CN112868641A - Cryopreservation method, resuscitation method and reconstruction method of islet cells - Google Patents

Cryopreservation method, resuscitation method and reconstruction method of islet cells Download PDF

Info

Publication number
CN112868641A
CN112868641A CN202110082783.2A CN202110082783A CN112868641A CN 112868641 A CN112868641 A CN 112868641A CN 202110082783 A CN202110082783 A CN 202110082783A CN 112868641 A CN112868641 A CN 112868641A
Authority
CN
China
Prior art keywords
islet
cell
cells
culture medium
islets
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202110082783.2A
Other languages
Chinese (zh)
Inventor
彭园征
周明
王慈慈
欧映廷
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Liver Biotechnology Shenzhen Co ltd
Original Assignee
Liver Biotechnology Shenzhen Co ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Liver Biotechnology Shenzhen Co ltd filed Critical Liver Biotechnology Shenzhen Co ltd
Priority to CN202110082783.2A priority Critical patent/CN112868641A/en
Publication of CN112868641A publication Critical patent/CN112868641A/en
Pending legal-status Critical Current

Links

Images

Classifications

    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0676—Pancreatic cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/34—Sugars
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/998—Proteins not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
    • C12N2502/1352—Mesenchymal stem cells
    • C12N2502/1358—Bone marrow mesenchymal stem cells (BM-MSC)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
    • C12N2502/1352—Mesenchymal stem cells
    • C12N2502/137—Blood-borne mesenchymal stem cells, e.g. Msc from umbilical cord blood
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
    • C12N2502/1352—Mesenchymal stem cells
    • C12N2502/1382—Adipose-derived stem cells [ADSC], adipose stromal stem cells

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Biomedical Technology (AREA)
  • Wood Science & Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Environmental Sciences (AREA)
  • Dentistry (AREA)
  • Cell Biology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The invention discloses a cryopreservation method, a recovery method and a reconstruction method of islet cells. The invention dissociates the islet into islet single cells, freezes the islet single cells, solves the problem that the islet single cells are not easy to freeze, and reconstructs the frozen islet single cells together with the human mesenchymal stem cells into reconstructed cell clusters after being recovered. The method for reconstructing the islet cells can enable the frozen islet single cells to be rapidly reconstructed into cell clusters after recovery, and is high in clustering rate (the highest rate can reach 100%) and good in repeatability; the reconstructed islet cell mass is spherical and solid, has a diameter of 100-750 μm, and is formed by 5-30 layers of cells; the reconstituted islet cell mass is capable of responding to glucose challenge in vitro culture. In the blood sugar level test of the transplanted nude mice, the reconstructed cell mass has a blood sugar regulation function equivalent to that of the islets of Langerhans.

Description

Cryopreservation method, resuscitation method and reconstruction method of islet cells
Technical Field
The invention relates to the technical field of biology, in particular to a cryopreservation method, a recovery method and a reconstruction method of islet cells.
Background
Islet cell transplantation is the most promising means for the eradication of type 1 diabetes. The islets are mainly separated and purified from the pancreas donor. After the pancreas is treated by collagenase solution with proper concentration, exocrine cells and structural components in the pancreas are digested and dispersed, the islets are released into the pancreatic digestive tissue fluid, and the purified islets can be obtained after the treatment by a cell separation fluid density gradient centrifugation method. The purified islets are transplanted into liver blood vessels of type 1 diabetes patients, and the blood sugar change of the patients can be physiologically regulated and controlled. The source of human islets (islets) is limited, mainly by the donation of a dead person, and the physical quality of the donation also greatly determines the clinical application possibility of the islets.
Long-term culture of islets in vitro can affect their function. The longer the in vitro culture, the less effective the regulation of blood glucose changes after transplantation. Typically, the islets are isolated and cultured in vitro for no more than 24 hours. Therefore, the temporal and spatial differences between pancreatic donors and islet transplant recipients also significantly limit the clinical utility of islet transplantation.
Cryopreservation of islets is not yet mature. The pancreatic islets are mainly cell clusters composed of four types of cells, namely, an alpha cell secreting glucagon, a beta cell secreting insulin, a delta cell secreting somatostatin and a PP cell secreting pancreatic polypeptide. Wherein the majority of cells are beta cells, accounting for 60% -80%; alpha cells account for 24% -40%, delta cells for 6-15%, and at a minimum, PP cells, about 1%. The four types of cells are not simply stacked together to form a cell mass, but interact with each other to achieve the purpose of accurately regulating and controlling the change of blood sugar. The current cell cryopreservation technology can not ensure that the pancreatic islet cryopreservation meets the quality requirement of clinical transplantation. The islet single cells are obtained by opening the close association between islet cells using a dissociation enzyme (e.g., pancreatin, etc.). This enzymatic action disrupts islet cell interactions and directly impairs islet cell response to glucose. Therefore, simple islet single cells (i.e., alpha cells, beta cells, delta cells, PP cells do not form a cell mass) have no or only weak glucose regulation ability. Therefore, it is necessary to develop a set of methods for cryopreservation and reconstitution of islet single cells.
Accordingly, the prior art is deficient and needs improvement.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: provides a cryopreservation method, a recovery method and a reconstruction method of islet cells, solves the problems of cryopreservation and recovery of islet cells, and solves the technical problem of reconstructing islet single cells into functional cell clusters.
The technical scheme of the invention is as follows: provided is a cryopreservation method of islet cells, comprising the following steps.
S1: taking a proper amount of purified pancreatic islets, namely cell mass obtained after pancreas purification.
S2: and (4) washing the filtered and purified pancreatic islets with a balanced salt solution, and centrifuging to remove the supernatant to obtain the precipitated pancreatic islets.
S3: adding a cell dissociation reagent to resuspend the precipitated pancreatic islets, and incubating for 5-60 minutes at 37 ℃ to dissociate the precipitated pancreatic islets into pancreatic islet single cells to obtain a pancreatic islet single cell suspension.
S4: adding islet cell culture medium into the islet single cell suspension to stop dissociation, centrifuging and removing supernatant to obtain precipitated islet single cells.
S5: and (4) cleaning the precipitated islet single cells by using an islet cell culture medium, centrifuging and removing supernatant to obtain the stored islet single cells.
S6: adding a cell freezing solution to resuspend and store the islet single cells to obtain a stored islet single cell suspension, and subpackaging the islet single cell suspension into the cell freezing tube.
S7: and (4) freezing and storing the cell freezing and storing tube filled with the islet single cell suspension in liquid nitrogen.
The purified islets are pre-treated with 5% CO in step S12And cultured in a 37 ℃ cell culture boxFiltering for 6-36 h, wherein the filtering net adopted for filtering is a stainless steel filtering net with 200-400 meshes; in step S3, the amount of the cell dissociation reagent is 5-15 times the volume of the precipitated islets; in step S4, the amount of the islet cell culture medium is 2-10 times of that of the cell dissociation reagent; in step S6, the cell density of the islet single cell suspension dispensed in the cell freezing tube is 0.1-10 × 106/mL。
The specific operation of step S7 is:
s71: and (3) placing the cell freezing tube filled with the islet single cell suspension into a cell program cooling box and sealing.
S72: putting the cell program cooling box into an environment with the temperature of minus 80 ℃ for incubation for 6h to 36 h;
s73: and transferring the freezing tube which is filled with the islet single cell suspension and is stored in the cell program cooling box into a liquid nitrogen tank for storage.
The balanced salt solution is D-hank's solution; the cell dissociation reagent is StemProTM AccutaseTMCell discovery Reagent, purchased from Gibco, usa under a lot number a 1110501; the islet cell culture medium is prepared by adding the following components into an F-10 culture medium: 10mmol/L HEPES, 1.2g/L sodium bicarbonate, 10% v/v fetal calf serum, 1% v/v penicillin-streptomycin; f-10 medium is commercially available, e.g., from Sigma, USA under the reference number N6635; the HEPES described above is commercially available, e.g., from sigma, USA under the designation V900477; the sodium bicarbonate described above is commercially available, for example from sigma, usa under the designation S5761; the fetal calf serum is commercially available, such as from BIOLOGICAL INDUSTRIES, Israel, under the reference REF 03-031-1B; the above-mentioned penicillin-streptomycin is commercially available, for example, from BIOLOGICAL INDUSTRIES, Israel under the cat number 04-001-1 ACS. The cell freezing medium is prepared by adding the following components into the islet cell culture medium: 40% v/v fetal bovine serum, 10% v/v dimethyl sulfoxide, 6% m/v hydroxyethyl starch. The fetal calf serum is commercially available, such as from BIOLOGICAL INDUSTRIES, Israel, under the reference REF 03-031-1B; the above-mentioned dimethyl sulfoxide is commercially available, for example, from Sigma, USA under the trade name D4540. The above hydroxyethyl starches are commercially available, e.g. fromSigma, USA, Cat # H6382. The cell cryopreservation tubes are commercially available, such as from the company of nai si (NEST) in china under the designation 607001.
The Cell program cooling box is a Cell Home box which does not use liquids such as isopropanol and the like to assist cooling and can be purchased in the market, for example, the Cell Home box is purchased from Chinese Kommers, has the product numbers of Cell Home-60 and Cell Home-12, respectively corresponds to different specifications, and can achieve the same effect in different specifications.
The invention also provides a resuscitation method which is characterized by comprising the following steps;
SS 1: and taking out the cell freezing tube filled with the islet single cell suspension from the liquid nitrogen, and quickly thawing in a water bath at 37 ℃ to obtain the islet single cell suspension.
SS 2: adding the stored islet single cell suspension into a resuscitation culture medium preheated at 37 ℃, inverting and uniformly mixing, standing at room temperature for 10-70min, centrifuging and removing supernatant to obtain resuscitation islet single cells; the resuscitation islet culture medium is prepared by adding the following components into an islet cell culture medium: 20-80g/L glucose, 10-70g/L bovine serum albumin.
SS 3: and adding the islet cell culture medium to the recovered islet single cells to resuspend the recovered islet single cells to obtain a recovered islet single cell suspension.
In step SS2, the amount of the resuscitated islet culture medium is 2-10 times that of the stored islet single cell suspension. In step SS3, a small amount of cell suspension was also counted by staining with 0.4% Trypan blue working solution and the proportion of viable cells was calculated.
The resuscitation culture medium is prepared by adding the following components into an islet cell culture medium: 52g/L glucose, 40g/L bovine serum albumin. The above glucose may be purchased commercially, for example, from Sigma, USA under the trade designation G7021; bovine serum albumin as described above is commercially available, for example, from Sigma, USA under the trade designation A1933. The 0.4% trypan blue working solution is commercially available, e.g., from Sigma, USA under the designation T8154.
The invention also provides a reconstruction method, which is characterized by comprising the following steps;
SSS 1: respectively taking out the above compoundsMixing the single cell suspension and the mesenchymal stem cell suspension at a density of 0.1 × 105/cm2-10×105/cm2Mixing uniformly, and adding into an ultra-low adsorption cell culture plate;
SSS 2: supplementing culture medium of islet cells with 5% CO2And culturing in a cell culture box at 37 ℃, and reconstructing the islet single cells into cell clusters.
In step SSS2, the cultivation time is 12-60 hours.
The preparation method of the mesenchymal stem cell suspension comprises the following steps: taking mesenchymal stem cells, and processing at a ratio of 0.5 × 103Viable cells/cm2-15×103Viable cells/cm2Inoculating the cells into a cell culture bottle which is treated by tissue culture, and adding a proper amount of mesenchymal stem cell culture medium for amplification culture; when the cell fusion degree is 70% -90%, pancreatin digestion is carried out for passage or mesenchymal stem cell suspension is obtained for standby.
The mesenchymal stem cell culture medium is DMEM/F12 culture medium added with the following components: 20% v/v fetal bovine serum, 1% v/v penicillin-streptomycin; the pancreatin is 0.25% Trypsin-EDTA solution.
The mesenchymal stem cells can be selected from human umbilical cord mesenchymal stem cells, human marrow mesenchymal stem cells and human adipose mesenchymal stem cells, in the scheme, the human umbilical cord mesenchymal stem cells are preferably selected from the human umbilical cord mesenchymal stem cells, and the human umbilical cord mesenchymal stem cells can be purchased commercially, such as from Wuhan Punuisal Life technologies, Inc., with the product number of CP-H165. The DMEM/F12 medium described above is commercially available, for example, from Gibco, USA under the accession number 12500096; the fetal calf serum is commercially available, such as from BIOLOGICAL INDUSTRIES, Israel, under the reference REF 03-031-1B; the above-mentioned penicillin-streptomycin is commercially available, for example, from BIOLOGICAL INDUSTRIES, Israel under the cat number 04-001-1 ACS.
The pancreatin is a 0.25% Trypsin-EDTA solution commercially available, e.g., from Sigma, USA under the designation T4049.
The tissue culture treated cell culture flasks, i.e., adherent (TC treated) culture flasks, are commercially available, for example, from the company Naist (NEST) in china, under the product numbers 707003, 708003, and 709003, and correspond to different specifications, respectively, and the same effect can be achieved with the different specifications.
The Ultra-low adsorption cell culture plate, namely Ultra-low Attachment, abbreviated as ULA, is commercially available, such as Corning corporation, USA, with a product number of 3474, 3473, 3471, respectively corresponding to different specifications, and can achieve the same effect.
By adopting the scheme, the invention provides a cryopreservation method, a resuscitation method and a reconstruction method of islet cells; the invention solves the problem of poor direct cryopreservation effect of the pancreatic islet, can ensure high activity of the frozen pancreatic islet cells, and can widely collect and store the pancreatic islet cells from different pancreatic donor sources to establish a pancreatic islet cell bank. The method for reconstructing the islet cells can enable the frozen islet single cells to be rapidly reconstructed into cell clusters after recovery, and is high in clustering rate (the highest rate can reach 100%) and good in repeatability; the reconstructed islet cell mass is spherical and solid, has a diameter of 100-750 μm, and is formed by 5-30 layers of cells; the reconstituted islet cell mass is capable of responding to glucose challenge in vitro culture. The invention uses mesenchymal stem cells to reconstitute cryopreserved recovered islet single cells into a functional cell mass, can be used for scientific research of islet cells, and can also be used for treating type 1 diabetes.
Drawings
FIG. 1 is a diagram of the morphology of a reconstituted cell mass formed by the reconstitution of single cells of pancreatic islets at different times under a phase-contrast microscope, wherein the scale is 100 μm according to an embodiment of the present invention;
FIG. 2 shows the insulin concentrations detected at high glucose in islets, single cells of islets, and reconstituted cell masses formed by 48h of culture;
FIG. 3 shows the insulin concentrations detected at low glycemia for islets, islet single cells, and reconstituted cell mass formed by 48h of culture;
FIG. 4 is the high/low carbohydrate stimulation index of islets, islet single cells, and reconstituted cell mass formed by 48h of culture;
FIG. 5 comparison of blood glucose levels in nude mice after transplantation of islets and reconstituted cell masses obtained by 48h culture into nude mice.
Detailed Description
The invention is described in detail below with reference to the figures and the specific embodiments.
The invention provides a cryopreservation method of islet cells, which comprises the following steps:
1. cryopreservation of islet single cells
Filtering purified islets cultured for 24 hours by using a 300-mesh stainless steel net, sucking 2000IEQ purified islets, washing the islets for 2 times by using a balanced salt solution, centrifuging to remove supernatant to obtain precipitated islets, adding 500 mu L of cell dissociation reagent into the precipitated islets to resuspend the precipitated islets, placing the islets in an environment at 37 ℃ for incubation for 15 minutes, and gently blowing and beating the islets by using a pipette tip for about 10 times to completely dissociate the precipitated islets into islet single cells to form islet single cell suspension; immediately adding 2.5mL of islet cell culture medium to the islet single cell suspension to stop dissociation; centrifuging and removing supernatant to obtain precipitated islet single cells; washing the single cells of the precipitated islets 1 times with 1mL of islet cell culture medium, and counting to obtain about 1X 106Centrifuging individual islet single cells, and removing supernatant to obtain stored islet single cells; adding 1mL of cell freezing solution into the islet single cells for resuspension, transferring the islet single cells to a 2mL cell freezing tube, then putting the islet single cells into a cell programmed cooling box cellHome-12, sealing the cooling box, then putting the islet single cells into a refrigerator with the temperature of minus 80 ℃ for incubation for 24 hours, and then transferring the freezing tube to a liquid nitrogen tank for storage. Wherein IEQ means the islet equivalent, a method of islet counting, corresponds to the number of islets with a diameter of 150 μm.
2. Islet single cell resuscitation
Taking the islet storage single cells stored in a liquid nitrogen tank, oscillating for about 90s in a water bath kettle at 37 ℃ gently until only a small amount of floating ice remains in a freezing tube, transferring the islet storage single cells to 5mL of resuscitation culture medium preheated at 37 ℃, reversing and mixing uniformly, standing for 30min at room temperature, centrifuging and discarding supernatant to obtain resuscitation islet single cells, adding 0.5mL of islet cell culture medium into the resuscitation islet single cells for resuspension, and performing dye counting on 0.4% trypan blue working solution to obtain about 8 × 105 total cells with the activity of about 90%. This resulted in a resuscitated single cell suspension of islets.
3. Human umbilical cord mesenchymal stem cell suspension
Taking human umbilical cord mesenchymal stem cells according to the ratio of 0.5 multiplied by 103Viable cells/cm2-15×103Viable cells/cm2Inoculating the cells into a cell culture bottle which is treated by tissue culture, and adding a proper amount of mesenchymal stem cell culture medium for amplification culture; when the cell confluence is 70% -90%, removing the culture medium, then gently washing the cells for 2 times by 5mL of Phosphate Buffered Saline (PBS) and completely removing the washing solution; adding 1mL of pancreatin, after uniformly spreading, incubating for 2 minutes at 37 ℃, tapping a culture bottle to completely separate cells, adding 5mL of mesenchymal stem cell culture medium to terminate digestion, transferring to a 15mL centrifuge tube, centrifuging to remove supernatant, washing for 1 time by using 5mL of mesenchymal stem cell culture medium, centrifuging to remove supernatant, and then re-suspending by using 0.5mL of mesenchymal stem cell culture medium; after 0.4% trypan blue working solution staining counting, about 7.5X 10 total cells were obtained5The activity was about 98%. Thus obtaining the human umbilical cord mesenchymal stem cell suspension.
The Phosphate Buffered Saline (PBS) is commercially available, e.g., from Hyclone, USA under the designation 10010023.
The 15mL centrifuge tube is commercially available, for example, from Nest corporation of China under the designation 601052. The mesenchymal stem cell culture medium is DMEM/F12 culture medium added with the following components: 20% v/v fetal bovine serum, 1% v/v penicillin-streptomycin; the pancreatin is 0.25% Trypsin-EDTA solution.
4. Reconstitution of islet single cells into reconstituted cell clusters
Taking 6.4X 10 from the resuscitated islet single cell suspension according to the number of living cells5Resuscitating single islet cells and taking 3.2 x 10 from human umbilical cord mesenchymal stem cell suspension5Mixing the human umbilical cord mesenchymal stem cells uniformly, supplementing the islet cell culture medium to 1.6mL, transferring the mixture into 8 holes of a 96-hole ultra-low adsorption cell culture plate, placing 0.2mL cell suspension in each hole, and placing the cell suspension in 5% CO2Culturing in a cell culture box at 37 ℃ for 48 hours; the resuscitated islet single cells are reconstituted into a reconstituted cell mass.
Referring to fig. 1, fig. 1 shows the reconstructed cell mass (islet cell mass) of the resuscitated islet single cells at 12h, 24h and 48h under the observation of a phase contrast microscope. As can be seen from FIG. 1, the reconstituted cell mass size becomes larger as the culture time increases.
Referring to FIGS. 2-4, FIGS. 2-4 show the respective sampling of 100IEQ islets (about 0.5X 10)5Single cell of islet), 0.8 × 105The single islet cells and the reconstituted cell mass formed by culturing 1 well for 48h were subjected to glucose-stimulated insulin secretion assay (GSIS), and the insulin levels released by each cell under high-glucose (fig. 2) and low-glucose (fig. 3) stimulation were measured separately, and the ratio of high-glucose to low-glucose stimulated insulin release (high-glucose/low-glucose stimulation index) was counted (fig. 4).
As can be seen in FIG. 2, the concentration of insulin produced by the islets is highest on high glucose stimulation, the concentration of insulin produced by the reconstituted cell mass is second to that produced by high glucose stimulation, and the concentration of insulin produced by the islet cells alone is lowest and very low on high glucose stimulation.
As can be seen in FIG. 3, the islet produced the highest concentration of insulin on the hypoglycaemic stimulation, the reconstituted cell mass produced the second highest concentration of insulin on the hypoglycaemic stimulation, and the islet single cells alone produced the lowest concentration of insulin on the hypoglycmic stimulation and was approximately zero. Therefore, the reconstructed cell mass obtained by reconstructing the islet single cells can generate insulin when being stimulated by sugar, and has practical application prospect.
It can be seen from fig. 4 that the ratio of high-glucose to low-glucose stimulated insulin release from islets, single islets, and reconstituted cell masses is relatively close, and in combination with fig. 2 and 3, the effect of single islet cells on glucose stimulated insulin is very poor.
The insulin secretion test (GSIS) under glucose stimulation refers to the level of insulin released from pancreatic islets, pancreatic islet single cells, or islet-like cell clusters, which are cultured in a medium containing high-concentration glucose and low-concentration glucose, respectively, for a certain period of time; the stimulation index is the ratio of the amount of insulin secreted by high-concentration glucose stimulation to the amount of insulin secreted by low-concentration glucose stimulation, and can reflect the response capability of corresponding cells to glucose in vitro. The specific implementation method of the GSIS of the experimental example in the present invention is as follows:
1) collecting 100IEQ pancreatic islets, 0.8 × 105The islet single cells and the reconstructed cell mass formed by culturing 1 well for 48h were placed in 1 cell chamber of a 12-well plate, and 1mL of low sugar stimulation medium (i.e., 2.8mmol/L glucose-Krebs buffer) was added to the corresponding well (outside the cell chamber) of the 12-well plate, and placed in 5% CO2Culturing in a cell culture box at 37 ℃ for 1 hour;
2) 1mL of low sugar stimulation medium was added to each of the other three wells of the 12-well plate. After three cell chambers containing cells were removed and the medium drained on sterile gauze, they were gently placed into three wells containing fresh 1mL of low sugar stimulation medium, respectively, and placed in 5% CO2Culturing in a 37 ℃ cell culture box for 1 hour, respectively collecting supernatant, and storing at minus 20 ℃ or directly detecting;
3) 1mL of high-sugar-stimulated medium (i.e., 28mmol/L glucose-Krebs buffer) was added to each of the other three wells of the 12-well plate. After three cell chambers containing cells were removed and the medium drained on sterile gauze, they were then gently placed into three wells containing fresh 1mL of high sugar stimulation medium, respectively, and placed in 5% CO2Culturing in a 37 ℃ cell culture box for 1 hour, respectively collecting supernatant, and storing at minus 20 ℃ or directly detecting;
4) detecting the content of the insulin in the collected supernatant according to an operation method of an insulin ELISA kit specification, and calculating the high-sugar/low-sugar stimulation index of each group of cells.
The glucose is commercially available, e.g., from Sigma, usa under the designation G7021;
the Krebs buffer solution comprises the following components: 25mmol/L HEPES, 115mmol/L NaCl, 5mmol/L KCl, 24mmol/L sodium bicarbonate, 1mmol/L MgCl2、2.5mmol/L CaCl20.1% m/v bovine serum albumin. The HEPES described above is commercially available, e.g., from sigma, USA under the designation V900477; the NaCl is commercially available, e.g., from Sigma, USA under the trade designation S5886; the above-mentioned KCl is commercially available, for example, from Sigma, USA under the trade designation P5405; the above sodium bicarbonate is commercially availablePurchased, e.g., from sigma, usa under the designation S5761; MgCl as described above2Commercially available, such as from sigma, usa under the designation M2393; above CaCl2Commercially available, such as from sigma corporation, usa under the designation C5670; bovine serum albumin as described above is commercially available, for example, from Sigma, USA under the trade designation A1933.
The 12-well plate cell chamber is commercially available, for example, from the firm of Chinese missing (NEST) under the designation 724301.
The insulin ELISA kit can be purchased commercially, for example, from Proteintetech, USA under the trade name KE 00045.
Please refer to fig. 5, fig. 5 is a graph showing that 1000IEQ of pancreatic islets and a cell mass formed by culturing 7 wells for 48h are taken to perform a transplantation experiment on a diabetic nude mouse recipient, and the blood glucose change of the diabetic nude mouse recipient is monitored after the transplantation, which reflects the capability of the graft to regulate and control the blood glucose change in vivo (the horizontal line in fig. 5 represents that the blood glucose value is 11.1mmol/L, the blood glucose value of the recipient mouse is lower than the value which indicates that the graft plays a blood glucose reducing function, and the value higher than the value which indicates that the graft does not have the blood glucose reducing function, and possible factors include insufficient amount of the graft, poor function of the graft, rejection of the graft by the.
As can be seen from FIG. 5, the cell mass reconstituted from the islet single cells exerted a better hypoglycemic effect after transplantation into the nude mice, although the blood glucose level of the nude mice recipients was slightly higher than that of the nude mice recipients directly transplanted with islets. But still shows that the reconstructed cell mass obtained by reconstructing the transplanted islet single cell has the effect of reducing blood sugar equivalent to that of transplanted islet and has excellent application prospect.
As can be seen from fig. 1 to 5, the reconstituted cell mass has the same function of producing insulin by glucose stimulation and regulating blood glucose changes as the islets, and the reconstituted cell mass graft has blood glucose regulating function comparable to that of the islet graft. The mesenchymal stem cells in the reconstructed cell mass not only serve as an adhesive to reconstruct the interaction among alpha cells, beta cells, delta cells and PP cells which are damaged by enzymolysis, thereby having the function of regulating blood sugar; meanwhile, the mesenchymal stem cells have excellent activity, can effectively improve the activity of the reconstructed cell mass, and can further improve the blood sugar reduction effect.
The reconstructed cell mass in the scheme is formed by the enzymolysis of the pancreatic islets into the pancreatic islet single cells, and the proportion of alpha cells, beta cells, delta cells and PP cells in the pancreatic islet single cells is basically consistent with that of the pancreatic islets, so that the alpha cells, the beta cells, the delta cells and the PP cells in the reconstructed cell mass are basically consistent with that of the alpha cells, the beta cells, the delta cells and the PP cells in the pancreatic islets, and the reconstructed cell mass has the effect of generating insulin by sugar stimulation similar to that of the pancreatic islets. Some existing researches completely adopt mesenchymal stem cells to directly differentiate into islet-like cells, the islet-like cells generated by differentiation only have a weak function of generating insulin by stimulating sugar, and whether alpha cells, delta cells and PP cells can be directly induced and differentiated by the mesenchymal stem cells is not concerned, so that the islet-like cells induced by the differentiation have a very poor function of regulating blood sugar change. The reconstructed cell mass in the scheme is formed by reconstructing islet single cells (namely alpha cells, beta cells, delta cells and PP cells) and mesenchymal stem cells, wherein the islet before enzymolysis has the functions of generating insulin and regulating blood sugar by sugar stimulation, so that the islet single cells also have the memory of generating the insulin by sugar stimulation, and the islet single cells can generate a small amount of insulin by sugar stimulation, as shown in fig. 2 and 3. The blood sugar change regulating function needs the interaction among alpha cells, beta cells, delta cells and PP cells, but the islet single cells are obtained by damaging the interaction among the alpha cells, the beta cells, the delta cells and the PP cells by pancreatin, so the islet single cells need to reconstruct and establish the mutual connection among the four types of cells to exert the blood sugar regulating function. After the islet single cells (alpha cells, beta cells, delta cells and PP cells) and the mesenchymal stem cells are reconstructed into clusters, the reconstructed cell clusters recover the interaction among the alpha cells, the beta cells, the delta cells and the PP cells and still have the memory of generating insulin by sugar stimulation, so that the reconstructed cell clusters can generate higher insulin concentration by sugar stimulation and play a better blood sugar regulation function in a type 1 diabetes receptor body. The transplantation experiment is that a certain amount of pancreatic islets or reconstructed islet cell mass (reconstructed cell mass) is transplanted to the left kidney tunica mucosa of a type 1 diabetic nude mouse induced by Streptozotocin (STZ), then the diabetic nude mouse receptor is normally bred, and the blood sugar value of the receptor is continuously monitored by using a blood sugar test paper and a glucometer. The specific implementation method of the transplantation experiment of the experimental example of the present invention is as follows:
1) taking two normally-fed 8-to-10-week-old BALB/c-nu nude mice, fasting for 12 hours without water prohibition, then carrying out intraperitoneal injection on 10mg/mL STZ solution according to the dose of 200mg/kg mouse body weight, feeding water normally after injection, and observing the physical conditions of the nude mice every day;
2) monitoring the blood sugar level of the nude mice at the same time every day after 3 days, wherein the nude mice with blood sugar higher than 16.7mmol/L for 2 times continuously can be regarded as diabetic nude mice and can be used as transplant recipient mice;
3) 1000IEQ islets and 7-well reconstructed cell masses cultured for 48h are transplanted under the left kidney tunica mucosa of a diabetic nude mouse recipient respectively, and the recipient mouse is normally fed with water for feeding after transplantation.
4) The blood glucose levels of the recipient mice were monitored beginning the day after transplantation. A blood glucose value of less than 11.1mmol/L reflects that the graft is functioning to regulate the blood glucose in the recipient mouse.
The BALB/c-nu nude mouse is purchased from a company or a public institution with the production license of experimental animals, such as the medical experimental animal center of Guangdong province;
the streptozotocin may be purchased commercially, e.g., from Sigma, usa under the serial number S0130;
the glucometer and glucose strips are commercially available, such as the steady luxury duo easy type ONETOUCH ultra easy glucometer and the steady luxury strip from qiangsheng corporation.
In summary, the present invention provides a cryopreservation method, a resuscitation method and a reconstitution method of islet cells; the invention solves the problem of poor direct cryopreservation effect of the pancreatic islet, can ensure high activity of the frozen pancreatic islet cells, and can widely collect and store the pancreatic islet cells from different pancreatic donor sources to establish a pancreatic islet cell bank. The method for reconstructing the islet cells can enable the frozen islet single cells to be rapidly reconstructed into cell clusters after recovery, and is high in clustering rate (the highest rate can reach 100%) and good in repeatability; the reconstructed islet cell mass is spherical and solid, has a diameter of 100-750 μm, and is formed by 5-30 layers of cells; the reconstructed islet cell mass can respond to glucose stimulation in vitro culture; the reconstructed islet cell mass transplanted into a type 1 diabetes receptor can regulate the blood sugar change of the receptor. The invention uses mesenchymal stem cells to reconstitute cryopreserved recovered islet single cells into a functional cell mass, can be used for scientific research of islet cells, and can also be used for treating type 1 diabetes.
The present invention is not limited to the above preferred embodiments, and any modifications, equivalent substitutions and improvements made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (10)

1. A cryopreservation method of islet cells is characterized by comprising the following steps:
s1: taking a proper amount of purified pancreatic islets;
s2: washing the purified islets with a balanced salt solution, centrifuging and removing supernatant to obtain precipitated islets;
s3: adding a cell dissociation reagent to resuspend the precipitated pancreatic islets, and incubating for 5-60 minutes at 37 ℃ to dissociate the precipitated pancreatic islets into pancreatic islet single cells to obtain a pancreatic islet single cell suspension;
s4: adding an islet cell culture medium into the islet single cell suspension to stop dissociation, centrifuging and removing supernatant to obtain precipitated islet single cells;
s5: cleaning the deposited islet single cells by using an islet cell culture medium, centrifuging and removing supernatant to obtain stored islet single cells;
s6: adding a cell freezing solution to resuspend and store the islet single cells to obtain a stored islet single cell suspension, and subpackaging the islet single cell suspension into cell freezing tubes;
s7: and (4) freezing and storing the cell freezing and storing tube filled with the islet single cell suspension in liquid nitrogen.
2. The method for cryopreserving pancreatic islet cells according to claim 1, wherein the purified pancreatic islets are pre-treated with 5% CO in step S12Culturing in a 37 ℃ cell culture box for 6-36 h, and then filtering, wherein a filter screen adopted for filtering is a stainless steel filter screen of 200-400 meshes; in step S3, the amount of the cell dissociation reagent is 5-15 times the volume of the precipitated islets; in step S4, the amount of the islet cell culture medium is 2-10 times of that of the cell dissociation reagent; in step S6, the cell density of the islet single cell suspension dispensed in the cell freezing tube is 0.1-10 × 106/mL。
3. The method for cryopreserving pancreatic islet cells according to claim 1, wherein the specific operation of step S7 is;
s71: loading the cell freezing tube filled with the islet single cell suspension into a cell program cooling box and sealing;
s72: putting the cell program cooling box into an environment with the temperature of minus 80 ℃ for incubation for 6h to 36 h;
s73: and transferring the freezing tube which is filled with the islet single cell suspension and is stored in the cell program cooling box into a liquid nitrogen tank for storage.
4. The cryopreservation method of islet cells according to claim 1, wherein the balanced salt solution is D-hank's solution; the cell dissociation reagent is StemProTMAccutaseTMCell discovery Reagent; the islet cell culture medium is prepared by adding the following components into an F-10 culture medium: 10mmol/L HEPES, 1.2g/L sodium bicarbonate, 10% v/v fetal calf serum, 1% v/v penicillin-streptomycin; the cell freezing medium is prepared by adding the following components into the islet cell culture medium: 40% v/v fetal bovine serum, 10% v/v dimethyl sulfoxide, 6% m/v hydroxyethyl starch.
5. A resuscitation method comprising the steps of;
SS 1: taking out the cell cryopreservation tube filled with the islet single cell suspension for storage according to any one of claims 1 to 4 from liquid nitrogen, and rapidly thawing in a water bath at 37 ℃ to obtain a storage islet single cell suspension;
SS 2: adding the stored islet single cell suspension into a resuscitation culture medium preheated at 37 ℃, inverting and uniformly mixing, standing at room temperature for 10-70min, centrifuging and removing supernatant to obtain resuscitation islet single cells; the resuscitation islet culture medium is prepared by adding the following components into an islet cell culture medium: 20-80g/L glucose, 10-70g/L bovine serum albumin;
SS 3: and adding the islet cell culture medium to the recovered islet single cells to resuspend the recovered islet single cells to obtain a recovered islet single cell suspension.
6. The resuscitation method according to claim 5, wherein said resuscitation islet culture medium is present in an amount of 2-10 times that of the stored islet single cell suspension in step SS 2.
7. A reconstruction method, comprising the steps of;
SSS 1: mixing the resuscitated single-cell islet suspension and the mesenchymal stem cell suspension according to claim 5 or 6 respectively according to any ratio, and the density is 0.1 x 105/cm2-10×105/cm2Mixing uniformly, and adding into an ultra-low adsorption cell culture plate;
SSS 2: supplementing culture medium of islet cells with 5% CO2And culturing in a cell culture box at 37 ℃, and reconstructing the islet single cells into cell clusters.
8. A reconstitution method according to claim 7, wherein the culturing period in step SSS2 is 12-60 hours.
9. A reconstitution method according to claim 7, wherein the mesenchymal stem cell suspension is prepared by the following steps: taking mesenchymal stem cells, and processing at a ratio of 0.5 × 103Viable cells/cm2-15×103Viable cells/cm2Inoculating into a cell culture flask treated by tissue culture, and adding appropriate amount of mesenchymeCarrying out amplification culture on the stem cell culture medium; when the cell fusion degree is 70% -90%, pancreatin digestion is carried out for passage or mesenchymal stem cell suspension is obtained for standby.
10. A method according to claim 9, wherein the mesenchymal stem cell culture medium is DMEM/F12 medium supplemented with the following components: 20% v/v fetal bovine serum, 1% v/v penicillin-streptomycin; the pancreatin is 0.25% Trypsin-EDTA solution.
CN202110082783.2A 2021-01-21 2021-01-21 Cryopreservation method, resuscitation method and reconstruction method of islet cells Pending CN112868641A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202110082783.2A CN112868641A (en) 2021-01-21 2021-01-21 Cryopreservation method, resuscitation method and reconstruction method of islet cells

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202110082783.2A CN112868641A (en) 2021-01-21 2021-01-21 Cryopreservation method, resuscitation method and reconstruction method of islet cells

Publications (1)

Publication Number Publication Date
CN112868641A true CN112868641A (en) 2021-06-01

Family

ID=76051532

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202110082783.2A Pending CN112868641A (en) 2021-01-21 2021-01-21 Cryopreservation method, resuscitation method and reconstruction method of islet cells

Country Status (1)

Country Link
CN (1) CN112868641A (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114107173A (en) * 2021-11-15 2022-03-01 北京赛拉达生物科技有限公司 Vascularized pancreatic islet micro-organ and construction method thereof
CN114868736A (en) * 2022-03-08 2022-08-09 四川中科奥格生物科技有限公司 Islet cell stabilizing solution and preparation method and application thereof

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102197802A (en) * 2011-03-25 2011-09-28 中国人民解放军***福州总医院 Pancreatic island freezing protective agent and using method thereof

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102197802A (en) * 2011-03-25 2011-09-28 中国人民解放军***福州总医院 Pancreatic island freezing protective agent and using method thereof

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
K. OHASHI 等: "Production of Islet Cell Sheets Using Cryopreserved Islet Cells", 《TRANSPLANTATION PROCEEDINGS》 *
M. MARUYAMA 等: "Simplified Method for Cryopreservation of Islets Using Hydroxyethyl Starch and Dimethyl Sulfoxide as Cryoprotectants", 《TRANSPLANTATION PROCEEDINGS》 *
刘玉溥 等: "大鼠胰岛B细胞分离与原代培养方法探讨", 《四川大学学报(医学版)》 *
王广宇 等: "脐带源间充质干细胞与大鼠胰腺细胞共培养向胰岛样细胞分化及移植治疗1 型糖尿病", 《细中国组织工程研究与临床康复》 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114107173A (en) * 2021-11-15 2022-03-01 北京赛拉达生物科技有限公司 Vascularized pancreatic islet micro-organ and construction method thereof
CN114868736A (en) * 2022-03-08 2022-08-09 四川中科奥格生物科技有限公司 Islet cell stabilizing solution and preparation method and application thereof
CN114868736B (en) * 2022-03-08 2022-12-09 四川中科奥格生物科技有限公司 Islet cell stabilizing solution and preparation method and application thereof

Similar Documents

Publication Publication Date Title
KR100374976B1 (en) In Vitro Growth and Functional Use of Functional Langerhans Island
Bonney Adult liver parenchymal cells in primary culture: Characteristics and cell recognition standards
CN103667187B (en) A kind of isolated culture method of human adipose-derived stem cell and the construction method of stem cell bank
CN106434557B (en) The method for preparing CD34 positive cell by umbilical cord mesenchymal stem cells
CN112868641A (en) Cryopreservation method, resuscitation method and reconstruction method of islet cells
CN110037010B (en) Temperature-sensitive gel preparation and method for preserving islet cells at low temperature for long time
CN105543169A (en) Preparation method and application of human TSCMs (T memory stem cells)
CN103422176A (en) Construction method of human amniotic mesenchymal stem cell bank
CN106798724A (en) A kind of mesenchymal stem cells MSCs parenteral solution and its preparation method and application
CN108135942A (en) Stem cell therapy based on adipose-derived stem cell
KR20080091874A (en) Medium for culturing autologous human progenitor stem cells and applications thereof
CN108728408A (en) Dog fetal membrane mescenchymal stem cell and preparation method and the culture medium used
CN102171332A (en) Methods for enhanced propagation of cells
RU2511395C2 (en) Artificial kidney precursor and method for manufacturing it
CN100418582C (en) Microcapsule for promoting bone regeneration and its use
CN103260631B (en) The method increasing cell products separation yield
Araki et al. Biohybrid Artificial Pancreas Long-Term Insulin Secretion by Devices Seeded with Canine Islets
Nielsen et al. Isolation, transplantation, and functional studies of adult porcine islets of Langerhans
Hicks et al. Transplantation of β cells from transgenic mice into nude athymic diabetic rats restores glucose regulation
CN102517248A (en) In-vitro induced pancreas-islet-like structure forming method
Zhou et al. Microencapsulation of rat islets prolongs xenograft survival in diabetic mice
CN104257690A (en) Stem cell preparation for treating diabetes mellitus and preparation method of preparation
Liu et al. Successful cryopreservation of fetal porcine proislets
CN108378019A (en) A kind of frozen stock solution of people's stem spermatogonium
Miyamoto et al. Development of a cryopreservation procedure employing a freezer bag for pancreatic islets using a newly developed cryoprotectant

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
RJ01 Rejection of invention patent application after publication

Application publication date: 20210601

RJ01 Rejection of invention patent application after publication