KR102628250B1 - Cryopreservation method of stem cell culture using high concentration of cryoprotective agents - Google Patents

Abstract

The present invention relates to a method of cryopreservation of two-dimensional (2D) and three-dimensional (3D) stem cell culture using a high concentration of cryoprotectant.

Description

Method for cryopreservation of stem cell cultures using high concentration of cryoprotectant {CRYOPRESERVATION METHOD OF STEM CELL CULTURE USING HIGH CONCENTRATION OF CRYOPROTECTIVE AGENTS}

The present invention relates to a method of cryopreservation of two-dimensional (2D) and three-dimensional (3D) stem cell cultures using a high concentration of cryoprotectant.

Cryopreservation methods are used for long-term stable preservation of tissues or cells.

The previously widely used method for such cryopreservation was the slow-freezing method using a low concentration of cryoprotective agent (CPA).

Recently, organoids that mimic human organs have been widely studied to recapitulate the tissue generation process in vitro. However, in the case of cryopreservation through conventional gentle freezing, there was a limitation that it was difficult for the cryoprotectant to penetrate into the organoid due to the strong adhesion between cells and the 3D structure as low concentrations of cryoprotectant were used. On the other hand, if a high concentration of cryoprotectant is used, an osmotic effect may occur during the freezing and thawing process, which may lead to cytotoxicity.

Moreover, the existing gentle freezing method had the problem that it could cause serious damage to cells and tissues due to the formation of ice crystals during the freezing process. Therefore, there was a problem that it was difficult to freeze-preserve the organoids without damaging them using the existing method.

Recently, the vitrification method has been used as a new rapid freezing method that can prevent damage caused by ice crystals during the freezing process. This is achieved through the process of rapidly freezing tissues or cells using liquid nitrogen. However, vitrification, which is a rapid freezing method, requires a high concentration (~8M) of cryoprotectant (CPA). However, as mentioned above, high concentration of cryoprotectant causes an osmotic effect during the freezing and thawing process, which causes the existing slow freezing (slow freezing). Compared to the -freezing) method, cytotoxicity may be a problem.

In order to solve the various problems mentioned above, continuous research on cryopreservation methods is needed.

Korean Patent No. 10-0411243

The purpose of the present invention is to provide a method for cryopreservation of single cells or spheroids using a high concentration of non-cytotoxic cryoprotectant.

In addition, another object of the present invention is to provide a method for vitrifying and thawing single cells or spheroids with excellent cell viability, shape maintenance, and function maintenance using a high concentration of cryoprotectant.

The present invention includes the steps of (a) treating cells in an equilibration solution for 5 to 15 minutes; (b) treating the cells in which step (a) has been completed in a vitrification solution for 10 to 120 seconds; and (c) placing the cells in which step (b) has been completed into liquid nitrogen at -196°C.

Specifically, the equilibration solution may be composed of ethylene glycol (EG) and 1.4 M DMSO based on DPBS (Dulbecco's phosphate buffered saline) containing 20% (v/v) FBS, but is not limited thereto.

In addition, the vitrification solution is composed of a high concentration of cryoprotective agent (CPA), is based on LM5 carrier solution, and may include PVP K12, DMSO, ethylene glycol (EG), and formamide, and may be composed of specific The vitrification solution may be composed of 70 g/L PVP K12, 2.8M DMSO, 2.7M EG, and 2.8M formamide based on the LM5 carrier solution. More specifically, the LM5 carrier solution may include glucose, It may be adjusted to contain 0.3M sucrose instead of mannitol and lactose, but is not limited thereto.

Additionally, specifically, the time for treating the cells with the equilibration solution may be 10 minutes, and the time for treating the cells with the vitrification solution may be 60 seconds, but are not limited thereto.

In another aspect, the present invention provides a method of thawing the cryopreserved cells while minimizing damage.

Specifically, the thawing method involves transferring cells stored in liquid nitrogen to a water bath at 37 °C, and then successively suspending them in sucrose solutions of 0.5, 0.25, and 0 M concentrations in DPBS containing 20% FBS for 5 minutes each. It may be done, but it is not limited to this.

According to the cryopreservation and thawing method of the present invention, cell viability, shape maintenance, and function maintenance are significantly increased and DNA damage is suppressed compared to conventional slow freezing methods.

In addition, cells that can be used in the cryopreservation and thawing method of the above cells include embryonic stem cells (ES), induced pluripotent stem cells (iPSC), adult stem cells (ASC), mesenchymal stem cells (MSC), and hematopoietic stem cells ( It may be one or more selected from the group consisting of HSC) and neural stem cells (NSC), and more specifically, it may be mesenchymal stem cell (MSC), but is not limited thereto.

Furthermore, it was confirmed that the cryopreservation method of the present invention can also successfully vitrify spheroids or organoids that form a three-dimensional structure by using a high concentration of cryoprotectant (CPA), especially the structure. It can be used even when the size is large, which has the advantage of being applicable to complex research.

The cryopreservation method according to the present invention does not exhibit cytotoxicity even when a high concentration of cryoprotectant is used, and has excellent cell shape and function maintenance properties.

The cryopreservation method according to the present invention is effective not only at the single cell level but also at the cryopreservation of spheroids, which have a three-dimensional structure.

The cryopreservation method according to the present invention has superior cryopreservation performance compared to the existing slow-freezing method.

The effects of the present invention are not limited to the above effects, and should be understood to include all effects that can be inferred from the configuration of the invention described in the detailed description or claims of the present invention.

Figure 1 shows the results of comparing the shape and growth pattern of MSCs by the cryopreservation method according to the present invention and the slow-freezing method, which is a conventional cryopreservation method (a: micrograph, b: MSC Graph comparing survival rates).
Figure 2 is a graph comparing the population doubling time of MSCs by the cryopreservation method according to the present invention and the slow-freezing method, which is a conventional cryopreservation method.
Figure 3 is a diagram showing TUNEL analysis performed using a fluorescence microscope to compare DNA fragmentation of each cell line by the cryopreservation method according to the present invention and the slow-freezing method, which is a conventional cryopreservation method.
Figure 4 is a diagram analyzing the intracellular level of reactive oxygen species after vitrification by the cryopreservation method according to the present invention.
Figure 5 is an analysis of cell viability by the cryopreservation method according to the present invention and the slow-freezing method, which is a conventional cryopreservation method, where a is quantitative real-time polymerase chain reaction (RT-PCR) at the single cell level. ) is a graph comparing the RNA expression levels of Bax/Bcl-2, Bcl-ti, Bid, p53, SOD1, and HSF-1, and b is a graph comparing Bax/Bcl-2 through quantitative real-time polymerase chain reaction in spheroids. This is a graph comparing the RNA expression levels of Bcl-T, Bid, p53, SOD1, and HSF-1.
Figure 6 is an analysis of the characterization of AD-MSCs by the cryopreservation method according to the present invention and the slow-freezing method, which is a conventional cryopreservation method, showing positive markers CD44, CD73, CD90, and CD105 and negative markers. This is a diagram analyzing the expression levels of CD31 and CD34.
Figure 7 is an analysis of the characterization of AD-MSCs by the cryopreservation method according to the present invention and the slow-freezing method, which is a conventional cryopreservation method. In order to investigate the differentiation potential of MSCs, osteoblasts and adipocytes , This is a diagram confirming osteogenesis, adipogenesis, and cartilage formation by differentiation of chondrocytes through oil red O staining, Alcian blue staining, and Von kossa staining.
Figure 8 is a diagram analyzing the viability of spheroids by size using a live-dead staining method after thawing by the cryopreservation method according to the present invention and the slow-freezing method, which is a conventional cryopreservation method (from the left, the viability of the spheroids is The sizes are 200, 300, 500, 700, and 900 ㎛, and the number of cells per spheroid is 10,000, 30,000, 50,000, 70,000, and 100,000 cells).

The present invention will be explained in more detail through the following examples, but it is not intended that the scope of the present invention be limited to the following examples.

Example 1. Cell derivation and culture

Mesenchymal stem cells (MSCs) purchased from Lonza (PT-5006; Walkersville, MD) were seeded at a density of 4500 cells/cm ² in polystyrene tissue culture dishes, and 10% bovine fetuses were added. Dulbecco's Modified Eagle's Medium (DMEM; Thermo Fisher Scientific, Waltham, MA, USA) containing GlutaMAX supplemented with serum (FBS; Thermo Fisher Scientific) and 1% penicillin streptomycin (P/S; Thermo Fisher Scientific). It was cultured in . At this time, cell culture conditions were maintained at 37°C in humid conditions containing 5% CO ₂ . The medium was changed every 2 days. Cells were subcultured in the culture medium described above until 90% confluency was reached. All experiments used cells subcultured five times.

Example 2. Generation of MSC spheroids

To generate spheroids, passage 5 MSCs were placed in suspension for up to 7 days using the hanging drop method in 25 μl of culture medium containing 10,000 to 100,000 cells/drop. Cells were cultured in MSC culture medium, and humid conditions were maintained at 37°C and 5% CO ₂ . Afterwards, MSC spheroids were confirmed using an inverted microscope (Nikon, Chiyoda-ku, Japan).

Example 3. Vitrification of cells

Vitrification of cells was performed in two steps using equilibration and vitrification solutions. Specifically, the equilibration solution was treated for 10 minutes, and the vitrification solution was treated for 1 minute.

The equilibration solution was EG (ethylene glycol, Sigma-Aldrich) based on Dulbecco's phosphate buffered saline (DPBS; Thermo Fisher Scientific) containing 20% (v/v) FBS and 1.4 M DMSO (Sigma-Aldrich, St. Louis). , MO, USA).

The vitrification solution consisted of 70 g/L PVP K12 (Thermo Fisher Scientific), 2.8 M DMSO, 2.7 M EG, and 2.8 M formamide (F;SigmaAldrich) based on LM5 carrier solution. At this time, the LM5 transport solution was slightly adjusted to contain 0.3M sucrose instead of glucose, mannitol, and lactose.

First, the cell pellet, approximately 500,000 cells per vial, separated with 0.25% trypsin-EDTA (Thermo Fisher Scientific), was suspended in the equilibration solution for 10 minutes and then mixed with the vitrification solution within 1 minute.

Suspended cells were immediately transferred to a 1.5 ml cryoglass (Nunc, Rochester, MN, USA) and directly injected into liquid nitrogen.

Likewise, vitrification of MSC spheroids was performed using the same two-step vitrification solution as described above. All spheroids were cryopreserved at approximately 10 spheroids per vial.

After 2 weeks, the cells were immediately thawed by immersing the vials in a water bath at 37 °C. Thawed cells were successively suspended in 0.5, 0.25, and 0 M sucrose solutions in DPBS containing 20% FBS for 5 min each. Finally, each cell line was resuspended and then cultured with culture medium.

Comparative Example 1. Freezing of cells using the existing gentle freezing method

To compare the survival rates of vitrified cells and non-vitrified cells using the conventional slow freezing method, freezing and thawing of cells using the commonly performed slow freezing method was performed as a control.

Specifically, the pellet separated by 0.25% trypsin-EDTA was transferred to a 1.5 ml cryovial containing 10% DMSO in FBS. Mr. Cryo vials were sealed and frozen at -80 °C in a Frosty controlled cooling rate device (Thermo Fisher Scientific). After 24 hours, the cryovial was transferred to liquid nitrogen. After 2 weeks, cryovials were warmed in a 37 °C water bath and agitated until only pea-sized ice cubes remained. Afterwards, the cells were centrifuged at 1000 rpm at 25 °C, resuspended with culture medium, and cultured.

Example 4. Cell viability and morphology evaluation

After sequentially thawing the cells, the survival rate was confirmed after staining with trypan blue (Thermo Fisher Scientific). Cells were plated at a density of 4500 cells/cm ² , and each cell line was observed with an inverted microscope (Nikon) after 1 day to confirm cell adhesion to the culture dish. Spheroid viability was confirmed using the LIVE-DEAD cell viability kit (Thermo Fisher Scientific). For LIVE-DEAD analysis, 2 μM calcein AM (green, live cells) and 4 μM ethidium homodimer-1 (red, dead cells) were added and 15 min later using a Zeiss 710 confocal microscope (Carl Zeiss, Germany). The spheroids were imaged.

As a result, as shown in Figure 1, after thawing in the vitrified group according to Example 3 and the non-vitrified group according to Comparative Example 1, the cells showed a shape and growth pattern similar to fibroblasts, similar to non-frozen preserved cells. Additionally, the survival rates of vitrified MSCs (v-MSCs) and non-vitrified MSCs (nMSCs) after thawing were 89.4 ± 4.2% and 93.2 ± 1.2%, respectively. The viability of vitrified MSCs was slightly decreased compared to the non-vitrified group, but no significant differences were observed between the two groups.

Example 5. Cell proliferation assay

MSCs were counted using a hemocytometer at the beginning and end of each passage. Population doubling time (PDT) was measured by the formula PDT = ln2 / ln(NT/N0). In the above formula, NT is the number of cells at the end of passage, N0 is the number of cells at seeding density, and T is the culture time.

As a result, as shown in Figure 2, the PDT of thawed cells also showed no significant difference between the vitrified and non-vitrified groups until passage 5.

Example 6. TUNEL assay for detection of apoptotic DNA fragmentation

DNA fragmentation was detected using terminal deoxynucleotidyl transferase 2-deoxyuridine 5-triphosphate (dUTP) nick end labeling (TUNEL; Roche, Indianapolis, IN, USA). Vitrified cells (v-cells) and non-vitrified cells (n-cells) were thawed as described in Example 3 and Comparative Example 1 above, fixed with 4.0% paraformaldehyde, and then subjected to Click-iT™Plus TUNEL assay (Thermo TUNEL analysis was performed using Fisher Scientific). Images of TUNEL+ cells were acquired using a fluorescence microscope (Nikon).

As a result, as shown in Figure 3, there was no significant difference in the number of TUNEL+ cells between the vitrified group and the non-vitrified group. This shows that vitrification does not damage cellular DNA.

In other words, these results suggest that, considering intracellular crystallization by conventional gentle freezing, the vitrification method using a high concentration of CPA may be a safer approach for cell preservation than the non-vitrification method.

Example 7. Measurement of intracellular active oxygen

To determine the toxicity of cryoprotectants (CPAs) that cause osmotic stress or oxidative stress, intracellular levels of cellular reactive oxygen species were examined.

Reactive oxygen species (ROS) were measured using the DCFDA cellular ROS detection assay kit (Abcam, Cambridge, MA, USA). That is, after thawing the vitrified cells (v-cells) and non-vitrified cells (n-cells), each pellet was incubated in 1x buffer containing 25 μM DCFDA for 45 min at 37 °C.

Reactive oxygen species (ROS) were measured by flow cytometry without a washing step, and 30,000 labeled cells were acquired and analyzed using a Becton Dickinson FACS Calibur.

As a result, as shown in Figure 4, there was no significant difference in ROS levels between the vitrified and non-vitrified groups in all cell lines. This proves that reactive oxygen species (ROS) are not generated by vitrification when cells are frozen and quickly thawed.

Example 8. Quantitative real-time polymerase chain reaction

Total RNA from each sample was extracted using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA). Next, 1 μg of total RNA was transcribed into complementary deoxyribonucleic acid (cDNA) using a high-capacity cDNA reverse transcription kit (Thermo Fisher Scientific). Real-time PCR was performed using FastStart Essential DNA Green Master (Roche, Pleasanton, CA, USA) on a LightCycler 96 instrument (Roche).

All PCR reactions were performed in triplicate. Average cycle threshold (Ct) values of triplicate wells for each sample were collected and expression data were normalized to the endogenous control glyceraldehyde 3-phosphate dehydrogenase (GAPDH).

Target genes and related primers are shown in Table 1 below.

target gene base sequence sequence number GAPDH sense 5'-GTCTGAACCATGAGAAGTATGA One GAPDH antisense 5'-CTTCCACGATACCAAAGTTGT 2 Bax sense 5'-GTCAGCTGCCACTCGGAAA 3 Bax antisense 5'-AGTAACATGGAGGCTGCAGAGGAT 4 Bcl-2 sense 5'-TCAGAGACAGCCAGGAGAAATCA 5 Bcl-2 antisense 5'-CCTGTGGATGACTGAGTACCTGAA 6 Bcl-XL sense 5'-ATGGCAGCAGTAAGCAAGC 7 Bcl-XL antisense 5'-CGGAAGAGTTCATTCACTACCTGT 8 Bid sense 5′-ACTGGTGTTTTGGCTTCCTCC 9 Bid antisense 5'-ATTCTTCCCAAGCGGGAGTG 10 SOD1 sense 5'-CTGAAGGCCTGCATGGATTC 11 SOD1 antisense 5'-CCAAGTCTCCAACATGCCTCTC 12 p53 sense 5'-CCCAAGCAATGGATGATTTGA 13 p53 antisense 5'-GGCATTCTGGGAGGCTTCATCT 14 HSF1 sense 5'-GCCTTCCTGACCAAGCTGT 15 HSF1 antisense 5'-AAGTACTTGGGCAGCACCTC 16

As a result, as shown in Figure 5a, there was no significant difference between the vitrified group and the non-vitrified group at the single cell level.

Figure 5b confirms the increased viability after vitrification of the spheroids by analyzing the expression of various genes in each spheroid group, showing that the Bax/Bcl-2 ratio is higher in non-vitrified spheroids compared to vitrified spheroids. Confirmed.

Bcl-xL, a member of the Bcl-2 family of proteins, was significantly upregulated in vitrified spheroids, and Bid, a pro-apoptotic Bcl-2 protein containing only the BH3 domain, was significantly upregulated between vitrified and non-vitrified spheroids. There was no difference. In addition, p53, which is associated with apoptosis, was significantly increased in non-vitrified spheroids, showing enhanced apoptosis by cryoinjury when cells were slowly frozen in cell aggregates. In addition, the occurrence of oxidative stress due to ROS removal was confirmed by analyzing the SOD1 gene, and both non-vitrified and vitrified spheroids were confirmed to have similar expression levels. HSF-1 showed the same trend as SOD1 expression.

Example 9. Characterization of MSCs by flow cytometry

The cell surface antigen profile of vitrified cells (v-MSC) and non-vitrified cells (n-MSC) was characterized by flow cytometry.

The cell pellet separated by trypsin-EDTA was resuspended in FACS buffer (DPBS solution containing 0.5% bovine serum albumin (BSA) and 2mM EDTA) and filtered using a pre-moistened 40 μm cell strainer. The cells were then labeled using each antibody of the MSC surface marker. Each antibody was a fluorochrome-conjugated antibody against CD44-APC, CD73-PE, CD90-APC, CD105-PE (BD Biosciences, Bedford, MA, USA) and the negative marker CD31, CD34 (BD Biosciences) conjugated to APC, PE. am. The corresponding IgG control was prepared identically and 30,000 labeled cells were obtained and analyzed using a Becton Dickinson FACS Calibur.

As shown in Figure 6, all groups showed a typical MSC antigenic profile showing expression of CD44, CD73, CD90 and CD105, but CD31 and CD34 for negative markers were not detected.

This indicates that both groups express all appropriate markers of MSCs without expressing differentiation markers.

Example 10. Evaluation of MSC differentiation potential

A commercial kit (Thermo Fisher Scientific) was used to induce differentiation of osteoblasts, chondroblasts and adipocytes. Under differentiation conditions, cells were maintained in 12-well plates. Osteogenesis and chondrogenesis were induced for 21 days and the adipogenic system was induced for 14 days. Appropriate staining was performed to evaluate each differentiation process, and intracellular lipid droplets were detected using Oil Red O staining. Von Kossa staining was performed to visualize the extracellular mineral matrix, and Alcian blue staining was used to confirm the formation of proteoglycan. Images were analyzed using an inverted microscope (Nikon, Chiyoda-ku, Japan).

As shown in Figure 7, it can be seen that there is no significant difference between vitrified cells (v-MSC) and non-vitrified cells (n-MSC) in terms of differentiation potential or characteristics.

Example 11. Viability of spheroids by size after thawing

Spheroids of various sizes (200-900 μm) were fabricated and viability between vitrified and non-vitrified groups was confirmed after thawing. After freezing and reheating steps, cell survival was visualized using a live-dead staining procedure.

As a result, as shown in Figure 8, in the control group, most cells were alive (green fluorescence), and almost no dead cells were observed (red fluorescence).

On the other hand, unlike normal 2D cell culture results, the non-vitrified (gently frozen) group showed excessive cell death in the core region. The vitrified group showed relatively mild cell death. This demonstrates that the viability of cell aggregates in the vitrification group after reheating was improved because a high concentration of cryoprotectant (CPA) could penetrate the spheroid core and protect cell death.

All statistical analyzes in the examples were performed using GraphPad Prism software version 5 (La Jolla, CA, USA). All statistical data were expressed as mean ± SEM, and statistical significance of experimental results was determined using one-way ANOVA. Differences between experimental groups were considered significant when p <0.05.

The description of the present invention described above is for illustrative purposes, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical idea or essential features of the present invention. will be. Therefore, the embodiments described above should be understood in all respects as illustrative and not restrictive. For example, each component described as unitary may be implemented in a distributed manner, and similarly, components described as distributed may also be implemented in a combined form.

The scope of the present invention is indicated by the claims described below, and all changes or modified forms derived from the meaning and scope of the claims and their equivalent concepts should be construed as being included in the scope of the present invention.

<110> Konkuk University Glocal Industry-Academic Collaboration Foundation <120> CRYOPRESERVATION METHOD OF STEM CELL CULTURE USING HIGH CONCENTRATION OF CRYOPROTECTIVE AGENTS <130> P208629 <160> 16 <170> KoPatentIn 3.0 <210> 1 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> GAPDH sense <400> 1 gtctgaacca tgagaagtat ga 22 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> GAPDH antisense <400> 2 cttccacgat accaaagttg t 21 <210> 3 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Bax sense <400> 3 gtcagctgcc actcggaaaa 19 <210> 4 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Bax antisense <400> 4 agtaacatgg agctgcagag gat 23 <210> 5 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Bcl-2 sense <400> 5 tcagagacag ccaggagaaa tca 23 <210> 6 <211> 24 <212> DNA <213> Artificial Sequence <220> <223>Bcl-2 antisense <400> 6 cctgtggatg actgagtacc tgaa 24 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Bcl-XL sense <400> 7 atggcagcag taaagcaagc 20 <210> 8 <211> 24 <212> DNA <213> Artificial Sequence <220> <223>Bcl-XL antisense <400> 8 cggaagagtt cattcactac ctgt 24 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Bid sense <400> 9 actggtgttt ggcttcctcc 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Bid antisense <400> 10 attcttccca agcgggagtg 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SOD1 sense <400> 11 ctgaaggcct gcatggattc 20 <210> 12 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> SOD1 antisense <400> 12 ccaagtctcc aacatgcctc tc 22 <210> 13 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> p53 sense <400> 13 cccaagcaat ggatgatttg a 21 <210> 14 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> p53 antisense <400> 14 ggcattctgg gagcttcatc t 21 <210> 15 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> HSF1 sense <400> 15 gccttcctga ccaagctgt 19 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223>HSF1 antisense <400> 16 aagtacttgg gcagcacctc 20

Claims (13)

(a) treating cells in an equilibration solution for 5 to 15 minutes;
(b) treating the cells in which step (a) has been completed in a vitrification solution for 10 to 120 seconds; and
(c) placing the cells in which step (b) has been completed into liquid nitrogen at -196°C; In the method of cryopreservation of cells, comprising:
The equilibration solution consists of ethylene glycol (EG) and 1.4 M DMSO based on DPBS (Dulbecco's phosphate buffered saline) containing 20% (v/v) FBS,
The vitrification solution is composed of a high concentration of cryoprotective agent (CPA),
Based on LM5 carrier solution, consisting of 70 g/L PVP K12, 2.8M DMSO, 2.7M EG, and 2.8M formamide;
A method of cryopreservation of cells, wherein the LM5 transport solution is adjusted to contain 0.3M sucrose instead of glucose, mannitol, and lactose. delete delete delete delete According to paragraph 1,
A method of cryopreservation of cells, wherein the time for treating the cells with the equilibration solution is 10 minutes, and the time for treating the cells with the vitrification solution is 60 seconds. In the method of thawing cells cryopreserved by the cryopreservation method of claim 1,
A cell thawing method that minimizes cell damage by thawing cryopreserved cells and then suspending them in a sucrose solution of gradually lower concentration. In clause 7,
Thawing of the cells is accomplished by transferring the cells stored in liquid nitrogen to a water bath at 37 °C and successively suspending them in 0.5, 0.25, and 0 M sucrose solutions in DPBS containing 20% FBS for 5 minutes each. Phosphorus, cell thawing method. According to clause 8,
Cells thawed by the thawing method maintain cell viability, shape maintenance, and function, and DNA damage is suppressed. According to any one of paragraphs 1 and 6,
The cells are embryonic stem cells (ES), induced pluripotent stem cells (iPSC), and adult stem cells (ASC), one of the group consisting of mesenchymal stem cells (MSC), hematopoietic stem cells (HSC), and neural stem cells (NSC). A method of cryopreservation of cells selected above. According to clause 10,
The cell is a collection of a plurality of cells, and is a spheroid or organoid that forms a three-dimensional structure. According to any one of claims 7 to 9,
The cells are embryonic stem cells (ES), induced pluripotent stem cells (iPSC), and adult stem cells (ASC), one of the group consisting of mesenchymal stem cells (MSC), hematopoietic stem cells (HSC), and neural stem cells (NSC). The cell thawing method selected above. According to clause 12,
The cell is a collection of a plurality of cells, and is a spheroid or organoid that forms a three-dimensional structure.