CN114794088B - Human pluripotent stem cell cryopreservation liquid and application thereof - Google Patents
Human pluripotent stem cell cryopreservation liquid and application thereof Download PDFInfo
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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
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- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
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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
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Abstract
The invention provides a human pluripotent stem cell frozen stock solution, which comprises a basal cell culture medium, a cryoprotectant and a signal path inhibitor composition, wherein the signal path inhibitor composition comprises CHIR-99021, ID-8 and Y-27632, and the human pluripotent stem cell frozen stock solution does not contain any protein component. The human pluripotent stem cell cryopreservation liquid recovers frozen cells under the condition of not containing protein components through the synergistic effect of 3 signal pathway inhibitors, and has higher cell survival rate after recovery. In addition, the human pluripotent stem cell freezing solution does not need programmed cooling during freezing, and is quicker, simpler and more convenient. The human pluripotent stem cell freezing medium has the advantages of full research on the quality, high product performance, low cost and suitability for different culture media.
Description
Technical Field
The invention relates to the technical field of cell cryopreservation resuscitation. More particularly, relates to a human pluripotent stem cell cryopreservation solution and application thereof.
Background
Human pluripotent stem cells (hpscs) including Embryonic Stem Cells (ESC), nuclear transfer embryonic stem cells (ntESC), Induced Pluripotent Stem Cells (iPSC), and the like have two potential of "self-renewal" and "three-way differentiation", i.e., the human pluripotent stem cells can be propagated indefinitely in vitro while maintaining "dryness", and have the potential to differentiate into all types of cells of three germ layers. The derived functional cells obtained by the directed differentiation of human pluripotent stem cells can supplement or replace damaged cells, tissues and organs, and are the core of regenerative medicine. The development of methods and reagents for cryopreservation of human pluripotent stem cells that meet key quality attributes for clinical applications and industrial production is at hand.
First, human pluripotent stem cell cryopreserved fluid is a raw material of cell therapy products. The Chinese pharmacopoeia 2020 edition clearly indicates that the selection principle of cell therapy product raw materials is based on the risk control principle, that is, in the selection of raw material grade, the medicinal grade is superior to the non-medicinal grade, the chemical source is superior to the biological source, and the non-animal source is superior to the animal source. The human pluripotent stem cell freezing medium prepared in a laboratory, disclosed in patent CN113025654A and patent CN108552156A at present contains fetal bovine serum or serum substitute with a certain concentration, and belongs to the freezing medium containing animal-derived components and undefined components. The patent CN112544613A discloses a human pluripotent stem cell frozen stock solution containing various cytokines including transferrin, basic growth factors and transforming growth factors, belonging to a frozen stock solution rich in biological components. The human pluripotent stem cell frozen stock solution has a large space for improving the risk control and the grade of the raw materials of cell therapy products, and the research and development of the human pluripotent stem cell frozen stock solution which has no animal-derived components, definite components and no protein can greatly improve the compliance of the human pluripotent stem cell frozen stock solution as the raw materials of the cell therapy products.
Secondly, the human pluripotent stem cell frozen stock solution should meet key quality indexes of industrial production, including low cost, high stability, simplicity, convenience and rapidness. In terms of cost control, human pluripotent stem cell cryopreservation solutions based on serum, serum substitutes and various protein components are expensive; the use of a combination of compounds instead of all protein components can reduce the cost by more than 85%. In the aspect of stability, the serum substitute and a plurality of protein components contained in the human pluripotent stem cell frozen stock solution have the defects of short storage period, severe storage conditions, poor stability among bioactive batches and the like, so that the cell survival rate of the frozen human pluripotent stem cells of different batches often exceeds the upper and lower control limits after the frozen human pluripotent stem cells are restored. In terms of simplicity and rapidness, the freezing solution disclosed in patent CN107494521A and the CTS-shaped multi-PSC freezing kit of Thermo Fisher company need to be permeated step by step according to concentration gradient and cooled to-80 ℃ in a programmed manner, and the operation is complicated and the time consumption is long. The research and development of the human pluripotent stem cell frozen stock solution which has low cost, high stability, simplicity, convenience and rapidness is the industrial basis for realizing the preservation of the general human pluripotent stem cells and the cell therapy products.
Referring to compliance documents such as cell therapy product research and evaluation technical guide principles (trial) and the like, the human pluripotent stem cells frozen by using the human pluripotent stem cell freezing solution are raw materials of clinical diagnosis and treatment and cell therapy products, and key quality attributes such as biological characteristics, safety, stability, effectiveness and the like of the human pluripotent stem cells can be kept for a certain time under the condition of liquid nitrogen freezing. The CTS (human pluripotent stem cell) PSC cryopreservation kit of Thermo Fisher company and the cryopreservation solution disclosed in patent CN108552156A, which are widely applied in the market, do not fully examine the key quality attributes of human pluripotent stem cells after cryopreservation recovery. The work of Thermo Fisher corporation does not disclose and provide critical quality attribute research data for CTS ™ PSC cryopreservation kits, and patent CN108552156A only investigates partial biological characteristics and partial stability indexes of human pluripotent stem cells after cryopreservation recovery, including only cell morphology, cell marker expression level and survival rate. The quality research lacking the compliance seriously restricts the industrial transformation and the clinical application of the human pluripotent stem cell frozen stock solution.
Therefore, the research and development of human pluripotent stem cell cryopreservation liquid which has high raw material level, clear components, no protein component, controllable cost, simplicity, rapidness and sufficient verification is urgently needed, and the increasingly urgent clinical application and industrial production requirements are met.
Disclosure of Invention
The invention aims to provide a human pluripotent stem cell frozen stock solution which does not contain protein components, does not need to be cooled by a program, is fully verified, overcomes the technical defects of the existing human pluripotent stem cell frozen stock solution, and meets the requirements of key quality attributes of clinical application and industrial production.
In order to achieve the first object, the invention provides a human pluripotent stem cell cryopreservation solution, which is characterized by comprising the following components: basal cell culture medium, cryoprotectant and signaling pathway inhibitor compositions; the signal pathway inhibitor composition comprises CHIR-99021, ID-8 and Y-27632, and further comprises CHIR-99021 with the volume molar concentration of 50-500 nM in the whole freezing solution, ID-8 with the volume molar concentration of 100 nM in the whole freezing solution and Y-27632 with the volume molar concentration of 1-30 mu M in the whole freezing solution; and the frozen stock solution of the human pluripotent stem cells does not contain any protein component.
Animal serum, transferrin, human serum albumin and the like belong to protein components, and the frozen stock solution does not contain the protein components, so that the raw material level is high, and the risk control is good.
The frozen stock solution does not contain protein components, and the cell survival rate is lower after recovery; if a proper signal channel inhibitor is added, the survival rate of the cells can be improved, but the survival rate is still low. However, the inventor of the application discovers that if CHIR-99021, ID-8 and Y-27632 are selectively added and respectively belong to a GSK3 beta signal channel inhibitor, a DYRK signal channel inhibitor and a ROCK signal channel inhibitor, a synergistic effect can be generated, and the cell viability rate after recovery is greatly improved under the condition that the protein component is not contained. In the performance test process, the inventor also finds that CHIR-99021, ID-8 and Y-27632 are added into the frozen stock solution at the same time, and during the freezing storage of the human pluripotent stem cells, the non-programmed cooling is opposite to the programmed cooling, and the cell survival rate after recovery is close to that of the human pluripotent stem cells, so that the freezing storage process is simpler, more convenient and faster.
Further, the volume molar concentration of the CHIR-99021 in the whole freezing solution is 100-250 nM; the molar concentration of ID-8 in the whole freezing solution is 250-750 nM; the molar concentration of Y-27632 in the whole frozen stock solution is 8-12 mu M. The unit "nM" is well-defined in the art and represents nmol/L; the unit "μ M" is well-defined in the art and represents μmol/L.
In the art, the basic cell culture medium is mainly composed of amino acids, vitamins, carbohydrates, inorganic salts and other auxiliary substances. Further, the basal cell culture medium is: one or more of DMEM, RPMI1640 and DMEM/F-12.
Preferably, the basic cell culture medium is DMEM/F-12, and accounts for 78.5-98.5% v/v of the whole freezing medium in percentage by volume;
more preferably, the DMEM/F-12 medium is 82-92.5% v/v by volume of the total frozen stock solution.
Further, the cryoprotectant is dimethyl sulfoxide and/or methyl cellulose.
Preferably, the volume percentage of the dimethyl sulfoxide in the whole frozen stock solution is 1-15% v/v, and the mass volume concentration of the methylcellulose in the frozen stock solution is 0.1-1.5 g/L; more preferably, the volume percentage of the dimethyl sulfoxide in the whole frozen stock solution is 5-12% v/v, and the mass volume concentration of the methylcellulose in the frozen stock solution is 0.5-1.2 g/L.
Furthermore, the human pluripotent stem cell frozen stock solution can be added with auxiliary components such as glucose, microorganisms, polysaccharide, inorganic salt and the like.
The second purpose of the invention is to provide the application of the human pluripotent stem cell freezing medium in freezing human pluripotent stem cells or in preparing human pluripotent stem cell freezing products.
The application of the human pluripotent stem cell frozen stock solution in any one of the following applications is also within the protection scope of the invention:
(1) the application in establishing a human pluripotent stem cell bank;
(2) application in cryopreservation of human pluripotent stem cells for research after resuscitation;
(3) the use in cryopreservation of human pluripotent stem cells for production following resuscitation;
(4) the application in frozen storage of human pluripotent stem cells for quality control and quality detection after resuscitation;
(5) the application of the cell culture medium in cryopreservation of human pluripotent stem cells for differentiation of the three germ layer cells and preparation of derived functional cells after recovery.
In the art, human pluripotent stem cells are stem cells that are self-renewing in vitro without limitation and have the potential to differentiate into tri-germ layer cells. The human pluripotent stem cells comprise fertilized embryo-derived embryonic stem cells, parthenogenetic embryo-derived embryonic stem cells, nuclear transfer embryonic stem cells, induced pluripotent stem cells, human embryonic stem cells in a naive state and the like.
In the present invention, the product may be a reagent or a kit.
In the invention, the quality control and quality detection items are key quality attributes, including biological characteristics, safety, stability and effectiveness. The biological characteristics include, but are not limited to, indicators of biological characteristics such as cell morphology, expression levels of cell markers, differentiation potential, genetics, and metabolic enzyme subtype profiles. The safety includes but is not limited to sterility tests (bacteria and fungi), mycoplasma detection, detection of intracellular exogenous pathogenic factors, endotoxin detection, abnormal immunological reaction, tumorigenicity detection, residual quantity of culture medium and related components and other safety indexes. The stability includes but is not limited to stability indexes such as stem cell density/concentration, survival rate, biological activity and the like. The effectiveness includes but is not limited to effectiveness indexes such as differentiation potential of stem cells in vivo and in vitro, morphological structure and physiological function of differentiated cells, expression of specific genes and proteins, secretion of specific cytokines and the like.
In the present invention, the three germ layers include endoderm, mesoderm and ectoderm. The said three germ layer cells and derived functional cells include but are not limited to: definitive endoderm (definitive endogerm) cells, and hepatic progenitor cells, hepatocytes, cholangiocytes, islet cells, and the like derived therefrom; early mesoderm (early mesoderm) cells, and mesenchymal stem cells, hematopoietic stem cells, cardiomyocytes, and the like derived therefrom; neuroectoderm (neuroectoderm) cells, and their derived mesenchymal stem/precursor cells, neurons, astrocytes, oligodendrocytes, and the like.
The invention also provides a preparation method of the pluripotent stem cell frozen stock solution, which comprises the following steps: preparing a methyl cellulose aqueous solution, sterilizing by high-pressure steam, and shaking overnight in a shaking table at room temperature; preparing a DMSO solution of ID-8, and uniformly mixing at room temperature; then all the components of the frozen stock solution are mixed, the pH value is finely adjusted, and the frozen stock solution is filtered, subpackaged and frozen.
The invention has the beneficial effects that:
(1) the raw material grade is high: the pluripotent stem cell freezing medium adopts full-chemical raw materials, has no animal-derived components, is clear in components and does not contain protein components.
(2) Cell viability rate after resuscitation: the pluripotent stem cell cryopreservation liquid comprises a basal cell culture medium, a cryoprotectant and a signal pathway inhibitor composition, and has higher cell viability rate after recovery, which can reach more than 90 percent and even reach 97 percent, under the condition of not containing protein components through the synergistic effect of the 3 signal pathway inhibitors.
(3) No need of programmed cooling: the pluripotent stem cell freezing solution of the invention can be directly frozen to-80 ℃ without programmed cooling, and the cell survival rate after recovery is only slightly lower than the programmed cooling, thereby meeting the industrial production requirement of freezing and storing cells in large scale.
(4) The cost is low: compared with the reagents/kits for cryopreserving human pluripotent stem cells on the market, the price of the cryopreserved liquid for the human pluripotent stem cells is reduced by more than 85%, and the requirements of common human pluripotent stem cell preservation and cell therapy products on raw material cost control are met.
(5) The universality is high: the cryopreservation liquid for the pluripotent stem cells is suitable for various types of embryonic stem cells and induced pluripotent stem cells cultured by different culture media (including but not limited to mTeSR1 and Essential 8) and different culture media (including but not limited to Vitronectin and Matrigel), and the density range of the cryopreserved cells is 1E5-2E7 living cells/mL of the cryopreservation liquid, so that the application range is wide.
(6) The quality research is sufficient, and the product performance is high. In the aspect of stability research, the human pluripotent stem cells can be stably frozen for up to 42 months under the condition of liquid nitrogen by using the pluripotent stem cell freezing medium of the inventor. In the aspect of biological attributes, the cell survival rate and the recovery rate after recovery are high.
Drawings
The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.
FIG. 1 shows the cell viability of human embryonic stem cells H1 frozen using samples 1-13 and controls 1-11 using programmed and unprogrammed cooling at 0 hours after recovery for 1 month, n.gtoreq.3, and before freezing for 95%.
Fig. 2 shows the cell morphology after cell recovery of cryopreserved human embryonic stem cells H1, H9 and human induced pluripotent stem cells ATCC-BXS0117 using sample 3, Scale bar = 100 μm.
FIG. 3 shows graphs of alkaline phosphatase-active cell staining after thawing of human embryonic stem cells H1, H9, and ATCC-BXS0117 cryopreserved using sample 12, mouse embryonic fibroblasts do not express alkaline phosphatase, and are negative controls for alkaline phosphatase-active cell staining, Scale bar = 100 μm.
FIG. 4 shows a diagram of karyotype analysis after thawing of cryopreserved human embryonic stem cells H1, H9 and human induced pluripotent stem cells ATCC-BXS0117 using sample 4.
FIG. 5 shows a section of teratoma triblast differentiated tissue and a HE staining pattern formed after thawing cryopreserved human embryonic stem cells H9 using sample 13.
Detailed Description
In order to more clearly illustrate the invention, the invention is further described below with reference to preferred embodiments and the accompanying drawings. Similar components in the figures are denoted by the same reference numerals. It is to be understood by persons skilled in the art that the following detailed description is illustrative and not restrictive, and is not to be taken as limiting the scope of the invention.
The antibodies and reagents used in the examples of the present invention were derived from the following sources:
CTS;. KnockOut;, Thermo Fisher, Cat. No. A1286101.
BenchStable ™ RPMI1640 medium, Thermo Fisher, Cat. No. A4192301.
CTS;. KnockOut;. DMEM/F-12 medium, Thermo Fisher Co., Ltd., Cat. No. A1370801.
Dimethyl sulfoxide (CAS number 67-68-5), WAK-Chemie Medical GmbH, Inc., cat # WAK-DMSO-70.
Methylcellulose (CAS number 9004-67-5), Sigma-Aldrich, cat # M7027.
CHIR-99021 (CAS number 1797989-42-4), Selleck, cat # S2924 (10 mM in DMSO).
ID-8 (CAS number 147591-46-6), Selleck, Inc., cat number S7327.
Y-27632 (CAS number 129830-38-2), Selleck, cat # S1049 (10 mM in DMSO).
Human serum albumin (CAS number 70024-90-7), Sigma-Aldrich, cat # A1653.
CTS ™ DPBS, Thermo Fisher, Cat. No. A1285602.
CTS; (VTN-N) Recombinant Human Protein, Truncated, Thermo Fisher, Cat. No. A27940.
Corning Matrigel hESC-Qualified Matri X, Corning Inc., goods number is 354277.
CTS.Versene.TM.solution, Thermo Fisher, Cat. A4239010.
StemPro ™ Accutase-responsive cell dissociation reagent, Thermo Fisher Co., Cat. No. A1110501.
CTS ™ Essential 8-chamber medium, Thermo Fisher, cat # A2656101.
mTeSR. sup.1 medium, STEMCELL Technologies, Inc., cat # 85850.
Alkaline phosphate Live Stain, Thermo Fisher Inc., Cat # A14353.
Alexa Fluor 488 Rabbit monoclonal [ EPR17929] to Oct4, Abcam Corp., cat # ab 283741.
Alexa Fluor 488 Rabbit monoclonal [ EPR2027(2) ] to Nanog, Abcam Corp., Cat. ab 196155.
Alexa Fluor 488 Rabbit IgG, monoconal [ EPR25A ] -Isotype Control, Abcam Corp., Cat # ab 199091.
Anti-Human TRA-1-81 Anti body, Clone TRA-1-81, Alexa Fluor 488-conjugated Antibody, STEMCELL Inc., Cat No. 60065 AD.
Mouse IgM, kappa isotope Control Antibody, Clone MM30, Alexa Fluor 488, STEMCELL Inc., Cat # 60069 AD.
Alexa Fluor 647 Mouse anti-human SSEA-4 antibody, BD Pharmingen, cat # 560796.
Alexa Fluor 647 Mouse IgG3, k Isotype control, BD Pharmingen, Cat 560803.
FITC Mouse anti-human CD3 antibody, BD Pharmingen, cat # 555339.
FITC Mouse IgG2, k Isotype control, BD Pharmingen, Cat 555573.
FITC Mouse anti-human CD56 antibody, BD Pharmingen, Cat 562794.
FITC Mouse anti-human CD45 antibody, BD Pharmingen, cat # 560976.
FITC Mouse IgG1, k Isotype control, BD Pharmingen, Cat 555748.
Cellometer-ViaStain ™ AOPI stabilizing Solution, Nexcelom, Inc., cat # CS 2-0106.
4% paraformaldehyde fixing solution, product number E672002, of Biotechnology engineering (Shanghai) Ltd.
STEMdiff. NK Cell Kit, STEMCELL, cat # 100-.
UltraPure ™ DNase/RNase-Free dispersed Water, Thermo Fisher Inc., cat # 10977023.
The invention relates to a programmed cooling cryopreservation method for human pluripotent stem cells, which comprises the following steps:
1) storing the frozen stock solution at-20 deg.C, and thawing at 2-8 deg.C before use;
2) discarding the old culture medium, adding 2 mL of CTS-DPBS to wash once;
3) adding CTS-Versene-antibody solution in an amount suitable for just covering the bottom of the culture dish, and digesting at 37 deg.C for 3 min;
4) when the cell colonies are digested to a relatively loosely adherent state, carefully discarding the CTS-Versene-like compartments, gently washing once with a basal cell culture medium (CTS-KnockOut-DMEM/F-12 medium);
5) resuspending cells in a basal cell culture medium (CTS-knock out-like DMEM/F-12 medium), centrifuging at 200g for 5min, and discarding the supernatant;
6) resuspending the cells with a 2-8 ℃ pre-cooled cryopreservation solution, subpackaging the cells into cryopreservation tubes, and recommending the density of 1E5-2E7 individual pluripotent stem cells per mL of the cryopreservation solution for cryopreservation;
7) placing the freezing tube into a refrigerator at 2-8 deg.C, standing for 30min, and transferring into a refrigerator at-20 deg.C;
8) placing the freezing tube into a refrigerator at the temperature of-20 ℃, standing for 2 hours, and transferring into the refrigerator at the temperature of-80 ℃;
9) the frozen tube was placed in a-80 ℃ freezer, left overnight and transferred to a liquid nitrogen tank.
The invention relates to a non-programmed cooling cryopreservation method for human pluripotent stem cells, which comprises the following steps:
1) the frozen stock solution was stored at-20 ℃. Melting at 2-8 deg.C before application;
2) discarding the old culture medium, adding 2 mL of CTS-DPBS to wash once;
3) adding CTS-Versene-antibody solution in an amount suitable for just covering the bottom of the culture dish, and digesting at 37 deg.C for 3 min;
4) when the cell colonies are digested to a looser adherent state, carefully discarding the CTS-Versene-like cells, gently washing once with a basal cell culture medium (CTS-KnockOut-DMEM/F-12 medium);
5) resuspending cells in a basal cell culture medium (CTS-knock out-like DMEM/F-12 medium), centrifuging at 200g for 5min, and discarding the supernatant;
6) resuspending the cells with a 2-8 ℃ pre-cooled cryopreservation solution, subpackaging the cells into cryopreservation tubes, and recommending the density of 1E5-2E7 individual pluripotent stem cells per mL of the cryopreservation solution for cryopreservation;
7) the frozen tube was placed directly in a-80 ℃ freezer overnight and transferred to a liquid nitrogen tank within a week.
The invention relates to a recovery method for human pluripotent stem cells, which comprises the following specific steps:
1) preheating a 6-well plate coated with a complete culture medium (CTS 8 or mTeSR1 culture medium) and a matrix (CTS viral protein (VTN-N) or Corning matrix) of human pluripotent stem cells at room temperature;
2) taking out the human pluripotent stem cells from the liquid nitrogen storage, and transferring the human pluripotent stem cells to a cell room by using dry ice;
3) the vial was immersed in a 37 ℃ water bath without the cap being submerged. Slightly rotating the freezing tube, taking out the freezing tube from the water bath pot when only one ice crystal is left, spraying 70% alcohol on the outer surface of the freezing tube, and then placing the freezing tube in a super clean bench;
4) the thawed cells were transferred to a 15 mL centrifuge tube, and 10 mL of complete human pluripotent stem cell medium (CTS 8-or mTeSR 1-medium) was slowly added thereto. Washing the cryopreserved tube with 1 mL of a complete culture medium (CTS 8 or mTeSR 1) for human pluripotent stem cells, and transferring the cryopreserved tube into a 15 mL tube containing cells;
5)200 g, centrifuged for 5min, the supernatant was discarded, and the cells were resuspended in 2 mL of a complete culture medium (CTS 8-or mTeSR 1-cell culture medium) for human pluripotent stem cells;
6) slowly adding the human pluripotent stem cell suspension into a preheated 6-hole plate coated with a matrix (CTS (VTN-N) or Corning Matrigel), and inoculating 1E6 living cells into each hole;
7) the plates were moved left and right rapidly to disperse the cells evenly in the wells, and then the plates were gently placed at 37 ℃ with 5% CO 2 The incubator of (1);
8) the following day, the old medium is aspirated away, and fresh human pluripotent stem cell complete medium (CTS 8-or mTeSR 1-medium) is added until the cell density reaches 85%.
The invention relates to a method for detecting the counting and the survival rate of human pluripotent stem cells, which comprises the following steps:
1) discarding the old culture medium, adding 2 mL of CTS-DPBS to wash once;
2) discarding CTS (total T-positive) mutant DPBS, and adding StemPro-Accutase in an amount suitable for completely covering cells;
3) incubating in an incubator at 37 ℃ for 2-5 min until single cells become round, and gently blowing and beating the cells to make the cells fall off;
4) transferring the cell suspension to a 15 mL centrifuge tube, adding a basic cell culture medium (CTS & lt & gtknock & lt & gtDMEM/F-12 culture medium) to resuspend cells, centrifuging for 5min at 200g, and discarding the supernatant;
5) adding a proper amount of basic cell culture medium (CTS. cnotkout. RTM. DMEM/F-12 medium) to resuspend the cells;
6) placing 20 mu L of cell suspension into a centrifuge tube, adding 20 mu L of AOPI stabilizing Solution, and gently blowing, beating and uniformly mixing;
7) adding 20 mu L of staining cell suspension into a counting plate, inserting the counting plate into an instrument, and selecting a proper AOPI activity determination method;
8) and previewing bright field and fluorescence images and counting.
The invention relates to a passage amplification method for human pluripotent stem cells, which comprises the following specific steps:
1) discarding the old culture medium, adding 2 mL of CTS-DPBS to wash once;
2) adding CTS-Versene-antibody solution in an amount suitable for just covering the bottom of the culture dish, and digesting at 37 deg.C for 3 min;
3) when the cell colonies are digested to a looser adherent state, carefully discarding the CTS-Versene-like cells, gently washing once with a basal cell culture medium (CTS-KnockOut-DMEM/F-12 medium);
4) resuspending cells in a human pluripotent stem cell complete medium (CTS 8 or mTeSR1 medium), and inoculating the cells into a 6-well plate previously coated with a matrix (CTS. inverter (VTN-N) or Corning matrix);
5) the old medium was aspirated every day, and fresh human pluripotent stem cell complete medium (CTS 8 or mTeSR1 medium) was added until the cell density reached 85%.
The method for calculating the doubling time of the cell population comprises the following specific steps:
1) counting the living cells of the human pluripotent stem cells at 3 days, 6 days, 9 days and 12 days after the cryopreservation recovery according to the method;
2) calculating cell Population Doubling Time (PDT), PDT = T 2-1 Log 2/(log N2-logN 1), wherein T 2-1 Time interval (h) for two cell counts; N2/N1 is the number of viable cells from the 2 nd/1 st cell count.
The method for detecting the expression of alkaline phosphatase by using the human pluripotent stem cells comprises the following specific steps (taking 1 plate hole with 24 holes as an example):
1) removing the cell culture solution from the well to be stained, washing the cells with 500 μ L of DMEM/F12 preheated to 37 ℃ each time for three times each time for 3 min (without shaking by a shaker);
2) diluting AP Live solution to 1 x with DMEM/F12 at a ratio of 1:500 to obtain AP staining solution, adding 150 μ L of the solution into cells to be stained, and adding CO at 37 deg.C 2 Incubating for 30min in the incubator;
3) discarding AP staining working solution, washing cells with 500 μ L DMEM/F12 each time for three times, each time for 3 min (shaking table is not required);
4) when 500. mu.L of DMEM/F12 was added and photographed under a fluorescence microscope, alkaline phosphatase positive cells were positive for green fluorescence and alkaline phosphatase negative cells were negative for green fluorescence.
The invention relates to a method for immunofluorescence staining and flow cytometry analysis of human pluripotent stem cells, which comprises the following specific steps:
1. detecting surface markers;
1) 200 μ L of cell suspension containing 1E6 viable cells was added to a 1.5 mL centrifuge tube, centrifuged and the supernatant discarded. Adding 1 mL of CTS-CAMBOLS DPBS into each tube for resuspension, centrifuging and discarding the supernatant;
2) each tube was filled with 100. mu.L of TRA-1-81/SSEA4/CD34/CD3/CD56/CD45 antibody or the corresponding Isotype control. Gently oscillating, mixing well, and incubating in a refrigerator at 4 deg.C in dark for 30 min;
3) adding 1 mL of CTS-DPBS per tube for centrifugation;
4) abandoning the supernatant, adding 1 mL of CTS-CAMBAGS into each tube for resuspension, and centrifuging;
5) supernatants were discarded and 500. mu.L of CTS-chamber DPBS were added to each tube for resuspension, and flow cytometry was used to detect the expression of TRA-1-81, SSEA4, CD34, CD3, CD56 and CD45 molecules on the cell surface of each group.
2. Nuclear marker detection
Cell preparation, fixation, permeabilization and sealing:
1) discarding the old medium, rinsing once with DMEM/F12;
2) adding 0.5 mM EDTA, wherein the addition amount is proper to just submerge the bottom of the culture dish, and digesting for 3 min at 37 ℃;
3) when the cells are digested to a loose adherent state, carefully removing EDTA, and gently rinsing with DMEM/F12 once;
4) resuspend cells with 1 mL of 4% PFA, collect in a 15 mL centrifuge tube, fix for 30min at room temperature;
5) discard 4% PFA, wash three times with 2 mL PBS, 5min each time with shaker (if fixed overnight or longer, need to extend the time of PBS wash on shaker to 30min each time);
6) PBS was discarded, and the tube was permeabilized with 1 mL of 0.3% Triton for 30min at room temperature, which was transferred to a 1.5 mL EP tube;
7) discard Triton and block with 1 mL 5% BSA for 1 hour at room temperature;
8) discard 5% BSA, add 100. mu.L OCT4 antibody/NANOG antibody/Isotype control to each tube, incubate 1 hour at room temperature with shaker;
9) discarding the OCT4 antibody/NANOG antibody/Isotype control, washing with 1 mL PBS for three times, each time shaking for 5 min;
10) supernatants were discarded and 500. mu.L of CTS-chamber DPBS was added per tube for resuspension and flow cytometry was used to detect OCT4 and NANOG molecule expression in each group of cells.
The invention relates to a method for detecting and analyzing karyotype of human pluripotent stem cells, which comprises the following specific steps:
1) removing cell culture supernatant, replacing with fresh culture solution containing 0.2 μ g/mL colchicine, and continuously culturing for 2-4 h;
2) abandoning the cell culture solution, cleaning the cell culture solution by using a CTS (human immunodeficiency virus) DPBS (double-stranded-polymerase chain reaction) with the volume of 1/2 culture solution, adding a StemPro (human actin) Accutase solution with the volume of 1/2 culture solution, digesting the solution at 37 ℃ for 5min, adding a CTS (human immunodeficiency virus) DPBS with the volume of 4 times, gently blowing the solution into single cells, collecting the single cells into a 15 mL centrifuge tube, and centrifuging the solution for 5min at 200 g;
3) discarding the supernatant, adding 4 mL of preheated 75 mM KCl hypotonic solution, gently blowing and beating the resuspended cell pellet by using a1 mL pipette until no cell pellet exists, and treating at 37 ℃ for 20 min;
4) to the hypotonic cell suspension was added 0.5 mL of a fixative (methanol: glacial acetic acid = 9: 4), 200g centrifuged for 5 min;
5) discarding the supernatant, adding 4 mL of stationary liquid, fixing at room temperature for 30min, centrifuging at 200g for 5min, and repeating the fixing for 1-2 times;
6) discarding the supernatant, adding 0.35 mL of fixing solution, and resuspending the precipitate for later use;
7) boiling a proper amount of boiled water, adding the boiled water into a 100 mL beaker, taking out the glass slide precooled by water from a refrigerator at 4 ℃, uniformly mixing the cell suspension obtained in the step 6), sucking about 80 mu L of the cell suspension, dripping the cell suspension into the center of the glass slide by 3-5 drops, and immediately putting the cell suspension at the mouth of the beaker to fumigate the glass slide for 5min by using steam;
8) drying the slide and receiving the slide rack; drying the slices at 60 ℃ for 2 h, and then drying and storing at room temperature;
9) taking out 1 slide, and placing into a dye vat filled with 0.005% pancreatin digestive juice for digestion for 4-5 min;
10) taking out the slide, putting the slide into a dye vat filled with CTS (total positive going lineage) DPBS (double positive lineage-specific cytoplasmic-repeat) and placing the slide for 30 s in an up-and-down extraction and insertion manner;
11) taking out the slide from CTS ™ DPBS, dipping residual liquid on toilet paper, putting the toilet paper into a dye vat filled with Giemsa dye solution working solution, and dyeing for 6-8 min;
12) after dyeing, washing the glass slide by tap water;
13) and throwing the washed slide for several times, putting the slide on a slide rack, airing the slide, observing the slide under an upright microscope, finding a visual field with a split phase by a 10X objective lens, converting a 100X oil lens to observe a banding effect, and scanning and analyzing the banding effect.
The method for directed differentiation of human pluripotent stem cells into NK cells of the present invention is carried out according to the steps of the STEMdiff & lt/EN & gt NK Cell Kit product technical manual.
The invention relates to an experimental method for formation of human pluripotent stem cell teratoma, which comprises the following specific steps:
1) a suspension of CTS-chamber DPBS cells containing 1.5E7 viable human pluripotent stem cells/100. mu.L was prepared as described above;
2) selecting NPG male mice with the age of more than 5 weeks, and injecting 100 mu L of CTS (human pluripotent stem) cell suspension containing 1.5E7 live human pluripotent stem cells under the testicular envelopes on two sides of each mouse respectively;
3) 6-10 weeks after cell injection, the experiment was terminated depending on tumor growth (a. mice lost more than 20% weight, b. tumors were larger than 1.5g or had a diameter of either dimension of more than 20 mm);
and after the tumor bearing is successful, dissecting the testis, stripping the tumor, and after the tumor is stripped, cutting the tumor tissue into blocks, embedding paraffin, slicing, HE dyeing and collecting pictures.
EXAMPLE 1 preparation of human pluripotent Stem cell cryopreservation solution
The preparation method comprises the following steps:
methyl cellulose (20 g/L, H) 2 O) solution preparation: weighing the powder, adding corresponding water to a final concentration of 20 g/L, sterilizing with high pressure steam, and shaking overnight in a shaker at room temperature. Preparation of ID-8 (10 mM in DMSO) solution: the powder was weighed, added to the corresponding DMSO to a final concentration of 10 mM and mixed well at room temperature. The other components are mixed according to a normal mixing method, and after the preparation is finished, the pH value is finely adjusted to 7.20-7.25. Filtering with 0.22 μm filter, packaging, and freezing at-20 deg.C.
In the frozen stock solution of the embodiment 1, the samples 1 to 13 are the frozen stock solution of the human pluripotent stem cells, and different dosage ratios are adopted; controls 1-6 differed from samples 1-13 by having only 1 or 2 signaling pathway inhibitors; the control groups 7-10 differed from the samples in that Human Serum Albumin (HSA) was also added; control 11 is a CTS-system PSC cryopreservation kit from Thermo Fisher, USA, comprising two components, a CTS-system PSC Cryomedium and a CTS-system RevitaCell-system supplement (100X).
The compositions and ratios of the frozen stocks are shown in Table 1, wherein the amounts of the culture media DMEM/F-12, DMEM and RPMI1640 are in v/v, the amount of dimethyl sulfoxide (DMSO) is in v/v, the amount of Methylcellulose (MC) is in g/L, the amount of the signal pathway inhibitor CHIR-99021 is nM, the amount of the signal pathway inhibitor ID-8 is nM, the amount of the signal pathway inhibitor Y-27632 is in μ M, and the amount of Human Serum Albumin (HSA) is mg/mL.
Table 1 example 1 composition and proportions of the frozen stock solution
Example 2 comparison of human pluripotent stem cell viability Rate after cryopreservation and recovery Using different cryopreservation solutions and different temperature reduction methods
Human pluripotent stem cells H1 were cryopreserved according to the programmed cooling and non-programmed cooling cryopreservation methods of the present invention, using human pluripotent stem cells H1 as test cells, using the cryopreservation solutions of samples 1 to 13 as test cryopreservation solutions, and using the cryopreservation solutions of controls 1 to 11 as control cryopreservation solutions. Thawing after 1 month of cryopreservation, detecting cell viability at 0 hour after thawing, and performing statistics to compare the cryopreservation effects of different cryopreservation solutions on human pluripotent stem cells, as shown in FIG. 1, the ordinate represents H1 cell viability (%) at 0 hour after thawing.
Using pairingstThe data in the figure 1 are analyzed statistically by examination, H1 cells are frozen by using the sample 1-13 cell frozen stock solution through programmed cooling and non-programmed cooling, the H1 cell survival rate is more than 93% after 0 hour of recovery, and the human pluripotent stem cell frozen stock solution can still achieve better technical effect even if protein components are not contained. The H1 cells are frozen by programmed cooling or non-programmed cooling by using the sample frozen liquid of 1-13 cells, and the survival rate of the H1 cells has no significant difference after 0 hour of recovery, so that the non-programmed cooling can be selected when the frozen liquid is frozen, thereby being simple, convenient and quick.
Compared with the control 7-10 cell frozen stock solution, the samples 5 and 11-13 are different in that human serum albumin is added into the control 7-10 cell frozen stock solution, and the result shows that the H1 cell survival rate after 0 hour of recovery has no significant difference no matter H1 cells are frozen by programmed cooling or non-programmed cooling. Demonstrating that the frozen stock solution of the present invention can avoid the use of protein components by the synergistic effect of 3 kinds of signal pathway inhibitors, and can achieve similar technical effects.
Compared with the use of the sample 1-13 cell cryopreservation solution, the use of the controls 1-6 and 11 significantly reduced the H1 cell viability rate 0 hours after resuscitation regardless of programmed or unprogrammed cryopreservation of H1 cells. It is demonstrated that the addition of only 1 or 2 signal pathway inhibitors to the frozen stock solution significantly reduced the technical effect of the frozen stock solution, especially the unprogrammed temperature reduction.
Example 3 detection of the survival Rate of human pluripotent Stem cells at different time points after cryopreservation Resuscitation
The human pluripotent stem cells H9 were cryopreserved by the non-programmed cryopreservation method of the present invention, using human pluripotent stem cells H9 as test cells, using the cryopreserved solutions of sample 5 and sample 13 as test cryopreserved solutions, and using the cryopreserved solution of control 11 as a control cryopreserved solution. And (4) performing resuscitation after freezing for 1 month, and performing cell viability detection at 0 hour, 24 hours, 48 hours and 72 hours after resuscitation.
As can be seen from Table 2, the H9 cells were cryopreserved using the cell cryopreservation solution of sample 5 or sample 13, and the H9 cell viability was over 90% at 0 hour and 24 hours after recovery with little/no delayed freezing injury.
TABLE 2 cell viability rates of human pluripotent stem cells H9 at different time points after cryopreservation recovery
(n is more than or equal to 3, and the cell survival rate before cryopreservation is more than or equal to 95%)
Example 4 cryopreservation cell Density study
And (3) taking the human induced pluripotent stem cells ATCC-BXS0117 as tested cells, taking the frozen stock solution of the sample 5 as the tested frozen stock solution, and freezing and storing the ATCC-BXS0117 cells with different densities according to the non-programmed cooling and freezing and storing method. And (4) recovering after freezing for 1 month, and detecting the cell viability rate 0 hour after recovery.
As can be seen from Table 3, the sample 5 frozen stock solution is used for freezing 1E5-2E7 viable cells/mL ATCC-BXS0117 cells, the cell viability can reach more than 90% in 0 hour after recovery, and the application range is wide.
TABLE 3 cell viability rates of human induced pluripotent stem cells ATCC-BXS0117 after different cell densities freeze recovery
(n is more than or equal to 3, and the cell survival rate before cryopreservation is more than or equal to 95%)
Example 5 morphological Observation of human pluripotent Stem cells after cryopreservation Resuscitation
Human embryonic stem cells H1 and H9 and human induced pluripotent stem cells ATCC-BXS0117 are used as tested cells, a sample 3 frozen stock solution is used as a tested frozen stock solution, the human pluripotent stem cells are frozen according to the non-programmed cooling freezing method, and are revived after being frozen for 12 months, and a cell light microscope picture is collected.
As can be seen from FIG. 2, the cells frozen with the samples 3, H1, H9 and ATCC-BXS0117, after recovery, grow in clonal clusters, have clear clonal margins, high nuclear-to-mass ratio and close contact between cells in the clones, and are in the typical morphology of human pluripotent stem cells.
Example 6 detection of doubling time of human pluripotent stem cell populations after cryopreservation recovery
Human embryonic stem cells H1 and H9 and human induced pluripotent stem cells ATCC-BXS0117 are used as tested cells, frozen stock solutions of a sample 3 and a sample 12 are used as tested frozen stock solutions, the human pluripotent stem cells are frozen according to the non-programmed cooling freezing method, and the frozen stock solutions are revived after being frozen for 12 months. The method is characterized in that unfrozen H1, H9 and ATCC-BXS0117 cells are used as control cells, frozen and recovered H1, H9 and ATCC-BXS0117 cells are used as test cells, viable cells are counted on the 3 rd day, the 6 th day, the 9 th day and the 12 th day after recovery according to the method, and the cell population doubling time is calculated.
Using pairingstTests statistical analysis of the data in Table 4 was performed, using samples 3 and 12 cryopreserved H1, H9 and ATCC-BXS0117 cells, the doubling time of the recovered cell population was not significantly different from that of the non-cryopreserved cells, and the cells could be expanded in vitro for a long period of time.
TABLE 4 cell population doubling time of human pluripotent stem cells at different time points after cryopreservation recovery
(n is more than or equal to 3, and the cell survival rate before cryopreservation is more than or equal to 95%)
Example 7 Long-term stability study of human pluripotent Stem cell cryopreservation solution-detection of human pluripotent Stem cell viability following cryopreservation Resuscitation
Human embryonic stem cells H1 and H9 and human induced pluripotent stem cells ATCC-BXS0117 are used as tested cells, frozen stock solutions of a sample 8 and a sample 11 are used as tested frozen stock solutions, the human pluripotent stem cells are frozen according to the non-programmed cooling freezing method, and the frozen stock solutions are recovered after 0, 12, 24 and 36 months. Cell viability assays were performed 0 hours after resuscitation according to the methods described above in the present invention.
Using pairingstStatistical analysis of the data in Table 5 showed that there was no significant difference in cell viability between H1, H9, and ATCC-BXS0117 cells thawed after different times of cryopreservation using the cryopreservation solutions from samples 8 and 11.
TABLE 5 cell viability Rate of human pluripotent Stem cells after thawing at different times (n.gtoreq.3)
Example 8 Long-term stability study of human pluripotent Stem cell cryopreservation solution-detection of human pluripotent Stem cell alkaline phosphatase expression levels after cryopreservation Resuscitation
Human embryonic stem cells H1 and H9 and human induced pluripotent stem cells ATCC-BXS0117 are used as tested cells, a sample 12 frozen stock solution is used as a tested frozen stock solution, the human pluripotent stem cells are frozen according to the non-programmed cooling freezing method, and the frozen cells are revived after being frozen for 24 months. Human pluripotent stem cell alkaline phosphatase was detected 72 hours after resuscitation according to the method described above in the present invention.
As can be seen from FIG. 3, the human embryonic stem cells H1, H9 and ATCC-BXS0117, which were recovered 24 months after the frozen storage of the sample 12, expressed the human pluripotent stem cell marker protein alkaline phosphatase at a high ratio.
Example 9 Long-term stability study of frozen stock solution of human pluripotent Stem cells-detection of marker protein of human pluripotent Stem cells after recovery from frozen stock
Human embryonic stem cells H1 and human induced pluripotent stem cells ATCC-BXS0117 are used as tested cells, frozen stock solutions of a sample 9 and a sample 10 are used as tested frozen stock solutions, the human pluripotent stem cells are frozen according to the non-programmed cooling freezing method, and the cells are recovered after being frozen for 24 months. Human pluripotent stem cell marker protein detection was performed 72 hours after resuscitation according to the methods described above in the present invention.
The results show that H1 and ATCC-BXS0117 cells recovered 24 months after cryopreservation express the human pluripotent stem cell marker proteins OCT4, NANOG, TRA-1-80 and SSEA4 in high proportion by using the cryopreservation solution of sample 9 and sample 10, and exceed the requirements of the group standard of human embryonic stem cells.
TABLE 6 detection of cell marker proteins of human pluripotent stem cells after long term cryopreservation recovery (flow cytometry, n. gtoreq.3)
Example 10 Long-term stability Studies of human pluripotent Stem cell cryopreservation solution-detection of human pluripotent Stem cell karyotype following cryopreservation Resuscitation
Human embryonic stem cells H1 and H9 and human induced pluripotent stem cells ATCC-BXS0117 are used as tested cells, the sample 4 frozen stock solution is used as tested frozen stock solution, the human pluripotent stem cells are frozen according to the non-programmed cooling freezing method, and the cells are recovered after being frozen for 24 months. Human pluripotent stem cell karyotype was detected according to the method described above in the present invention.
The results are shown in FIG. 4, where the karyotypes of human embryonic stem cells H1, H9 and human induced pluripotent stem cells ATCC-BXS0117 recovered 24 months after cryopreservation using sample 4 were normal, 46 XY, 46 XX and 46 XX, respectively.
Example 11 Long-term stability study of human pluripotent Stem cell cryopreservation solution-detection of the Directional differentiation Capacity of human pluripotent Stem cells after cryopreservation recovery
Human embryonic stem cells H1 and human induced pluripotent stem cells ATCC-BXS0117 are used as tested cells, frozen stock solutions of a sample 6 and a sample 7 are used as tested frozen stock solutions, the human pluripotent stem cells are frozen according to the non-programmed cooling freezing method, and the cells are recovered after being frozen for 24 months. NK cells are directionally differentiated and cell marker protein detection is carried out according to the method disclosed by the invention.
The results showed that H1 and human-induced pluripotent stem cell ATCC-BXS0117 cells could be directionally differentiated into CD34 with high efficiency after 24 months of cryopreservation using the frozen stock solution of sample 6 + And CD3 - CD56 + CD45 + Has mesoderm differentiation potential.
TABLE 7 detection of cell marker proteins for directed differentiation of human pluripotent stem cells after long-term cryopreservation recovery
(flow cytometry, n.gtoreq.3)
Example 12 Long-term stability study of human pluripotent Stem cell cryopreservation solution-detection of the ability of human pluripotent Stem cell to form teratoma after cryopreservation Resuscitation
Human embryonic stem cells H9 are used as test cells, the frozen stock solution of the sample 13 is used as a test frozen stock solution, the human pluripotent stem cells are frozen according to the non-programmed cooling freezing method, and the cells are recovered after being frozen for 24 months. The teratoma formation ability in vivo was examined according to the method of the present invention as described above.
The results are shown in fig. 5, and H9 cells after 24 months of cryopreservation formed teratomas with a three germ layer structure in nude mice with in vivo three germ layer differentiation potential using the cryopreservation solution of sample 13.
Example 13 Long-term stability Studies of human pluripotent Stem cell frozen stock solution-appearance assay of human pluripotent Stem cell frozen stock solution
And (3) taking the sample frozen stock solution 5-10 as the frozen stock solution to be tested, freezing the human pluripotent stem cell frozen stock solution according to the non-programmed cooling freezing method, and recovering after freezing for 0, 12, 24 and 36 months. Appearance and pH measurements were performed according to the methods described above in the present invention.
The results show that after the frozen stock is recovered at different times, the appearance of the frozen stock solution of the samples 5-10 is stable, and the phenomena of agglomeration, floccule and the like do not occur.
TABLE 8 appearance of frozen stock solution of human pluripotent stem cells after thawing at different times
Example 14 Long-term stability Studies of human pluripotent Stem cell frozen stock solution-pH assay of human pluripotent Stem cell frozen stock solution
And (3) taking the sample frozen stock solution 5-10 as the frozen stock solution to be tested, freezing the human pluripotent stem cell frozen stock solution according to the non-programmed cooling freezing method, and recovering after freezing for 0, 12, 24 and 36 months. The pH value was measured according to the method described above in the present invention.
The results show that the pH of the frozen stock solution of samples 5-10 was 7.2. + -. 0.2 at 25 ℃ after thawing at different times of freezing.
TABLE 9 pH of frozen stock solution of human pluripotent stem cells after thawing at different times
Example 15 Long-term stability Studies of human pluripotent Stem cell frozen stock solution-detection of bacterial endotoxin in human pluripotent Stem cell frozen stock solution
The frozen stock solution of the sample 1-10 is used as the frozen stock solution to be tested, the frozen stock solution of the human pluripotent stem cells is frozen according to the method of the invention, and the frozen stock solution is recovered after 0, 12, 24 and 36 months. Bacterial endotoxin detection was performed according to the above-described method of the present invention.
The results show that after the samples are frozen and restored at different times, the bacterial endotoxin of the frozen stock solutions of the samples 1 to 10 is less than or equal to 0.5 EU/mL.
TABLE 10 endotoxin content of human pluripotent stem cell cryopreserved fluid after different thawing periods
It should be understood that the above-described embodiments of the present invention are merely examples for clearly illustrating the invention and are not to be construed as limitations of the embodiments of the present invention, and various changes, modifications, substitutions and alterations based on the present invention can be made by those skilled in the art without departing from the principle and spirit of the present invention.
Claims (10)
1. The human pluripotent stem cell cryopreservation liquid is characterized by comprising the following components: basal cell culture medium, cryoprotectant and signaling pathway inhibitor compositions; the signal channel inhibitor composition comprises CHIR-99021, ID-8 and Y-27632, wherein the molar concentration of the CHIR-99021 in the whole freezing solution is 50-500 nM, the molar concentration of the ID-8 in the whole freezing solution is 100-1000 nM, and the molar concentration of the Y-27632 in the whole freezing solution is 1-30 mu M; and the human pluripotent stem cell frozen stock solution does not contain any protein component.
2. The cryopreservation solution for human pluripotent stem cells of claim 1, wherein the molar concentration of CHIR-99021 in the total cryopreservation solution is 100-250 nM, the molar concentration of ID-8 in the total cryopreservation solution is 250-750 nM, and the molar concentration of Y-27632 in the total cryopreservation solution is 8-12 μ M.
3. The human pluripotent stem cell cryopreservation solution of claim 1, wherein the basal cell culture medium is: one or more of DMEM, RPMI1640 and DMEM/F-12, in a volume percentage of 78.5-98.5% v/v of the whole frozen stock solution.
4. The human pluripotent stem cell lysate of claim 1, wherein the basal cell culture medium is DMEM/F-12 at 78.5-98.5% v/v by volume of the total lysate.
5. The human pluripotent stem cell cryopreservation liquid according to claim 1, wherein the cryoprotectant is dimethyl sulfoxide and/or methyl cellulose, the volume percentage of the dimethyl sulfoxide to the whole cryopreservation liquid is 1-15% v/v, and the mass volume concentration of the methyl cellulose to the cryopreservation liquid is 0.1-1.5 g/L.
6. The human pluripotent stem cell cryopreservation liquid according to claim 1, wherein the cryoprotectant is dimethyl sulfoxide and/or methyl cellulose, the volume percentage of the dimethyl sulfoxide to the whole cryopreservation liquid is 5-12% v/v, and the mass volume concentration of the methyl cellulose to the cryopreservation liquid is 0.5-1.2 g/L.
7. Use of the frozen stock solution of human pluripotent stem cells according to any one of claims 1 to 6 for freezing human pluripotent stem cells or for preparing frozen products of human pluripotent stem cells.
8. Use according to claim 7, wherein the product is a reagent or kit.
9. Use of the human pluripotent stem cell cryopreserved of any one of claims 1 to 6, in any one of:
(1) the application in establishing a human pluripotent stem cell bank;
(2) application in cryopreservation of human pluripotent stem cells for research after resuscitation;
(3) the use in cryopreservation of human pluripotent stem cells for production following resuscitation;
(4) the application in frozen storage of human pluripotent stem cells for quality control and quality detection after resuscitation;
(5) the application of the cell in the frozen storage of the human pluripotent stem cells for the differentiation of the three germ layer cells and the preparation of the derived functional cells after recovery.
10. The method for preparing a frozen stock solution of human pluripotent stem cells according to any one of claims 1 to 6, wherein the method comprises the following steps: preparing a DMSO solution of ID-8, and uniformly mixing at room temperature; mixing all components of the human pluripotent stem cell frozen stock solution, finely adjusting the pH value to 7.20-7.25, filtering by a filter, subpackaging and freezing.
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