIG. 1 The cell viability of human keratinocytes during 5 week storage at
  • FIG. 2 The number of cells (normalized) during 5 week storage at 4°C in medium formulations from Examples 1, 2, and 3, and Hypothermosol.
  • FIG. 3 Flow chart for experiment repeatability used in cryogenic study.
  • FIG. 4 Cell recovery results for medium formulas D, E, G, and H.
  • FIG. 5 Cell recovery results for medium formulas D, E, G, and H.
  • FIG. 6 Combined recovery results for medium formulas D, E, G, and H, and
  • FIG. 7A Morphology micrographs (100X and 25X) at p4 of PIK41
  • FIG. 7B Morphology micrographs (100X and 25X) at p4 of PIK41 Keratinocytes Post Thaw, formula G and formula H.
  • FIG. 8 Harvest results at p5 of the medium formulas D, E, G, and H, and
  • FIG. 9 Thaw results of cryogenic study with recovery, viability and LDH values for PIK 41 keratinocytes in medium formulas D, E, G, and H, and Synth-a-freeze.
  • FIG. 10 Keratinocyte cell numbers in medium formulas D, E, G, and H, and
  • FIG. 11 The cell viability of human keratinocytes after 7 days storage at minus 80°C for medium formulas J, K, and L, and Synth-a-freeze.
  • FIG. 12 The number of viable cells after 7 days storage at minus 80°C for medium formulas J, K, and L, and Synth-a-freeze.
  • FIG. 13 The cell recovery results for medium formulas J, K, and L, and
  • FIG. 14 LDH concentration for medium formulas J, K, and L, and Synth-a freeze used in cryogenic study.
  • FIG. 15 The cell viability of human keratinocytes after 7 days storage at minus 80°C for medium formulas M, N, and P, and Synth-a-freeze.
  • FIG. 16 The number of viable cells after 7 days storage at minus 80°C for medium formulas M, N, and P, and Synth-a-freeze.
  • FIG. 17 The cell recovery results for medium formulas M, N, and P, and
  • FIG. 18 LDH concentration for medium formulas M, N, and P, and Synth-a freeze used in cryogenic study.
  • FIG. 19 Viscosity results of medium formulas J, K, and L.
  • FIG. 20 Cell recovery as a function of viscosity of medium formulas J, K, and L, and Synth-a freeze.
  • the present invention provides for a biopreservation medium composition and methods of use thereof.
  • the biopreservation medium of the present invention comprises a basal medium, one or more poloxamers, such as poloxamer 407, and one or more cryoprotectants, such as glycerin, and is suitable for the cryopreservation phase, thawing phase, and hypothermic preservation phase (cold storage) of biological materials such as cells, cell preparations, biological tissues and organs.
  • the advantage of the biopreservation medium of the present invention is that it can be used for all phases of biopreservation including cryopreservation, thawing, and hypothermic preservation carried out in succession without the need to change the medium between phases.
  • biopreservation medium composition useful for the cryopreservation, thawing, and hypothermic preservation of a cell preparation or a plurality of cells, the biopreservation medium comprising a basal medium, one or more poloxamers, and one or more cryoprotectants
  • the biopreservation medium of the present invention is also suitable for use in each phase of biopreservation separately and independent of the other phases.
  • the biopreservation medium of the present invention can be used solely as a cryopreservation medium, or solely as a thawing medium, or solely as a hypothermic (cold storage) medium.
  • the biopreservation medium is used solely as a cryopreservation medium. In one embodiment, the biopreservation medium is used solely as a thawing medium. In one embodiment, the biopreservation medium is used solely as a hypothermic (cold storage) medium. In another embodiment, the biopreservation medium is used as the cryopreservation medium, the thawing medium, and the hypothermic (cold storage) medium.
  • the biopreservation medium of the present invention has also demonstrated the ability to preserve cells at hypothermic conditions for up to 5 weeks while maintaining at least 50% cell viability.
  • the viability of cells stored at hypothermic conditions for 5 weeks in the biopreservation medium of the present invention can be at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, or at least 85% of the cells initially present (in the thawed state).
  • the viability of cells stored at hypothermic conditions for 2 weeks in the biopreservation medium of the present invention can be at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% of the cells initially present (in the thawed state).
  • the biopreservation mediums of the present invention can be made by blending the one or more poloxamers and the one or more cryoprotectants with a basal medium until a homogenous mixture is obtained using mixing and blending techniques and equipment known in the art.
  • suitable industrial mixing equipment include Lightnin' propeller-type mixers, Silverson rotor/stator homogenizers, and MorehouseCowles dissolvers. Use of aseptic techniques are desirable when producing the mediums. Animal Origin Free
  • the biopreservation medium of the present invention can be animal origin free
  • OEF i.e., a medium substantially free from animal derived components.
  • a medium containing serum and/or animal derived components e.g. high cost, batch-to-batch variability of the serum and/or animal derived components, the risk of introducing viruses and pathogenic contaminants, and the difficulty in removing serum proteins and/or animal derived proteins from the biomaterials.
  • An exemplary formulation of the present invention is AOF.
  • the basal medium can be AOF.
  • the basal medium can be obtained through commercial means from a manufacture or it can be manufactured in situ.
  • EPILIFE® Basal Medium commercially available from Life Technologies Corp., is suitable for use in the present invention.
  • EPILIFE Basal Medium is especially compatible with human keratinocytes and human corneal epithelial cells.
  • EPILIFE Basal Medium is AOF and is available with calcium chloride (60 ⁇ ) or without calcium chloride. It is also available with or without phenol red. The components of EPILIFE Basal Medium are shown in Table 1 below.
  • the biopreservation medium of the present invention comprises one or more poloxamers, which are anhydrous, hydrophilic, nonionic, synthetic block copolymers of ethylene oxide and propylene oxide conforming to the general formula (I) (I) HO(C 2 H 4 O) a (C 3 H 6 OMC 2 H 4 O) Q H.
  • polyxamer is the nonproprietary name of the copolymer.
  • Poloxamers are also known as PEO-PPO-PEO tri-block copolymers and can have various block lengths. Poloxamers are available in several types which have various physical forms and various average molecular weights. Each specific poloxamer type is identified by the nonproprietary name "poloxamer" followed by a three digit number, the first two digits of which when multiplied by 100 correspond to the approximate average molecular weight of the polyoxypropylene portion of the copolymer; and the third digit, when multiplied by 10, corresponds to the percentage by weight of the polyoxyethylene portion. [00102] Poloxamers are commercially available from BASF under the trade names
  • Poloxamers are available in solid and liquid forms. Solid forms are available as, but not limited to, flakes, prills, and pastes. Poloxamers are available in pharmaceutical, cosmetic, and industrial grades. Pharmaceutical grade poloxamers are listed in recognized pharmaceutical compendia such as USP/NF and European Pharmacopeia (PhEur). According to the USP/NF and PhEur, a suitable antioxidant may be added.
  • Table 2 below. Also shown are the physical form, average molecular weight, and a and b values corresponding to formula (I) for each poloxamer.
  • Poloxamer 407 also known by its trade name PLURONIC F 127, is suitable for use in the present invention.
  • the data reveal that Poloxamer 188, also known by its trade name PLURONIC F68, was not as effective as Poloxamer 407 in maintaining cell viability after storage of cells at hypothermic conditions in the biopreservation medium formulation of the present invention.
  • the concentration of poloxamers in the biopreservation medium of the present invention can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25% w/w or any percentage derivable therein of the total composition weight.
  • the concentration of poloxamers can be 2% w/w, or 5% w/w, or 6 % w/w, or 7% w/w, or 8% w/w, or 10% w/w, or 11% w/w, or 12 % w/w, or 20 % w/w of the total composition weight.
  • the concentration of poloxamers can be 2-20 % w/w, or 2-12% w/w, or 5-20 % w/w, or 5-12% w/w, or 5-11% w/w, or 5-10% w/w, or 6-20% w/w, or 6-12% w/w, or 6-l l%w/w, or 6-10% w/w of the total composition weight.
  • cryoprotectants known in the art may be used in the present invention as long as they are not toxic to human tissue.
  • DMSO is widely used as a cryoprotectant of cells, but it is toxic (irritating) to the skin and mucous membranes; thus, DMSO is not a suitable cryoprotectant for use in the present invention.
  • Glycerin also known as glycerol, is a suitable cryoprotectant for use in the present invention.
  • a non-animal source of glycerin is preferred, such as a synthetic or vegetable source of glycerin both of which are widely available commercially.
  • the concentration of the cryoprotectant in the biopreservation medium of the present invention can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25% w/w or any percentage derivable therein of the total composition weight.
  • the concentration of the cryoprotectant can be 4% w/w, or 8% w/w, or 16% w/w of the total composition weight.
  • the concentration of the cryoprotectant can be 4-16% w/w, or 4-8% w/w, or 8-16% w/w.
  • the biopreservation medium of the present invention is suitable for the biopreservation of a variety of cell types including animal (mammalian and non-mammalian) and plant cells.
  • the biopreservation medium of the present invention is suitable for the biopreservation of one cell, a plurality of cells which can be of one cell type or of mixed cell types, a population of cells of one cell type, or a population of mixed cells.
  • Non-limiting examples of mammalian animal cell types include fibroblasts, keratinocytes, dermal cells, melanocytes, hair cells, outer root sheath cells, epithelial cells, corneal epithelial cells, progenitor cells, stromal cells, lymphocytes including plasma cells, B cells, T cells, cytotoxic T cells, natural killer T cells, regulatory T cells, T helper cells, myeloid cells, granulocytes, basophil granulocytes, eosinophil granulocytes, neutrophil granulocytes, hypersegmented neutrophils, monocytes, macrophages, reticulocytes, platelets, mast cells, thrombocytes, megakaryocytes, dendritic cells, thyroid cells, thyroid epithelial cells, parafollicular cells, parathyroid cells, parathyroid chief cells, oxyphil cells, adrenal cells, chromaffin cells, pineal cells, pinealocytes, glial cells, glioblasts,
  • Non limiting examples of stem cells include adult stem cells, fetal stem cells, embryonic stem cells, pluripotent stem cells, induced pluripotent stem cells, multipotent stem cells, totipotent stem cells, oligopotent stem cells, unipotent stem cells, mesenchymal stem cells, amniotic stem cells, umbilical cord blood stem cells, bone marrow stem cells, hematopoietic stem cells, endothelial stem cells, adipose stem cells, dental pulp stem cells, cancer stem cells.
  • the biological material is a plurality of cells.
  • the plurality of cells comprises fibroblasts and/or keratinocytes.
  • the plurality of cells comprises fibroblasts and/or keratinocytes, wherein the fibroblasts and/or keratinocytes are mitotically inactivated.
  • the plurality of cells comprises stem cells.
  • the biopreservation medium of the present invention is suitable for the biopreservation of cell preparations.
  • Cell preparations are known in the art and are useful for a variety of medical and clinical uses including, but not limited to, tissue regeneration, wound healing, and cellular therapy.
  • Cell preparations include, but are not limited to, cells in or on a carrier, substrate or matrix; a suspension of cells in a culture medium or carrier; skin equivalents; and skin substitutes.
  • An example of a cell preparation is DERMAGRAFT®, available from Organogenesis, Inc.
  • cell preparations are the cell preparations disclosed in US patents US 6673603, US 7144729, US 7449333, US 7700351, US 7879605, US 8137965, US 8323638, and US 8679475 all of which are herein incorporated by reference.
  • the biological material is a cell preparation.
  • the cell preparation comprises a carrier, fibroblasts, and/or keratinocytes.
  • the cell preparation comprises a carrier, fibroblasts, and/or keratinocytes, wherein the fibroblasts and/or keratinocytes are mitotically inactivated.
  • the cell preparation comprises a carrier and stem cells.
  • the biopreservation medium of the present invention is suitable for the biopreservation of a variety of biological tissues and organs including animal (mammalian and non-mammalian) tissues and organs.
  • mammalian animal tissues include chondral tissue, cartilage, tendons, ligaments, vertebral discs, osteochondral tissue, islet tissue, osteogenic tissue, neural tissue, skin, mucous membranes, bone tissue, bone marrow, adipose tissue, muscle tissue, blood, corneas, lens, ocular tissue, meniscus, hair follicles, striated muscle, smooth muscle, cardiac muscle, connective tissue, blood vessels, functional spine units, muscle-tendon grafts.
  • organs include heart, liver, and kidney.
  • the biopreservation method of the invention relates to the cryopreservation, thawing, and hypothermic preservation (cold storage) of cells, cell preparations, biological tissues and organs, carried out in succession using the biopreservation medium composition of the invention without changing the biopreservation medium composition at any time throughout the biopreservation method.
  • the biopreservation method of the invention relates to the cryopreservation of cells, cell preparations, biological tissues and organs using the biopreservation medium composition of the invention.
  • the biopreservation method of the invention relates to the thawing of cells, cell preparations, biological tissues and organs from cryogenic conditions using the biopreservation medium composition of the invention.
  • the biopreservation method of the invention relates to the hypothermic preservation (cold storage) of cells, cell preparations, biological tissues and organs using the biopreservation medium composition of the invention. It was demonstrated that the biopreservation medium of the present invention retained a significantly higher cell viability percentage after 5 weeks of hypothermic storage than the commercially available hypothermic storage medium HYPOTHERMASOL®.
  • a method for cryopreservation, thawing, and subsequent hypothermic storage of a cell preparation or a plurality of cells comprising: a. combining the cell preparation or a plurality of cells with a biopreservation medium composition comprising a basal medium, one or more poloxamers, and one or more cryoprotectants, to form a mixture;
  • viability of the cells is at least 50% of the viable cells initially present in the thawed mixture after storage at hypothermic conditions for 5 weeks.
  • the viability of the cells is at least 85% of the viable cells initially present in the thawed mixture after storage at hypothermic conditions for 5 weeks.
  • the viability of the cells is at least 60% of the viable cells initially present in the thawed mixture after storage at hypothermic conditions for 2 weeks. In another embodiment, the viability of the cells is at least 90% of the viable cells initially present in the thawed mixture after storage at hypothermic conditions for 2 week. Cryopreservation
  • cryopreservation techniques for cryopreserving cells, cell preparations, biological tissues and organs are known in the art. Generally, the cryopreservation temperatures are between minus 20°C to minus 196°C. Cryogenic freezers are commonly used for cryopreservation and are commercially available. The use of liquid nitrogen in storage tanks is another known technique for cryopreservation.
  • Hypothermic storage Cold Storage
  • Techniques for hypothermic storage (cold storage) of biological materials are known in the art and include the transport and storage of the materials. Hypothermic conditions include temperatures at 0°C to 10 °C or 2°C to 8°C. Ordinary household or commercial grade refrigerators can be used for hypothermic storage. Care must be exercised during the transport of biological materials to prevent exposure of the materials to temperatures above hypothermic conditions.
  • Biological materials can be shipped via refrigerated trucks or rail cars. Insulated shipping containers with cold packs can be used for shipment by mail or express delivery services. Ice chests with cold packs or ice can also be employed for transport in automobiles.
  • Poloxamer 407 was added to the basal medium and mixed at 4°C until dissolved. Glycerin was added and blended until homogenous at 4°C. The solution was filtered through a 0.2 ⁇ filter into a sterile container.
  • Poloxamer 407 was added to the basal medium and mixed at 4°C until dissolved. Glycerin was added and blended until homogenous at 4°C. The solution was filtered through a 0.2 ⁇ filter into a sterile container.
  • Human keratinocytes from the fifth passage of cell line PIK 48 were seeded into T75 flasks and collected after 5 days. The collected cells were then suspended into 30 mL of 2H media, which is a medium comprising EpiLife® Basal Medium + HKSdaFreeTM + HKGETM. HKSdaFree and HKGE are available from AvantBio Corp, Vancouver, Washington. Cell counts were obtained (1.0 x 10 6 total cells per mL) and 2 mL aliquots were placed into 15 mL tubes. Cells were spun down in the centrifuge and the media was aspirated away. The cells were added to 1 mL of each biopreservation medium of Example 1, Example 2, and Example 3 and transferred to 2 mL Nunc vials.
  • 2H media which is a medium comprising EpiLife® Basal Medium + HKSdaFreeTM + HKGETM. HKSdaFree and HKGE are available from AvantBio Corp, Vancouver, Washington. Cell counts were obtained (1.0 x 10 6 total cells per m
  • Human keratinocytes from the fifth passage of cell line PIK 48 were seeded into T75 flasks and collected after 5 days. The collected cells were then suspended into 30 mL of 2H media. Cell counts were obtained (1.0 x 10 6 total cells per mL) and 2 mL aliquots were placed into 15 mL tubes. Cells were spun down in the centrifuge and the media was aspirated away. The cells were added to 1 mL of each medium in Table 3 and transferred to 2 mL Nunc vials. Human keratinocytes were also added to Hypothermasol®, EpiLife® Basal medium w/60 ⁇ calcium, and 2H Media. All samples were stored at 2-8°C for 14 days (two weeks). The cell viability and cell count of each sample was determined at 7 and 14 days using a NucleoCounter® NC-3000 cell analyzer. Samples were run twice and the average values were used in the results shown in Table 7 below.
  • Fig. 9 depicts each result obtained from the thaw performed from cells harvested at p5. The supernatant from the cells was measured on the Cedex Bio Analyzer to determine LDH (lactate dehydrogenase) levels in the freezing media. LDH has been shown to reflect the amount of dead cells in a cell suspension. Fig. 9 confirms that higher LDH reflects more dead cells, resulting in lower recovery. Both Synth- a-Freeze® and formulation E had undesirably high LDH levels, reflecting the lower recovery. [00134] Fig. 10 depicts the cells at each stage of the experiment.
  • LDH lactate dehydrogenase

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Abstract

L'invention concerne des milieux de bioconservation contenant des poloxamères et des agents cryoprotecteurs utilisés pour chacune des phases de la bioconservation, notamment la cryoconservation, la décongélation et l'hypothermie (conservation par le froid) de matières biologiques comme les cellules, les préparations cellulaires, les tissus biologiques et les organes. L'invention concerne également les utilisations de ces milieux. Chacune des phases de la bioconservation peut être effectuée avec un seul milieu de bioconservation.
PCT/US2016/023587 2015-03-26 2016-03-22 Milieu de bioconservation et ses utilisations pour la bioconservation de matières biologiques WO2016154206A1 (fr)

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CN114007416A (zh) * 2019-04-30 2022-02-01 明尼苏达大学董事会 用于细胞的冷冻保存的组合物和方法
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