CN112544613A - Pluripotent stem cell cryopreservation liquid, application thereof and cryopreservation method - Google Patents
Pluripotent stem cell cryopreservation liquid, application thereof and cryopreservation method Download PDFInfo
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Abstract
The invention discloses a pluripotent stem cell cryopreservation liquid, application thereof and a cryopreservation method, and relates to the technical field of biology. The frozen stock solution comprises inorganic salt, vitamins, chemical small molecule inhibitors, cytokines, DMSO and other components, has definite components, and does not contain serum or animal-derived components. The stem cell cryopreservation liquid greatly improves the cell cryopreservation activity, is simple and convenient in cryopreservation process, enables cells to recover after long-term cryopreservation, and is stable in various characteristics. Therefore, the cryopreservation solution greatly expands the clinical application of the human pluripotent stem cells.
Description
[ technical field ] A method for producing a semiconductor device
The invention relates to the technical field of biology, and particularly relates to a pluripotent stem cell cryopreservation solution, application thereof and a cryopreservation method.
[ background of the invention ]
Regenerative medicine refers to an emerging science that utilizes a variety of novel technical disciplines to reconstruct tissues and organs that are aged or functionally lost, and to treat related diseases through a variety of medical means. Important research directions of regenerative medicine are mechanisms of normal tissue characteristics and functions, biological bases of post-traumatic repair, regeneration mechanisms of tissues and organs and differentiation mechanisms of various stem cells, so that an effective biological treatment method is finally obtained. The research method integrates a plurality of means, including the principles and methods of the subjects of life science, material science, clinical medicine and the like, and comprehensively solves the clinical solution scheme for replacing, repairing, reconstructing or regenerating various tissues and organs of the human body. In the development of regenerative medicine, there are a number of core problems that need to be addressed. An ideal regenerative medicine product, especially a cell therapy product, should have several characteristics: first, the cell source must be a single immortalizable cell type; second, the cells must be amenable to further modification, such as modification of disease-causing genes using tools such as gene editing; thirdly, the final product is uniform in properties. How to obtain a primary source of cells that can be used for cell therapy is an important research topic in the early regenerative medicine field. Among them, Embryonic stem cells (ESCs, abbreviated as ES, EK or ESC cells) are the most attractive cell type in early regenerative medicine research. Embryonic stem cells are a class of cells isolated from early embryos (pre-gastrulation) or primitive gonads that have the property of immortalization, self-renewal, and multidirectional differentiation in vitro cultures. ES cells can be induced to differentiate into almost all cell types in the body, both in vitro and in vivo. However, the availability and use of this cell is highly ethical because the study of embryonic stem cells must be carried out to destroy the embryo, which is the life form of a human in the uterus when it is not yet formed. This ethical debate has greatly hindered the advancement and application of regenerative medicine.
In 2006, the Ministry of Onchida, the Ministry of Shanzhou, invented a "cocktail" method consisting of four transcription factors including OCT4, SOX2, KLF4 and c-Myc, which successfully reprograms terminally differentiated dermal fibroblasts into stem cells with differentiation pluripotency, which are called induced pluripotent stem cells (Takahashi K, et al., Cell, 2006, 126(4) pp.663-676; Takahashi K and Yamanaka S, Cell, 2007, 131(5) pp.861-872). These stem cells have a differentiation potential similar to that of embryonic stem cells (embryonic stem cells), and are capable of forming the three germ layers most essential for human development: ectoderm, mesoderm and endoderm, and eventually form a variety of adult cells. The invention breaks through the ethical limitation of using human embryonic stem cells in medicine, can solve the problem of immunological rejection in cell transplantation treatment, and greatly expands the application potential of stem cell technology in clinical medicine. The induction differentiation of ectoderm cells by using totipotent stem cells or pluripotent stem cells including embryonic stem cells and iPSCs as raw materials can be used as a new idea of clinical treatment, and the application potential of the ectoderm cells in clinical medicine is greatly expanded. Only in the field of regenerative medicine, the fields to which the application of induced pluripotent stem cells to the stage of coming into clinical use has been directed include neurodegenerative diseases, spinal cord injury, type I diabetes, tumors, and the like.
Pluripotent stem cells represented by induced pluripotent stem cells are a hot spot of stem cell research in the world as a core technology platform of regenerative medicine. Cell cryopreservation is one of the main methods for cell preservation. In order to be able to store, apply and transport pluripotent cells over long periods of time, an effective cell cryopreservation solution and cryopreservation method should be used, and the characteristics of the cells should be maintained after repeated resuscitation. Cryopreservation can help to use the cells at any time in time, while reducing the risk of microbial contamination and reducing the risk of cross-contamination with other cell lines. Therefore, the research on the cryopreservation method of pluripotent stem cells is particularly important. "vitrification cryopreservation" of cells and tissues allows the cells and their protectant solutions to be cooled at a rate fast enough to convert the biological material to a fully vitrified state and allow long term storage at low temperatures. In the process, icing inside and outside the cells is avoided, so that cell damage is avoided. The key to realize vitrification is a cell cryopreservation agent which can reduce the formation of ice crystals in the process of cryopreservation of cells or tissues, thereby playing a role in protecting the cells. Commonly used cell cryopreservatives include dimethyl sulfoxide (DMSO), Ethylene Glycol (EG) and Propylene Glycol (PG) (Almanosoori KA et al, Cryobiology.2012Jun; 64(3): 185-91). At present, the human pluripotent stem cells generally adopt DMSO (dimethyl sulfoxide) as a cryopreservation agent, the DMSO is a low-molecular compound, the molecular weight is small, the solubility is high, the permeability is high, the freezing point can be lowered, the permeability of a cell membrane to water is improved, the chance of forming ice crystals in cells is reduced in the cooling process, the damage of the ice crystals to the cells is reduced, and the cells are protected. High concentrations of DMSO are cytotoxic and can interact with hydrophobic groups of intracellular proteins, resulting in denaturation of the proteins, resulting in cell damage or inactivation; furthermore, in clinical experience with hematopoietic stem cells, DMSO residues cause certain side effects (ZambelliA et al., Anticancer Res 1998; 18(6B): 4705-. 10% DMSO is a safe concentration for use in cells, and therefore, currently, human pluripotent stem cells are generally cryopreserved using 90% (fetal) bovine serum plus 10% DMSO as a cryopreservation solution (Hanna J and HubelA, Organogenesis,01Jul2009,5(3): 134-. Although many scientists reduce the concentration of serum components through various media containing serum components, this method of protecting the activity of stem cells by using a large amount of animal serum is not suitable for the development of regenerative medicine products, and the serum components can carry various animal-derived components or viruses, which can cause serious infection and complications.
Therefore, how to develop a cryopreservation reagent which is serum-free, has definite chemical components and can ensure the activity of the pluripotent stem cells is an important tool for expanding the clinical use and application prospects of the pluripotent stem cells.
[ summary of the invention ]
The invention aims to provide a pluripotent stem cell cryopreservation liquid, application thereof and a cryopreservation method.
In view of the above, the technical solution adopted to achieve the above object of the present invention is:
a pluripotent stem cell cryopreservation liquid comprising a minimal medium and additives, wherein the additives comprise Optiferrin, DMSO and Y-276322 HCl.
In particular embodiments, the additives include 0.5-10 μmol/L Optiferrin, 2.5-10 v/v% DMSO, and 10nmol/L-10 μmol/L Y-276322 HCl. Wherein, the DMSO is preferably 5-10 v/v%.
In particular embodiments, the additive further comprises an inorganic salt, an amino acid, and a cytokine.
In a specific embodiment, the vitamins include 1.2 μmol/L vitamin B12, 64mg/L vitamin C.
In a specific embodiment, the inorganic salts include 0.5g/L sodium chloride, 13.6. mu.g/L sodium selenite.
In specific embodiments, the growth factor comprises 22. mu.g/mL IGF, 50ng/mL plant-derived recombinant human basic growth factor, 1.74ng/mL transforming growth factor TGF-b.
In a specific embodiment, the additive in the frozen stock solution of pluripotent stem cells further comprises 6.3ng/ml progesterone and 23 μ g/ml putrescine.
In a specific embodiment, the minimal medium is DMEM medium.
The invention also aims to provide application of the frozen stock solution in freezing and storing the human pluripotent stem cells.
Still another object of the present invention is to provide a cryopreservation method of human pluripotent stem cells, comprising the steps of: and centrifuging the expanded and passaged pluripotent stem cell suspension, removing the supernatant, mixing the induced pluripotent stem cell frozen stock solution, subpackaging and freezing.
Compared with the prior art, the invention has the beneficial effects that:
1. the invention provides a cell cryopreservation solution with clear chemical components without using animal-derived components, which not only greatly improves the cell activity after cell recovery, but also has complete cell functions after long-time cryopreservation compared with commercial cryopreservation solutions.
2. The invention has simple and convenient configuration and convenient use, and greatly expands the clinical application prospect of the pluripotent stem cells.
[ description of the drawings ]
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 is a cell morphology map of human pluripotent stem cells after resuscitation with iRePlur-F cryopreservation according to the invention.
Figure 2 is a graphical representation of the effect of different concentrations of Optiferrin in cryopreservation on cell viability according to the invention.
FIG. 3 is a schematic diagram showing the effect of different concentrations of Y-27632 hydrochloride freezing medium on the apoptosis rate of human pluripotent stem cells after resuscitation.
FIG. 4 is a schematic diagram showing the result of detecting the apoptosis rate of the human pluripotent stem cells cryopreserved by iRePlur-F cell cryopreserved solutions with different DMSO concentrations and a control group CELLBANKER cryopreserved solution.
FIG. 5 is a schematic diagram showing the results of detecting the resuscitation apoptosis rate of iRePlur-F cell cryopreserved fluid and CELLBANKER cryopreserved fluid after human pluripotent stem cells are cryopreserved.
FIG. 6 is a schematic diagram of the result of identifying the cell pluripotency by resuscitation of human pluripotent stem cells.
[ detailed description ] embodiments
The invention provides a pluripotent stem cell liquid frozen stock solution which comprises a basic culture medium and additives, wherein the additives comprise 0.5-10 mu mol/L Optiferrin, 2.5-10 v/v% DMSO and 10nmol/L-10 mu mol/L Y-276322 HCl.
According to one embodiment of the invention, the DMSO is preferably between 5 and 10 v/v%.
According to the pluripotent stem cell cryopreservation liquid provided by the invention, the additive further comprises vitamins, inorganic salt and cytokines according to needs.
According to one embodiment of the invention, the vitamins include 1.2 μmol/L vitamin B12, 64mg/L vitamin C. The vitamins may also be selected from other commonly used vitamins such as vitamin B6, vitamin B2, etc., according to common knowledge in the art.
According to one embodiment of the invention, the inorganic salt comprises 0.5g/L sodium chloride, 13.6ug/L sodium selenite. Other commonly used inorganic salts may also be selected according to common general knowledge in the art.
According to one embodiment of the invention, the cytokine comprises 22ug/mL IGF, plant derived recombinant human basic growth factor, 50ng/mL, transforming growth factor 1.74 ng/mL. Other commonly used cytokines may also be selected according to common knowledge in the art.
According to one embodiment of the invention, the cryopreservation solution further comprises 6.3ng/ml progesterone and 23ug/ml putrescine.
According to one embodiment of the invention, the minimal medium is DMEM medium.
The following examples are intended to illustrate the invention without limiting its scope. It is intended that all modifications or alterations to the methods, procedures or conditions of the present invention be made without departing from the spirit and substance of the invention.
EXAMPLE 1 preparation of human pluripotent Stem cell liquid cryopreservation solution iRePlur-F
The iRePlur-F cell cryopreservation solution is prepared according to the following formula, and is called iRePlur-F for short:
duchenne modified eagle's medium (DMEM medium), vitamin B12 (1.2. mu. mol/L), vitamin C (L-ascorbic acid, 64mg/L), Progesterone (Progesterone, 6.3ng/mL), Putrescine (Putrescine, 23ug/mL), Sodium Chloride (Sodium Chloride, 0.5g/L), Sodium selenite (13.6ug/L), IGF (22ug/mL), Optiferrin (0.5-10. mu. mol/L), plant-derived recombinant human basic growth factor (OsrbFGF, 50ng/mL), TGF-B (1.74ng/mL), 2.5% -10% DMSO, Y-276322HCl (10 nmol-10. mu. mol/L).
Wherein the control test is performed
DMSO Free GMP grade (ZENOAQ) frozen stock solution is hereinafter referred to as CELLBANKER.
Example 2 cryopreservation of human pluripotent Stem cells
Human pluripotent stem cells include embryonic pluripotent stem cells such as the H9 cell line and human induced pluripotent stem cells. Wherein the human induced pluripotent stem cells are obtained by reprogramming CD34+ cells according to a reprogramming medium and a culture method of reprogramming induced pluripotent stem cells (ZL 201910050800.7).
Human pluripotent stem cells T25 cell culture flasks were coated with matrigel (STEMCELL technologies), plated, and incubated in a 37 ℃ incubator for more than one hour. According to 1 × 106The cell number per flask was inoculated in T25 flasks for expansion and passaging. Cultured in a commercial TeSRTM-e8(STEMCELL Tech nologices) medium at 37 ℃ in 5% CO2Culturing in the medium. When the cell growth reaches 70% coverage, 0.05% trypsin/EDTA is used, digestion is carried out by incubating at 37 ℃ for 5min, and the digested cells are washed by centrifugation and then are washed according to the proportion of 1 × 106Resuspending in iRePlur-F cell freezing medium at a density of one milliliter, putting the cells into a programmed cooling freezing box, placing the box in a refrigerator at minus 80 ℃ for 24 hours, and then transferring the box into liquid nitrogen for long-term storage. Control experiments were performed with the same batch of cells, which were cryopreserved using cellbank.
Example 3 Resuscitation of human pluripotent Stem cells
4.1 Effect of DMSO concentration on cell viability after human pluripotent Stem cell recovery
Human pluripotent stem cells T25 cell culture flasks were coated with matrigel (STEMCELL technologies), plated, and incubated in a 37 ℃ incubator for more than one hour. Cells frozen by using iRePlur-F frozen stock solution and control group CELLBANKER frozen stock solution are quickly thawed in water bath at 37 ℃, cells after thawing are washed by a DMEM culture medium to remove DMSO, and digested cells are centrifugally washed according to the proportion of 1 × 106Per 10 ml of the culture medium was inoculated into a T25 cell culture plate at 37 ℃ with 5% CO2Culturing in a cell culture box. The morphology of the recovered pluripotent stem cells is shown in figure 1. Wherein, FIG. 1a, FIG. 1b, FIG. 1c show the cell morphology of iRePlur-F cell frozen stock (10% DMSO) at day 1, day 2, and day 4 after the recovery of human pluripotent stem cells; FIG. 1d, FIG. 1e, FIG. 1F show the cell morphology of iRePlur-F cells frozen in culture (7.5% DMSO) on days 1, 2, and 4 after recovery of human pluripotent stem cells; FIG. 1g, FIG. 1h, FIG. 1i show iRePlur-F thinCell morphology diagrams of 1 st day, 2 nd day and 4 th day after cell frozen stock solution (5% DMSO) frozen human pluripotent stem cells are recovered; FIGS. 1j, 1k, and 1l show the cell morphology of iRePlur-F frozen human pluripotent stem cells on days 1, 2, and 4 after recovery from frozen human pluripotent stem cells (2.5% DMSO), respectively; FIG. 1m, 1n, and 1o show the cell morphology of the control group CELLBANKER frozen stock solution on day 1, day 2, and day 4 after the recovery of the human pluripotent stem cells. It can be seen that cells cryopreserved with iRePlur-F cryopreserved medium show better cell morphology at elevated DMSO concentrations than cells cryopreserved with the control CELLBANKER cryopreserved medium.
1) Cells are frozen by iRePlur-F frozen stock solutions with different concentrations of Optiferrin, and after the cells are recovered according to the method, a Cyquant test is used for carrying out quantitative cell viability detection, so that the influence of the frozen stock solutions with different concentrations of Optiferrin on the cell viability in the process is compared. Coating a 96-hole opaque cell culture plate, and after coating, arranging the cells according to the 5 multiplied by 104The number of cells per well was seeded separately and three replicates were set (the average of the three sets was calculated as data). Samples were taken 24 hours after resuscitation and cell viability assays were performed using the CyQuant Kit (Invitrogen, X12223) and data reads were performed using the SpectraMax i3 Multi-Mode Microplate Reader (VWR, model ID3-STD) following the instructions. The results are shown in FIG. 2, in which FIGS. 2a-2b show the morphology of the cells after 24 hours of resuscitation, respectively, and FIG. 2e shows the results of the Cyquant assay, which represents the number of cells. It can be seen that the viability of the cells after recovery was positively correlated with the concentration of Optiferrin. In addition, as can be seen from the cell morphology of fig. 2d, the cell recovery has better activity already at the Optiferrin concentration of 10. mu. mol/L, and the Optiferrin concentration is preferably 0.5-10. mu. mol/L based on the cost.
2) Cells were cryopreserved using iRePlur-F cryopreserved at various concentrations of Y-276322HCl and quantified for cell viability using the Cyquant assay as described above. The results are shown in FIG. 3, where viability after cell recovery is positively correlated with Y-276322HCl concentration. In which FIGS. 3a-3b show the morphology of the cells after 24 hours of resuscitation, respectively, and FIG. 3e shows the results of the Cyquant assay, which represents the number of cells.
Example 4 Activity detection after Resuscitation of human pluripotent Stem cells
4.1 Activity Difference between different cryopreservations on short-term Resuscitation
The cells stored in the liquid nitrogen tank for one week were thawed by following the procedure of example 3, and the supernatant was collected on day 1 after the thawing, and the culture solution of human pluripotent stem cells and the cells were collected on day 3. The viability of the cells was determined by a Lactate Dehydrogenase (LDH) assay (cloudy days). The apoptosis rate is calculated by detecting the release concentration of Lactate Dehydrogenase (LDH) in the supernatant of the culture solution and the total LDH concentration of adherent cells according to the following formula: the LDH concentration of the supernatant/(LDH concentration of the supernatant + LDH concentration of adherent cells) × 100%. The results of the detection are shown in FIG. 4. The result shows that the apoptosis rate of the cells after iRePlur-F recovery is obviously lower than that of a control group, the optimal range of DMSO concentration in iRePlur-F is 5% -10%, and the apoptosis rate of the cells after recovery is less than 10% in the range.
4.2 comparison of Long-term cryopreservation viability between different cryopreservation solutions
Referring to example 3 and example 4.1, cells were thawed after 1 week, 2 weeks, 4 weeks, 8 weeks, and 12 weeks after cryopreservation, and samples were collected and assayed for cell viability after thawing. The results of the detection are shown in FIG. 5. The results show that the difference of apoptosis rate of the human pluripotent cells using iRePlur-F after being recovered at different time periods (1w, 2w, 4w, 8w and 12w) is not significant, and the influence on the cell survival rate is small; at the same time, the iRePlur-F series produced less apoptosis than the control.
Example 5 identification of cell pluripotency by resuscitating human pluripotent Stem cells
Subjecting the prepared induced pluripotent stem cells to endodermal (Endoderm) differentiation: the clones in the reprogramming plate were scooped up with a pipette tip under a dissecting microscope, placed in 48-well plates, one clone per well, and cultured in a commercial TeSRTM-e8(STEMCELL Technologies) medium at 37 ℃ in 5% CO2Culturing in the medium. When the induced pluripotent stem cells reached 70% coverage, they were treated with 0.05% trypsin/EDTA at 37 ℃ for 5 minutes, and cell digestion was stopped using DMEM. After washing and centrifuging the cells, according to1×105The mixture was re-seeded in 24-well plates at 37 ℃ in 5% CO2Incubate for 48 hours in ambient. Endoderm differentiation was then performed using the following medium: DMEM/F12 basal medium was supplemented with 1% N-2supplement (Invitrogen), 2% B-27supplement (Invitrogen), no N-essential amino acids 0.1mM, GlutaMAX 1mM, β -mercaptoethanol 0.1mM, Activin 10 μ g/ml (Nat Biotechnol,2005,23(12): 4-. The differentiation culture conditions are as follows: 37 ℃ and 5% CO2In (1). The identification was carried out after 4 days of culture.
The prepared induced pluripotent stem cells were subjected to mesodermal (Mesoderm) differentiation: when induced pluripotent stem cells reached 70% coverage, cells were treated with 0.05% trypsin/EDTA for 5 minutes at 37 ℃ and cell digestion was stopped using DM EM. After washing and centrifuging the cells, the ratio of the cells to the total volume of the cells is 1X 105The mixture was re-seeded in 24-well plates at 37 ℃ in 5% CO2Incubate for 48 hours in ambient. Mesoder m differentiation was performed using the following medium: 1xB-27supplement (Invitrogen) was added to RPMI basal medium, CHIR99021 at 5 μ g/ml (Lian et al, Proc Natl Acad Sci USA, 2012). The differentiation culture conditions are as follows: 37 ℃ and 5% CO2In (1). The identification was carried out after 4 days of culture.
The prepared induced pluripotent stem cells were subjected to ectodermal (entoderm) differentiation: when the induced pluripotent stem cells reached 70% coverage, they were treated with 0.05% trypsin/EDTA at 37 ℃ for 5 minutes, and cell digestion was stopped using DMEM. After washing and centrifuging the cells, the ratio of 5X 105The mixture was re-seeded in 24-well plates at 37 ℃ in 5% CO2Incubate for 48 hours in ambient. The endoserm differentiation was performed using the following medium: DMEM/F12 basal medium was supplemented with 1% N-2supplement (Invitrogen), 2% B-27supplement (Invitrogen), non-essential amino acids at 0.1mM, GlutaMAX at 1mM, beta-mercaptoethanol at 0.1mM, 10. mu. mol/L SB431542 and 100nmol/L LDN. The differentiation culture conditions are as follows: 37 ℃ and 5% CO2In (1). The culture was carried out for 21 days, and the culture was changed every day, followed by identification.
And (3) identifying the marker after the revival of the human pluripotent stem cells by adopting an immunofluorescence experiment, and identifying the markers of different germ layers obtained by a differentiation experiment. Fixing the cells with 4% paraformaldehyde at room temperature for 40 minutes, and washing twice with DPBS buffer solution; then permeabilizing with 0.1% Triton X-100 for 5 minutes, and washing twice with DPBS buffer solution; cells were then incubated overnight at 4 ℃ with DPBS buffer containing 10% horse serum and 0.1% Triton X-100; then, the antibody diluted with DPBS buffer was added, incubated at 37 ℃ for 2 hours, washed three times with DPBS buffer, and photographed. Details of antibody use are shown in table 1. The results are shown in FIG. 6, which shows that the human pluripotent stem cell frozen stock solution used in the present invention does not change the pluripotency of the pluripotent stem cells after the cells are frozen, and the cells still have the ability to differentiate into three different germ layers.
Table 1: antibodies for use in differential germ layer fluorescence immunoassay
It should be noted that the above-mentioned embodiments are only preferred embodiments of the present invention, and do not limit the present invention in any way, and all technical solutions obtained by replacing or changing the technical solutions according to the present invention are covered in the protection scope of the present invention.
Claims (10)
1. A pluripotent stem cell cryopreservation liquid comprising a minimal medium and additives, wherein the additives comprise Optiferrin, DMSO and Y-276322 HCl.
2. The cryopreservation liquid of claim 1, wherein the additives comprise 0.5-10 μmol/L Optiferrin, 2.5-10 v/v% DMSO, and 10nmol/L-10 μmol/LY-276322 HCl.
3. The cryopreservation solution of claim 1, wherein the DMSO is 5-10 v/v%.
4. The cryopreservation liquid of claim 1, wherein the additive further comprises inorganic salts, amino acids, cytokines.
5. The frozen stock solution of claim 3, wherein the vitamins comprise 1.2 μmol/L vitamin B12, 64mg/L vitamin C.
6. The frozen stock solution of claim 3, wherein the inorganic salts comprise 0.5g/L sodium chloride and 13.6ug/L sodium selenite.
7. The cryopreservation solution of claim 3, wherein the cytokines comprise 22 μ g/mL IGF, plant-derived recombinant human basic growth factor, 50ng/mL, transforming growth factor TGF-b 1.74 ng/mL.
8. The cryopreservation solution of claim 1, further comprising 6.3ng/ml progesterone and 23 μ g/ml putrescine.
9. The use of the frozen stock solution of claim 1 for freezing human pluripotent stem cells.
10. A cryopreservation method of human pluripotent stem cells comprises the following steps: centrifuging the expanded and passaged pluripotent stem cell suspension, removing supernatant, mixing with the induced pluripotent stem cell frozen stock solution of any one of claims 1 to 8, subpackaging, and freezing.
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| CN114794088A (en) * | 2022-06-24 | 2022-07-29 | 北京细胞治疗集团有限公司 | Human pluripotent stem cell cryopreservation liquid and application thereof |
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