NL2034495A

NL2034495A - Stem cell cryopreservation protective agent, preparation method, and application thereof

Info

Publication number: NL2034495A
Application number: NL2034495A
Authority: NL
Inventors: Li Qingfeng; Zhou Shuangbai
Original assignee: Shanghai 9Th Peoples Hospital Shanghai Jiaotong Univ School Medicine
Priority date: 2023-04-04
Filing date: 2023-04-04
Publication date: 2023-06-29

Abstract

A stem cell cryopreservation protective agent, which includes, based on the total volume of stem cell cryopreservation protective agent, the following components: glycerol with a volume fraction of 10%-30%‚ 0.6 mol/L-l.5 mol/L of trehalose, and a physiological saline or phosphate buffer serving as the solvent. The stem cell cryopreservation protective agent of the present disclosure can effectively retain the activity and function of stem cells, and has high efﬁciency, no toxicity, and xenoantigens, therefore, resulting in higher biosafety and clinical application possibilities. No polymer or biotoxic substances are employed, that is all the used reagents are non-cytotoxic, and all the used reagents are easy to elute. In addition, no xenogeneic-derived blood products and no xenoantigens are included, which guarantees the low possibility of hypersensitivity reaction. The stem cell cryopreservation protective agent is less costly and easier for operation, therefore, having a high possibility in large dose application and promotion.

Description

1 002142P-NL

STEM CELL CRYOPRESERVATION PROTECTIVE AGENT, PREPARATION

METHOD, AND APPLICATION THEREOF

FIELD OF DISCLOSURE

[0001] The present disclosure relates to the field of medicine, and in particular, to a stem cell cryopreservation protective agent.

DESCRIPTION OF RELATED ARTS

[0002] Stem cells have the potential to differentiate into mesodermal-derived cells such as adipogenic cells, osteoblast cells, and chondrocytes, and stem cells can also secrete bioactive molecules, such as growth factors and cytokines, that stimulate tissue regeneration through paracrine signal transduction mechanisms, thus, providing alternative treatments for soft tissue filling, diabetes, liver injury, difficult-to-heal wounds, autoimmune diseases, and even allogeneic transplantation. It has become one of the most cutting-edge and hottest research areas in the medical field. Long-term preservation of stem cells is beneficial to overcome the limitation of surgery timing and can provide a sutficient number of cells with stable genetic characteristics for future clinical applications. Cryopreservation is the typical method for long-term preservation, however, some processes, such as the formation of intracellular ice crystals, can cause cryodamage to cells, which has a significantly negative impact on the activity and function of stem cells. The addition of a cryopreservation protective agent can protect cells from cryodamage during cryopreservation and can better retain the biological properties of cells.

[0003] Currently, the exemplary cryopreservation protective agent for stem cells contains 10% dimethyl sulfoxide (DMSO) and 90% fetal bovine serum (FBS), where DMSO is cytotoxic at room temperature, and no residual DMSO is difficult to ensure after elution when cryopreserved stem cells are used in clinical use. In addition, the fetal bovine serum contains xenogeneic antigens, which leads to the risk of zoonotic diseases and immune

2 002142P-NL response. Therefore, both DMSO and FBS may have adverse effects on patients. Other cryopreservation protective agents are complicated or costly to use, thereby they are not suitable for the application.

SUMMARY OF THE PRESENT INVENTION

[0004] In view of the above-described disadvantages of the prior art, the present disclosure provides a stem cell cryopreservation protective agent, preparation method, and application thereof, which solves the problems of high toxicity and poor cryopreservation effect of the cryopreservation protective agent in the prior art.

[0005] A first aspect of the present disclosure provides a stem cell cryopreservation protective agent, which includes, based on the total volume of the stem cell cryopreservation protective agent, the following components:

[0006] glycerol with a volume fraction of 10%-30%,

[0007] 0.6 mol/L-1.5 mol/L of trehalose, and

[0008] a physiological saline or phosphate buffer serving as the solvent.

[0009] A second aspect of the present disclosure provides the use of the aforementioned stem cell cryopreservation protective agent for stem cell cryopreservation.

[0010] A third aspect of the present disclosure provides a method for preparing the stem cell cryopreservation protective agent, including the following operations:

[0011] 1) adding trehalose into physiological saline or phosphate buffer to prepare trehalose solution;

[0012] 2) preparing the stem cell cryopreservation protective agent using glycerol as the solute and the trehalose solution as the solvent.

[0013] A fourth aspect of the present disclosure provides a method for cryopreservation of

3 002142P-NL stem cell, including the following operations:

[0014] 1) mixing the stem cell cryopreservation protective agent with a stem cell at a volume ratio of 1:1 to obtain a mixture 1;

[0015] 2) placing the mixture 1 in a programmed cooling box and performing programmed cooling at -80°C; and

[0016] 3) placing the resulting mixture 1 obtained in operation 2) in liquid nitrogen for cryopreservation, to obtain a cryopreserved stem cell.

[0017] As described above, the stem cell cryopreservation protective agent, preparation method, and application thereof of the present disclosure has the following beneficial effects:

[0018] The stem cell cryopreservation protective agent of the present disclosure can effectively retain the activity and function of stem cells, and has high efficiency, no toxicity, and xenoantigens, therefore, resulting in higher biosafety and clinical application possibilities.

No polymer or biotoxic substances are employed in the stem cell cryopreservation protective agent of the present disclosure, that is all the used reagents are non-cytotoxic, and all the used reagents are easy to elute. In addition, no xenogeneic-derived blood products and no xenoantigens are included in the stem cell cryopreservation protective agent of the present disclosure, which guarantees the low possibility of hypersensitivity reaction. The stem cell cryopreservation protective agent of the present disclosure is less costly and easier for operation, therefore, having a high possibility in large dose application and promotion.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019] FIG. 1 shows a diagram of the cell viability of adipose-derived stem cells through

Trypan Blue assay, where the adipose-derived stem cells are first cryopreserved under the protection of different cryopreservation protective agents at -196°C for 180 days and then revived.

[0020] FIG 2 shows a diagram of the cell proliferation of adipose-derived stem cells

4 002142P-NL through CCK8 assay, where the adipose-derived stem cells are first cryopreserved under the protection of different cryopreservation protective agents at -196°C for 180 days and then revived.

[0021] FIG 3 shows a diagram of the cell migration of adipose-derived stem cells through cell scratch test, where the adipose-derived stem cells are first cryopreserved under the protection of different cryopreservation protective agents at -196°C for 180 days and then revived.

[0022] FIG 4 shows a diagram of cell three-directional differentiation of adipose-derived stem cells, where the adipose-derived stem cells are first cryopreserved under the protection of different cryopreservation protective agents at -196°C for 180 days and then revived.

[0023] FIG 5 shows a diagram of cell viability immediate after thawing of adipose stem cells cryopreserved under the protection of the cryopreservation protective agents in

WO2009/115581A2 and the stem cell cryopreservation protective agents in the present disclosure.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] In the prior art, there is no clinically applicable cryopreservation protective agent for adipose-derived stem cells, and only a few formulations with low toxicity are used in the experiment, such as 97% human platelet lysate (PL) + 3% DMSO disclosed in Wang C et al. 2017 for cryopreservation of adipose-derived stem cells and 90% human albumin/90% human serum/90% serum-free medium + 10% DMSO disclosed in Seah P et al. 2018 for cryopreservation of adipose-derived stem cells.

[0025] The existing cryopreservation protective agents have the following disadvantages:

[0026] (1) poor permeability and poor cell cryoprotection effect;

[0027] (2)biotoxicity which is caused by the use of polymers or DMSO and partial residual thereof after elution in a clinical application;

002142P-NL

[0028] the risk of hypersensitivity response which is resulted from the use of blood products containing allogeneic antigens, such as fetal bovine serum;

[0029] (4)high cost because of the use of biological agents, such as blood products (human albumin), and the risk of blood-borne diseases; 5 [0030] (5)complicated technology, difficulty in the promotion and the use of nanomaterials, such as sodium alginate, to encapsulate the cells.

[0031] The embodiments of the present disclosure will be described below through specific exemplary implementations. Those skilled in the art can easily understand other advantages and effects of the present disclosure according to the contents disclosed by the specification.

The present disclosure may also be implemented or applied through other different specific implementation modes. Various modifications or changes may be made to all details in the specification based on different points of view and applications without departing from the spirit of the present disclosure.

[0032] Before the detailed description of the embodiments of the present disclosure, it needs to understand that the protection scope of the present disclosure is not limited to the specific exemplary embodiments described below. It should further understand that the specific terms used in the embodiments are just for the description of the present disclosure, rather than limiting the protection scope of the present disclosure. Unless otherwise stated, the terms, for example, “a”, “an”, “this” etc., in a singular form in the specification and claim of the present disclosure also indicate plural form.

[0033] When numerical ranges are given in the embodiments, it should understand that, unless otherwise indicated herein, both endpoints of each numerical range and any number between the two endpoints may be selected for use. Unless otherwise defined, all technical and scientific terms used in this disclosure have the same meaning as understood by those skilled in the art from the prior art. And except for the specific methods, devices, and materials utilized in the embodiments, the present disclosure can also be implemented by any methods, devices, and materials of the prior art similar or equivalent to those described in the

6 002142P-NL embodiments according to the knowledge of the prior art acquired by those skilled in the art and the contents of the present disclosure.

[0034] Unless otherwise stated, the laboratory procedures, tests, and preparation methods disclosed in the present disclosure are those well-known and commonly used in the field of molecular biology, biochemistry, chromatin biology, analytical chemistry, cell culture, recombinant DNA, and other routine techniques.

[0035] In an embodiment, the stem cell cryopreservation protective agent, includes, based on the total volume of the stem cell cryopreservation protective agent, the following components:

[0036] glycerol with a volume fraction of 10%-30%,

[0037] 0.6 mol/L- 1.5 mol/L of trehalose, and

[0038] a physiological saline or phosphate buffer serving as the solvent.

[0039] The combination of two types of protective agents with different effects in the stem cell cryopreservation protective agent can improve the cell protection efficiency because of the following reasons: (1) the use of an osmotic protective agent (i.e, glycerol) facilitates the transfer of a non-osmotic protective agent (i.e, trehalose) into the cells, thus increasing the efficiency of the non-osmotic protective agent; (2) the complementation between the action of the osmotic and non-osmotic protective agents.

[0040] Optionally, the volume fraction of glycerol in the stem cell cryopreservation protective agent may be 10%-15%, 15%-20%, 20%-25%, or 25%-30%.

[0041] Optionally, the concentration of trehalose in the stem cell cryopreservation protective agent may be 0.6 mol/L -0.65 mol/L, 0.65 mol/L-0.75 mol/L, 0.75 mol/L-0.85 mol/L, 0.85 mol/L-0.95 mol/L, 0.95 mol/L-1.0 mol/L, 1.0 mol/L-1.1 mol/L, 1.1 mol/L-1.2 mol/L, 1.2 mol/L-1.3 mol/L, 1.3 mol/L-1.4 mol/L, or 1.4 mol/L-1.5 mol/L.

[0042] The physiological saline is a sodium chloride solution with a concentration of 0.9%.

7 002142P-NL

[0043] The phosphate buffer is conventional and known to those skilled in the art.

[0044] And any phosphate buffer that does not affect parameters, such as osmotic pressure, of stem cells can be employed in the present disclosure. For example, in an embodiment, the phosphate buffer includes, based on the total volume of the phosphate buffer, 1.4-2.0 mmol/L of potassium dihydrogen phosphate, 3.5-10 mmol/L of disodium hydrogen phosphate, 137 mmol/L of sodium chloride, and 2.7 mmol/L of potassium chloride.

[0045] Further, there is no biotoxic substance, polymer, xenogeneic antigen, or xenogeneic-derived blood product included in the stem cell cryopreservation protective agent.

[0046] The biotoxic substance may be dimethyl sulfoxide (DMSO).

[0047] The polymer may be sodium alginate, dextran, chitosan, or polyvinylpyrrolidone.

[0048] The xenogeneic antigen may be fetal bovine serum.

[0049] The xenogeneic-derived blood product may be bovine blood albumin (BSA), platelet lysate, etc.

[0050] Further, the stem cell cryopreservation protective agent enables stable storage of stem cells at -196°C for at least 180 days.

[0051] In an embodiment, the stem cells may be adipose-derived stem cells.

[0052] The aforementioned stem cell cryopreservation protective agent can be used for stem cell cryopreservation.

[0053] Further, the stem cell cryopreservation protective agent can be used for retaining the activity and function of the cryopreserved stem cells. The function may be the cell migration ability as well as the cell multidirectional differentiation ability.

[0054] In an embodiment, the present disclosure provides a method for preparing the stem cell cryopreservation protective agent, including the following operations:

8 002142P-NL

[0055] 1) adding trehalose into physiological saline or phosphate buffer to prepare trehalose solution; and

[0056] 2) preparing the stem cell cryopreservation protective agent using glycerol as the solute and the trehalose solution as the solvent.

[0057] The concentration of trehalose in the stem cell cryopreservation protective agent, based on the total volume of the stem cell cryopreservation protective agent, is 0.6 mol/L-1.5 mol/L.

[0058] Further, step 1) includes autoclaving the obtained trehalose solution.

[0059] The physiological saline is a sodium chloride solution with a concentration of 0.9%.

[0060] The phosphate buffer contains, based on the total volume of the phosphate buffer, 1.8 mmol/L of potassium dihydrogen phosphate, 10 mmol/L of disodium hydrogen phosphate, 137 mmol/L of sodium chloride, and 2.7 mmol/L of potassium chloride.

[0061] The stem cell cryopreservation protective agent includes, based on the total volume of the stem cell cryopreservation protective agent, glycerol with a volume fraction of 10%-30%.

[0062] Further, step 2) is performed under an aseptic condition.

[0063] The glycerol is medically sterilized glycerol.

[0064] In an embodiment, the present invention provides a method for cryopreservation of stem cell, including the following operations:

[0065] 1) mixing the stem cell cryopreservation protective agent with a stem cell at a volume ratio of 1:1 to obtain a mixture 1;

[0066] 2) placing the mixture 1 in a programmed cooling box and performing programmed cooling at -80° C; and

[0067] 3) placing the resulting mixture 1 obtained in operation 2) in liquid nitrogen for

9 002142P-NL cryopreservation, to obtain a cryopreserved stem cell.

[0068] Optionally, the stem cells are adipose-derived stem cells.

[0069] Further, step 1) includes sealing the mixture 1.

[0070] In step 2), the programmed cooling time is at least 12 hours.

[0071] The method for cryopreservation of stem cell according to the present disclosure does not involve the purpose of diagnosis and treatment. The method of the present disclosure may be used for healthcare purposes or basic research purposes.

Embodiment 1

[0072] The stem cell cryopreservation protective agent is prepared according to the following formula:

[0073] glycerol with a volume fraction of 20%;

[0074] 1.0 mol/ml of trehalose; and

[0075] the physiological saline serving as the solvent.

Control Example 1

[0076] The stem cell cryopreservation protective agent is prepared according to the following formula:

[0077] glycerol with a volume fraction of 10%;

[0078] 0.3 mol/ml of trehalose; and

[0079] the physiological saline serving as the solvent.

Embodiment 2

[0080] The stem cell cryopreservation protective agent is prepared according to the following

10 002142P-NL formula:

[0081] glycerol with a volume fraction of 15%;

[0082] 0.6 mol/ml of trehalose; and

[0083] phosphate buffer (2.0 mmol/L of potassium dihydrogen phosphate, 10 mmol/L of disodium hydrogen phosphate, 137 mmol/L of sodium chloride, 2.7 mmol/L of potassium chloride ) serving as the solvent.

Embodiment 3

[0084] The stem cell cryopreservation protective agent is prepared according to the following formula:

[0085] glycerol with a volume fraction of 25%;

[0086] 1.2 mol/ml of trehalose; and

[0087] the physiological saline serving as the solvent.

Embodiment 4

[0088] The stem cell cryopreservation protective agent is prepared according to the following formula:

[0089] glycerol with a volume fraction of 30%;

[0090] 1.5 mol/ml of trehalose; and

[0091] the physiological saline serving as the solvent.

Control Example 2

[0092] The stem cell cryopreservation protective agent is prepared according to the following formula:

11 002142P-NL

[0093] glycerol with a volume fraction of 20%;

[0094] 0.25 mol/ml of trehalose; and

[0095] phosphate buffer (1.4 mmol/L of potassium dihydrogen phosphate, 3.5 mmol/L of disodium hydrogen phosphate, 137 mmol/L of sodium chloride, 2.7 mmol/L of potassium chloride) serving as the solvent.

Embodiment 5

[0096] 1. Lipids were extracted, then digested to SVF through the enzyme digestion method under an aseptic condition, and afterward cultured to P3 generation in a cell culture incubator.

When fusing to 90%, the P3 generation adipose-derived stem cells were collected by trypsin digestion, and then the adipose-derived stem cells (110° cells/mL) were mixed with (1) the protective agent in Embodiment 1 of the present disclosure, (2) 10%, 20%, 30%, 40%, or 50% of glycerol (Gly), (3) 0.3 mol/L, 0.6 mol/L, 1.0 mol/L, or 1.25 mol/L of trehalose (Tre), and (4) 10% DMSO + 90% FBS (the classical protective agent), respectively, at a volume ratio of 1:1. Equal amount of physiological saline was added as a control, and the cryopreservation tube was turned upside down for mixing well.

[0097] 2. The cryopreservation tube after mixing was placed in a programmed cooling box, which was then put into a refrigerator at -80°C. After cooling for 12 hours, the cryopreservation tube was transferred to liquid nitrogen at -196°C.

[0098] 3. The cryopreservation tube was taken out from the liquid nitrogen after being cryopreserved for 180 days, and then placed into a 37°C water bath for 2 minutes for rewarming.

[0099] 4. The cryopreservation tube was transferred to an aseptic condition, the mixture in the cryopreservation tube was placed in an aseptic centrifuge tube, the mixture was first eluted with hyperosmotic sodium chloride solution (9%) for 3 minutes and then centrifuged at 500 rpm for 5 minutes to obtain a lower-layer precipitate. The lower-layer precipitate was eluted once more with 0.9% sodium chloride solution, and then centrifuged at 1500 rpm for 5

12 002142P-NL minutes, where the supernatant was discarded to obtain another lower-layer precipitate.

[00100] 5. The cell viability of the eluted adipose-derived stem cells at the lower layer was tested by trypan blue staining and CCKS assay, and the cell migration ability was detected by scratch test.

[00101] The results in FIG 1A showed that the cell viability of the adipose-derived stem cells under the protection of the 20% Gly group was significantly higher than those of the adipose-derived stem cells under the protection of the 10% Gly group, 30% Gly group, and 50% Gly group after 180 days of cryopreservation. FIG 1B showed that the cell viability of the adipose-derived stem cells under the protection of the 1.0 M Tre group was significantly higher than those of the adipose-derived stem cells under the protection of the 0.3 M Tre group, 0.6 M Tre group, and 1.25 M Tre group after 180 days of cryopreservation. FIG 1C showed that the cellular viability of the adipose-derived stem cells under the protection of the cryopreservation protective agent of the present disclosure was significantly higher than those of the adipose-derived stem cells under the protection of the 20% Gly group and 1.0 M Tre group, and there was no significant difference between the cellular viability of the adipose-derived stem cells under the protection of the cryopreservation protective agent of the present disclosure and that of the adipose-derived stem cells under the protection of the classical protective agent group (10% DMSO + 90% FBS) after 180 days of cryopreservation.

As can be seen from FIG 1, compared to other stem cell cryopreservation protective agents, for example, the 50% Gly group in FIG. 1A and the 1.25 mol/L Tre group in FIG 1B, the stem cell cryopreservation protective agent of the present disclosure had a superior cell preservation effect (FIG 1C), and the cell viability of the adipose-derived stem cells under the protection of the cryopreservation protective agent of the present disclosure was the same as that of the adipose-derived stem cells under the protection of the classical protective agent group (FIG 1C). In addition, the cryopreservation protective agent of the present disclosure had no potential biotoxicity.

[00102] As shown in FIG 2, the cell proliferation capacity of the adipose-derived stem cells under the protection of the cryopreservation protective agent of the present disclosure

13 002142P-NL was significantly higher than those of the adipose-derived stem cells under the protection of the 20% Gly group and 1.0 M Tre group, and there is no significant difference between the cell proliferation capacity of the adipose-derived stem cells under the protection of the cryopreservation protective agent of the present disclosure and that of the adipose-derived stem cells under the protection of the classical protective agent group (10% DMSO + 90%

FBS) after 180 days of cryopreservation. FIG. 3 showed that the cell migration capacity of the adipose-derived stem cells under the protection of the cryopreservation protective agent of the present disclosure was significantly higher than that of the adipose-derived stem cells under the protection of the classical protective agent group (10% DMSO + 90% FBS) after 180 days of cryopreservation, and there was no significant difference between the cell migration capacity of the adipose-derived stem cells under the protection of the cryopreservation protective agent of the present disclosure and that of fresh cells. As shown in FIG 4, the adipose-derived stem cells under the protection of the cryopreservation protective agent of the present disclosure still could differentiate into adipogenic cells, osteoblastic cells, and cartilage cells, and the differentiation ability of the adipose-derived stem cells under the protection of the cryopreservation protective agent of the present disclosure had no difference compared to those of the adipose-derived stem cells under the protection of the classical protective agent group and fresh cells.

[00103] The results of FIGs. 2, 3, and 4 showed that the revived cells under the protection of the cryopreservation protective agent of the present disclosure had a higher cell proliferation capacity as well as similar cell migration capacity and multidirectional differentiation capacity to those of fresh cells, indicating the cryopreservation protective agent of the present disclosure had a better effect on retaining the cell functions.

[00104] The cryopreservation protective agents prepared in Embodiments 2-4 of the present disclosure were also subjected to the above same cell experiments, and the results showed that the cryopreservation protective agents of the present disclosure had excellent cell cryopreservation effects, the cell viability of the cells under the protection of the cryopreservation protective agents of the present disclosure was the same as that of fresh cells, and the cryopreservation protective agents of the present disclosure had no potential

14 002142P-NL biotoxicity. In addition, the revived cells under the protection of the cryopreservation protective agents of the present disclosure had higher cell proliferation capacity as well as similar cell migration capacity and multidirectional differentiation capacity to those of fresh cells, indicating the cryopreservation protective agents of the present disclosure had better effects on retaining the cell functions.

Control Example 3

[00105] A cryopreservation protective agent disclosed in WO2009/115581A2 contains 20 wt% of glycerol, 0.2 M of trehalose, and adequate physiological saline, and the cryopreservation protective agent was used to cryopreserve adipose tissues.

[00106] In order to compare the effect of the cryopreservation protective agent in

WO2009/115581A2 and the stem cell cryopreservation protective agent in the present disclosure, 2 groups of the cryopreservation protective agent in WO2009/115581A2 and 2 groups of the stem cell cryopreservation protective agent in the present disclosure were prepared for performing cryopreservation of adipose stem cells.

[00107] Specifically, the cryopreservation protective agents in WO2009/115581A2 were 20% glycerol + 0.2M sucrose and 20% glycerol + 0.2M trehalose, respectively, and the stem cell cryopreservation protective agents in the present disclosure were 20% glycerol + 0.6M trehalose and 20% glycerol + 1.0M trehalose, respectively. The above four groups of cryopreservation protective agents were used to cryopreserve adipose stem cells, after being cryopreserved for 180 days, the adipose stem cells were thawed and resuscitated, and the cell viability immediate after thawing was examined. The corresponding results are shown in FIG 5.

[00108] As can be seen from FIG 5, the cell viability immediate after thawing and resuscitation of the adipose stem cells cryopreserved under the protection of the stem cell cryopreservation protective agents in the present disclosure is higher than that of the adipose stem cells cryopreserved under the protection of the cryopreservation protective agents in

WO2009/115581A2, i.e, the adipose stem cells cryopreserved under the protection of the

15 002142P-NL cryopreservation protective agents in WO2009/115581A2 cannot achieve the cell viability immediate after thawing as the adipose stem cells cryopreserved under the protection of the stem cell cryopreservation protective agents in the present disclosure.

[00109] The adipose stem cells cryopreserved under the protection of the stem cell cryopreservation protective agents in the present disclosure maintain their functions after thawing and resuscitation, for example, strong cell proliferation ability, good cell migration ability, and the cells still have multi-directional differentiation ability, such as differentiating into adipogenic cells, osteoblastic cells, and cartilage cells (see FIGs 2-4). Therefore, the stem cell cryopreservation protective agents in the present disclosure have better performance in terms of maintaining the functions of adipose stem cells.

[00110] The above embodiments are intended to illustrate the implementations of the present disclosure and should not be construed as a limitation of the present disclosure. In addition, the various modifications listed herein, as well as variations in the methods and compositions of the present disclosure, will be apparent to those skilled in the art without departing from the scope and spirit of the present disclosure. While the present disclosure has been specifically described according to various specific preferred embodiments of the present disclosure, it should be understood that the present disclosure should not be limited to these specific embodiments. In fact, various modifications as described above that would be obvious to one skilled in the art should be included within the scope of the present disclosure.

Claims

16 002142P-EN CONCLUSIONS

1. Use of a stem cell protectant in stem cell cryopreservation, wherein the stem cell protectant in cryopreservation contains the following components based on the total volume of the stem cell protectant: glycerol with a volume fraction of 10%-30%, 0.6 mol/L-1.5 mol/L trehalose, and physiological saline or phosphate buffer as solvent; in which the protectant for the cryopreservation of stem cells does not contain any biotoxic substances, polymers, xenoantigens or blood products derived from xenobiotics; and wherein the stem cells are selected from adipose-derived stem cells.

2. Use of the stem cell protectant in stem cell cryopreservation according to claim 1, wherein the stem cell cryopreservation protectant allows stable storage of stem cells at -196°C for at least 180 days.

A method for the preparation of a protectant for stem cell cryopreservation, comprising the steps of: 1) adding trehalose to a physiological saline solution or phosphate buffer to prepare a trehalose solution; and 2) preparing the protectant for the cryopreservation of stem cells using glycerol as a solvent and the trehalose solution as the solvent, wherein a concentration of trehalose in the protectant for the cryopreservation of stem cells is 0.6 mol/L-1.5 mol/L, based on the total volume of protection fluid for stem cell cryopreservation; and wherein a volume fraction of glycerol in the stem cell cryopreservation agent is 10%-30%, based on the total volume of the stem cell cryopreservation agent.

17 002142P-EN

4. A method for the cryopreservation of stem cells, comprising the following operations: 1) mixing the protectant for cryopreservation of stem cells according to requirement 1 with a stem cell in a volume ratio of 1:1 to obtain a mixture 1; 2) placing the mixture 1 in a programmed cool box and programmed cooling at -80°C; and 3) placing the resulting mixture 1, obtained in operation 2), in liquid nitrogen for cryopreservation, to obtain a cryopreserved stem cell; and wherein the stem cell is an adipose-derived stem cell.