CN112544613B - Pluripotent stem cell cryopreservation liquid, application thereof and cryopreservation method - Google Patents

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 solution greatly improves the cell cryopreservation activity, is simple and convenient in cryopreservation process, and is revived after being subjected to long-term cryopreservation, so that various characteristics of the cells are stable. Therefore, the cryopreservation solution greatly expands the clinical application of the human pluripotent stem cells.

Description

A kind of pluripotent stem cell cryopreservation liquid, its application and cryopreservation method

【Technical field】

The invention relates to the field of biotechnology, in particular to a pluripotent stem cell cryopreservation solution, its application and a cryopreservation method.

【Background technique】

Regenerative medicine refers to an emerging science that uses a variety of new technical disciplines to rebuild aging or functionally lost tissues and organs, and to treat related diseases through a variety of medical means. The important research direction of regenerative medicine is the mechanism of normal tissue characteristics and functions, the biological basis of post-traumatic repair, the regeneration mechanism of tissues and organs, and the differentiation mechanism of various stem cells, so as to finally obtain effective biological treatment methods. Its research method integrates a variety of means, including principles and methods of life science, material science, clinical medicine and other disciplines, and comprehensively solves clinical solutions for replacing, repairing, reconstructing or regenerating various tissues and organs of the human body. In the development of regenerative medicine, there are several core issues that need to be addressed. Ideal regenerative medicine products, especially cell therapy products, should have the following characteristics: first, the source of cells must be a single cell type that can proliferate indefinitely; second, the cells must be easy to further modify, such as using tools such as gene editing. The disease-causing gene is corrected; thirdly, the properties of the final product are uniform. How to obtain the initial cell source that can be used for cell therapy is an important research topic in the early stage of regenerative medicine. Among them, embryonic stem cells (ESCs, referred to as ES, EK or ESC cells) are the cell type that has attracted the most attention in early regenerative medicine research. Embryonic stem cells are a type of cells isolated from early embryos (before gastrulation stage) or primordial gonads, which have the characteristics of unlimited proliferation, self-renewal and multidirectional differentiation in vitro. ES cells can be induced to differentiate into almost all cell types in the body, both in vitro and in vivo. However, the acquisition and use of this cell is a big ethical controversy, because to conduct embryonic stem cell research, it is necessary to destroy the embryo, and the embryo is the life form in the womb when the human has not yet formed. This ethical controversy has greatly hindered the advancement and application of regenerative medicine.

In 2006, Yamanaka Shinya's team invented a "cocktail" method composed of four transcription factors OCT4, SOX2, KLF4 and c-Myc, which can successfully reprogram terminally differentiated skin fibroblasts into differentiated pluripotent cells. stem cells, which are called induced pluripotent stem cells (induced pluri potent 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 similar differentiation potential to embryonic stem cells, and can form the three most basic germ layers in human development: ectoderm, mesoderm and endoderm, and eventually form a variety of adult cells. This invention breaks through the ethical limitations of using human embryonic stem cells in medicine, can solve the problem of immune rejection in cell transplantation therapy, and greatly expand the application potential of stem cell technology in clinical medicine. The use of totipotent stem cells or pluripotent stem cells including embryonic stem cells and iPSCs as raw materials to induce differentiation of ectodermal cells can be a new idea for clinical treatment, which greatly expands the application potential of ectodermal cells in clinical medicine. Only in the field of regenerative medicine, the applications of induced pluripotent stem cells that are about to enter the clinical stage include neurodegenerative diseases, spinal cord injury, type I diabetes, and tumors.

Pluripotent stem cells represented by induced pluripotent stem cells, as the core technology platform of regenerative medicine, are the hotspot of current global stem cell research. Cryopreservation of cells is one of the main methods of cell preservation. In order to store pluripotent cells for a long time, use them and transport them over long distances, effective cell cryopreservation solutions and cryopreservation methods should be used, and the characteristics of cells should remain unchanged after repeated recovery. Cryopreservation can facilitate timely use of cells at all times while reducing the risk of microbial contamination and 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, which allows the cells and their protectant solutions to cool down at a rate fast enough to convert the biomaterial to a fully vitrified state, where it can be used for long periods of time at low temperatures save. Cell damage is avoided due to the avoidance of icing both inside and outside the cell during this process. The key to achieving "vitrification" is cell cryopreservation, which can reduce the formation of ice crystals in cells or tissues during cryopreservation, thereby protecting cells. Commonly used cell cryopreservation agents include dimethyl sulfoxide (DMSO), ethylene glycol (EG) and propylene glycol (PG) (Almansoori KA et.al., Cryobiology. 2012 Jun;64(3):185-91). At present, human pluripotent stem cells generally use DMSO as a cryopreservation agent. DMSO is a low-molecular-weight compound with small relative molecular weight, high solubility and strong permeability, which can lower the freezing point and improve the permeability of the cell membrane to water. It reduces the chance of forming ice crystals in cells and reduces the damage of ice crystals to cells, thereby achieving a protective effect on cells. High concentrations of DMSO are cytotoxic and can interact with hydrophobic groups of intracellular proteins, resulting in protein denaturation, cell damage or inactivation; and in the clinical experience of hematopoietic stem cells, DMSO residues can cause certain side effects (ZambelliA et al. al., Anticancer Res 1998;18(6B):4705-4708). 10% DMSO is a safe concentration for cells. Therefore, human pluripotent stem cells generally use 90% (fetal) bovine serum plus 10% DMSO as a cryopreservation solution for cell cryopreservation (Hanna J and Hubel A, Organogenesis, 01 Jul 2009, 5(3):134-137). Although many scientists use a variety of medium containing serum components to reduce the concentration of serum components, this method of protecting stem cell activity through a large amount of animal serum is not suitable for the development of regenerative medicine products, and serum components will carry a variety of animal-derived components ingredients, or viruses, can cause serious infections and complications.

Therefore, how to develop a serum-free, clear chemical composition, and a cryopreservation reagent that can ensure the viability of pluripotent stem cells is an important tool to expand the clinical use and application prospects of pluripotent stem cells.

[Content of the invention]

The problem to be solved by the present invention is to provide a pluripotent stem cell cryopreservation solution, its application and cryopreservation method. On the premise that the cryopreservation solution of the present invention can ensure the viability of pluripotent stem cells, the chemical composition is clear, and the transmission of serum to animals is avoided. risk of pathogens, ensuring the safety of cryopreservation.

In view of this, the technical scheme adopted to realize the above-mentioned purpose of the present invention is:

A pluripotent stem cell cryopreservation solution includes a basic medium and additives, the additives include Optiferrin, DMSO and Y-276322HCl.

In a specific embodiment, the additives include 0.5-10 μmol/L Optiferrin, 2.5-10 v/v% DMSO and 10 nmol/L-10 μmol/L Y-276322HCl. Wherein, the DMSO is preferably 5-10 v/v%.

In specific embodiments, the additives further include inorganic salts, amino acids and cytokines.

In a specific embodiment, the vitamins include 1.2 μmol/L vitamin B12, 64 mg/L vitamin C.

In a specific embodiment, the inorganic salts include 0.5 g/L sodium chloride and 13.6 μg/L sodium selenite.

In a specific embodiment, the growth factors include 22 μg/ml IGF, 50 ng/mL plant-derived recombinant human basic growth factor, 1.74 ng/ml transforming growth factor TGF-b.

In a specific embodiment, the additives in the pluripotent stem cell cryopreservation solution of the present invention further include 6.3 ng/ml progesterone and 23 μg/ml putrescine.

In specific embodiments, the minimal medium is DMEM medium.

Another object of the present invention is to provide the application of the above-mentioned cryopreservation solution in cryopreservation of human-derived pluripotent stem cells.

Another object of the present invention is to provide a cryopreservation method for human-derived pluripotent stem cells, comprising the steps of: centrifuging the expanded and passaged pluripotent stem cell suspension, removing the supernatant, and then Induced pluripotent stem cell cryopreservation solution is mixed, aliquoted, and cryopreserved.

Compared with the prior art, the beneficial effects of the present invention are:

1. The present invention provides a cell cryopreservation solution that does not use animal-derived components and has a clear chemical composition. Compared with the commercial cryopreservation solution, it not only greatly improves cell viability after cell recovery, but also has complete cell function after long-term cryopreservation. .

2. The present invention is easy to configure and easy to use, and greatly expands the clinical application prospect of pluripotent stem cells.

【Description of drawings】

In order to illustrate the technical solutions of the embodiments of the present invention more clearly, the following briefly introduces the accompanying drawings used in the embodiments. It should be understood that the following drawings only show some embodiments of the present invention, and therefore do not It should be regarded as a limitation of the scope, and for those of ordinary skill in the art, other related drawings can also be obtained according to these drawings without any creative effort.

Fig. 1 is a cell morphological diagram of human pluripotent stem cells after recovery using the iRePlur-F cryopreservation solution of the present invention.

Fig. 2 is a schematic diagram showing the effect of cryopreserved solutions of Optiferrin in different concentrations of the present invention on cell viability.

Fig. 3 is a schematic diagram showing the effect of cryopreserved solutions of different concentrations of Y-27632 hydrochloride according to the present invention on the apoptosis rate of human pluripotent stem cells after resuscitation.

Figure 4 is a schematic diagram showing the detection results of apoptosis rate of human-derived pluripotent stem cells cryopreserved in iRePlur-F cell cryopreservation solution of different concentrations of DMSO and control group CELLBANKER cryopreservation solution after resuscitation.

Fig. 5 is a schematic diagram showing the detection results of the apoptosis rate of human pluripotent stem cells after cryopreservation by iRePlur-F cell cryopreservation solution and CELLBANKER cryopreservation solution of the present invention.

FIG. 6 is a schematic diagram showing the results of pluripotency identification of human-derived pluripotent stem cells recovered and identified.

【Detailed ways】

The invention provides a pluripotent stem cell liquid cryopreservation solution, which includes a basic medium and additives, the additives include 0.5-10 μmol/L Optiferrin, 2.5-10 v/v% DMSO and 10 nmol/L-10 μmol/L Y-276322HCl.

According to an embodiment of the present invention, the DMSO is preferably 5-10 v/v%.

In the pluripotent stem cell cryopreservation solution provided by the present invention, the additives further include vitamins, inorganic salts and cytokines as required.

According to an embodiment of the present invention, the vitamins include 1.2 μmol/L vitamin B12 and 64 mg/L vitamin C. According to common knowledge in the art, the vitamins can also be selected from other commonly used vitamins, such as vitamin B6, vitamin B2, and the like.

According to an embodiment of the present invention, the inorganic salt includes 0.5g/L sodium chloride and 13.6ug/L sodium selenite. Other common inorganic salts can also be selected according to common knowledge in the art.

According to an embodiment of the present invention, the cytokines include 22ug/ml IGF, plant-derived recombinant human basic growth factor, 50ng/mL, and transforming growth factor 1.74ng/ml. Other commonly used cytokines can also be selected according to common knowledge in the art.

According to an embodiment of the present invention, the cryopreservation solution provided by the present invention further includes 6.3 ng/ml progesterone and 23 ug/ml putrescine.

According to an embodiment of the present invention, the minimal medium is DMEM medium.

The following examples are intended to illustrate the present invention, but not to limit the scope of the present invention. Modifications or substitutions made to the methods, steps or conditions of the present invention without departing from the spirit and essence of the present invention all belong to the scope of the present invention.

Example 1 Preparation of human-derived pluripotent stem cell liquid cryopreservation solution iRePlur-F

Prepare iRePlur-F cell cryopreservation solution according to the following recipe, hereinafter referred to as iRePlur-F:

Dulbecco's modified Eagle's medium (DMEM medium), vitamin B12 (1.2μmol/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 (0.5-10μmol/L), plant-derived recombinant human Basic growth factor (OsrbFGF, 50ng/mL), TGF-b (1.74ng/ml), 2.5%-10% DMSO, Y-276322HCl (10nmol/L-10μmol/L).

DMSO Free GMP grade(ZENOAQ)冻存液，以下简称CELLBANKER。Among them, the control experiment used

DMSO Free GMP grade (ZENOAQ) freezing solution, 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. Among them, human induced pluripotent stem cells were obtained by reprogramming from CD34+ cells according to "Reprogramming medium and culture method for reprogramming induced pluripotent stem cells" (ZL201910050800.7).

Human pluripotent stem cells were used to coat T25 cell culture flasks with Matrigel (STEMCELL Technologies), and then placed in a 37°C incubator for more than one hour after plating. The cells were seeded in T25 culture flasks at 1×10 ⁶ /flask for expansion and passage. The cells were cultured using a commercialized TeSRTM-e8 (STEMCELL Technologies) medium at 37°C in 5% CO ₂ . When the cell growth reaches 70% coverage, use 0.05% trypsin/EDTA and incubate at 37°C for 5 min for digestion. The digested cells are washed by centrifugation and resuspended in iRePlur-F cells at a density of 1×10 ⁶ /ml. The cells were then placed in a programmed cooling freezer box and placed in a -80°C refrigerator for 24 hours, and then transferred to liquid nitrogen for long-term storage. The control experiment was carried out with the same batch of cells, and the cells were cryopreserved using CELLBANKER.

Example 3 Recovery of human pluripotent stem cells

4.1 The effect of DMSO concentration on cell viability after recovery of human pluripotent stem cells

Human pluripotent stem cells were coated with Matrigel (STEMCELL Technologies) on T25 cell culture flasks, plated and incubated in a 37°C incubator for more than one hour. The cells cryopreserved using the iRePlur-F cryopreservation solution of the present invention and the control group CELLBANKER cryopreservation solution were rapidly thawed in a water bath at 37°C. After thawing, the cells were washed with DMEM medium to remove DMSO, and the digested cells were centrifuged and washed according to 1 × 10 T25 cell culture plates were seeded at a density of ^6/10 ml and cultured in a 37°C, 5% CO ₂ cell incubator. The morphology of pluripotent stem cells after recovery is shown in Figure 1. Among them, Figure 1a, Figure 1b, Figure 1c show the cell morphology of the human pluripotent stem cells cryopreserved in iRePlur-F cell cryopreservation solution (10% DMSO) on the 1st, 2nd, and 4th days after resuscitation, respectively. ; Figure 1d, Figure 1e, and Figure 1f respectively show the cell morphology of the human pluripotent stem cells cryopreserved in iRePlur-F cell cryopreservation solution (7.5% DMSO) on day 1, day 2, and day 4 after resuscitation; Figure 1g, Figure 1h, and Figure 1i show the cell morphology diagrams on the first day, the second day, and the fourth day after the recovery of the human pluripotent stem cells cryopreserved in iRePlur-F cell cryopreservation solution (5% DMSO) respectively; Figure 1j, Fig. 1k, Fig. 1l respectively show the cell morphology of the human pluripotent stem cells cryopreserved in iRePlur-F cell cryopreservation solution (2.5% DMSO) on day 1, day 2, and day 4 after resuscitation; Fig. 1m , 1n and 1o respectively show the cell morphology of the human pluripotent stem cells cryopreserved in CELLBANKER cryopreservation solution in the control group on the 1st, 2nd, and 4th days after resuscitation. It is not difficult to see that compared with the cells cryopreserved in CELLBANKER cryopreservation solution of the control group, when the concentration of DMSO is increased in iRePlur-F cryopreservation solution, the cryopreserved cells show better cell morphology.

1) Use the iRePlur-F cryopreservation solution of different concentrations of Optiferrin to freeze the cells. After resuscitation according to the above method, use the Cyquant test to perform quantitative detection of cell viability, so as to compare the effect of different concentrations of Optiferrin cryopreservation solution on cell viability during this process. Impact. A 96-well opaque cell culture plate was coated, and after the coating was completed, the cells were seeded according to the number of cells per well of 5×10 ⁴ , and three groups of parallel replicates were set (the average of the three groups was calculated as the data). Samples were taken 24 hours after recovery, and cell viability was detected using CyQuant Kit (Invitrogen, X12223), following the instructions for use, and using SpectraMax i3 Multi-Mode Microplate Reader (VWR, model ID3-STD) for data reading. The results are shown in Figure 2, wherein Figures 2a-2b respectively represent the morphology of the cells after resuscitation for 24 hours, and Figure 2e shows the results of the Cyquant test representing the number of cells. It is not difficult to see that the viability of cells after recovery is positively correlated with the Optiferrin concentration. In addition, according to the cell morphology in Figure 2d, it can be seen that when the Optiferrin concentration is 10 μmol/L, the cell recovery has good viability, and the Optiferrin concentration of 0.5-10 μmol/L is more appropriate based on cost considerations.

2) Freeze cells with iRePlur-F cryopreservation solution of different concentrations of Y-276322HCl, and perform quantitative detection of cell viability by Cyquant test according to the above method. The results are shown in Figure 3, and the viability of the cells after recovery was positively correlated with the concentration of Y-276322HCl. Among them, Figures 3a-3b respectively show the morphology of the cells after 24 hours of recovery, and Figure 3e shows the results of the Cyquant test representing the number of cells.

Example 4 Viability detection of human pluripotent stem cells after resuscitation

4.1 Differences in the viability of short-term resuscitation between different cryopreservations

The cells stored in the liquid nitrogen tank for one week were revitalized according to the steps of Example 3, and the supernatant was collected on the 1st day after the revival, and the human-derived pluripotent stem cell culture medium and cells were collected on the 3rd day. The detection of cell viability was carried out by lactate dehydrogenase (LDH) assay (Biyuntian). By detecting the concentration of lactate dehydrogenase (LDH) released in the culture supernatant and the total LDH concentration of adherent cells, the formula for calculating the apoptosis rate is: supernatant LDH concentration/(supernatant LDH concentration + adherent cells Cell LDH concentration) × 100%. The detection results are shown in Figure 4. The results showed that the apoptosis rate of iRePlur-F after recovery was significantly lower than that of the control group. The optimal range of DMSO concentration in iRePlur-F was 5%-10%. Within this range, the apoptosis rate after recovery was less than 10%. .

4.2 Comparison of the viability of long-term cryopreservation between different cryopreservation solutions

Referring to Example 3 and Example 4.1, cell recovery was performed sequentially after 1 week, 2 weeks, 4 weeks, 8 weeks, and 12 weeks after cryopreservation, and samples were collected and tested for cell viability after recovery. The detection results are shown in Figure 5. The results showed that the apoptosis rate of human pluripotent cells using iRePlur-F after resuscitation in different time periods (1w, 2w, 4w, 8w, 12w) was not significantly different, and the effect on cell viability was small; , the iRePlur-F series produced a smaller apoptosis rate than the control group.

Example 5 Human-derived pluripotent stem cells are recovered to identify cell pluripotency

Endoderm differentiation of the prepared induced pluripotent stem cells: Under a dissecting microscope, the clones in the reprogramming plate were aspirated with a pipette tip, placed in a 48-well plate, and one clone was placed in each well. TeSRTM-e8 (STEMCELL Technologies) medium was cultured at 37°C in 5% CO ₂ . When the induced pluripotent stem cells reached 70% coverage, they were treated with 0.05% trypsin/EDTA at 37°C for 5 minutes, and DMEM was used to stop cell digestion. After the cells were washed and centrifuged, they were re-seeded in a 24-well plate at a ratio of 1×10 ⁵ per well, and cultured at 37°C in a 5% CO ₂ environment for 48 hours. Endoderm differentiation was then performed using the following medium: DMEM/F12 basal medium supplemented with 1% N-2supplement (Invitrogen), 2% B-27supplement (Invitrogen), no n-essentialamino acids at 0.1 mM, GlutaMAX at 1 mM, β -mercaptoethanol at 0.1 mM and Activin at 10 μg/ml (Nat Biotechnol, 2005, 23(12):1534-1541). Differentiation culture conditions were: 37°C, 5% CO ₂ . Identification was performed after 4 days of culture.

The prepared induced pluripotent stem cells were subjected to Mesoderm differentiation: when the induced pluripotent stem cells reached 70% coverage, they were treated with 0.05% trypsin/EDTA at 37°C for 5 minutes, and DM EM was used to stop cell digestion. After the cells were washed and centrifuged, they were re-seeded in a 24-well plate at a ratio of 1×10 ⁵ per well, and cultured at 37° C. and 5% CO ₂ for 48 hours. Mesoder m differentiation was performed using the following medium: RPMI basal medium supplemented with IxB-27supplement (Invitrogen) at 5 μg/ml CHIR99021 (Lian et al., Proc Natl Acad Sci USA, 2012.). Differentiation culture conditions were: 37°C, 5% CO ₂ . Identification was performed after 4 days of culture.

The prepared induced pluripotent stem cells were subjected to Ectoderm differentiation: when the induced pluripotent stem cells reached 70% coverage, they were treated with 0.05% trypsin/EDTA at 37°C for 5 minutes, and DMEM was used to stop cell digestion. After the cells were washed and centrifuged, they were re-seeded in a 24-well plate at a ratio of 5×10 ⁵ per well, and cultured at 37° C. and 5% CO ₂ for 48 hours. Endoderm differentiation was performed using the following medium: DMEM/F12 basal medium supplemented with 1% N-2supplement (Invitrogen), 2% B-27supplement (Invitrogen), 0.1 mM non-essential aminoacids, 1 mM GlutaMAX, β-mercaptoethanol 0.1 mM, 10 μmol/L SB431542 and 100 nmol/L LDN. Differentiation culture conditions were: 37°C, 5% CO ₂ . The culture was carried out for 21 days, and the medium was changed once a day, after which the identification was carried out.

Immunofluorescence experiments were used to identify markers of human pluripotent stem cells after recovery, and markers of different germ layers obtained from differentiation experiments. Cells were fixed with 4% paraformaldehyde at room temperature for 40 minutes and washed twice with DPBS buffer; then permeabilized with 0.1% Triton X-100 for 5 minutes and washed twice with DPBS buffer; then with 10% horse serum and The cells were incubated overnight at 4°C with 0.1% Triton X-100 in DPBS buffer; antibodies diluted with DPBS buffer were then added, incubated at 37°C for 2 h, washed three times with DPBS buffer, and then photographed. Antibody usage details are shown in Table 1. The identification results are shown in Figure 6. The results show that the human pluripotent stem cell cryopreservation solution used in the present invention does not change the pluripotency of the pluripotent stem cells after freezing the cells, and the cells still have the ability to differentiate into three different germ layers.

Table 1: Antibodies used for fluorescent immunoassays of different germ layers

It should be noted that the above-mentioned embodiments are only preferred specific implementations of the present invention, and do not limit the present invention in any form. within the scope of protection.

Claims (8)

1. The human pluripotent stem cell frozen stock solution is characterized by comprising a basic culture medium and additives, wherein the additives comprise 0.5-10 mu mol/LOPTIFERRIN, 5-10 v/v% DMSO and 10nmol/L-10 mu mol/L Y-276322 HCl.

2. The cryopreservation liquid of claim 1, wherein the additives further comprise inorganic salts, vitamins, cytokines.

3. The frozen stock solution of claim 2, wherein the vitamins comprise 1.2 μmol/L vitamin B12, 64mg/L vitamin C.

4. The cryopreservation liquid of claim 2, wherein the inorganic salts comprise 0.5g/L sodium chloride and 13.6 μ g/L sodium selenite.

5. The cryopreservation solution of claim 2, 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.

6. The cryopreservation solution of claim 1, further comprising 6.3ng/ml progesterone and 23 μ g/ml putrescine.

7. The use of the frozen stock solution of claim 1 for freezing human pluripotent stem cells.

8. A cryopreservation method of human pluripotent stem cells comprises the following steps: centrifuging the expanded and passaged human pluripotent stem cell suspension, removing supernatant, mixing with the human pluripotent stem cell frozen stock solution of any one of claims 1 to 6, subpackaging, and freezing.

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