阅读说明：本技术 一种干细胞冻存保护剂及其使用方法 (Stem cell cryopreservation protective agent and application method thereof ) 是由 于兵 朱梦梅 汪超 胡以平 于 2021-08-10 设计创作，主要内容包括：本发明专利公开了一种干细胞冻存保护剂及其使用方法,具体涉及干细胞的技术领域。该干细胞冻存保护剂,包含如下浓度的组分：甘油：15-30％(v/v),蔗糖：0.25-0.5Mol,非必需氨基酸混合液：1×,N-乙酰-L半胱氨酸：1-5mM。本发明的干细胞冻存保护剂的成分明确,不含生物毒性物质,无异种动物蛋白或血液制品,无异种抗原,发生超敏反应的概率较低,具有较高的生物安全性和广泛的临床应用前景。该干细胞冻存保护剂不仅原料价格低廉,且操作方便,易于推广,可实现低成本大量冻存干细胞。(The invention discloses a stem cell cryopreservation protective agent and a using method thereof, and particularly relates to the technical field of stem cells. The stem cell cryopreservation protective agent comprises the following components in concentration: glycerol: 15-30% (v/v), sucrose: 0.25-0.5Mol, non-essential amino acid mixture: 1 ×, N-acetyl-L cysteine: 1-5 mM. The stem cell cryopreservation protective agent disclosed by the invention is definite in components, free of biological toxic substances, free of xenogenic animal protein or blood products, free of xenogenic antigens, low in probability of generating hypersensitivity, high in biological safety and wide in clinical application prospect. The stem cell cryopreservation protective agent has the advantages of low raw material price, convenient operation, easy popularization and capability of realizing low cost and mass cryopreservation of stem cells.)

1. A stem cell cryopreservation protective agent is characterized in that: comprising the following components in the following concentrations:

glycerol: 15-30% (v/v),

sucrose: 0.25 to 0.5Mol of,

non-essential amino acid mixture: 1X of a first reaction solution and a second reaction solution,

N-acetyl-L cysteine: 1-5 mM.

2. The stem cell cryopreservation protective agent according to claim 1, wherein: the solvent of the stem cell freezing protective agent is DMEM, DMEM/F12 and RPMI-1640 basic culture solution.

3. A use method of a stem cell cryopreservation protective agent is characterized in that: the method comprises the following steps:

s1, preparing the cells growing adherent to the skin into single cell suspension, and counting the number of the cells;

s3, centrifuging and collecting the single cell suspension in the S1, wherein the single cell suspension needs 500g and is centrifuged for 5 minutes, and removing a supernatant and keeping cell precipitates;

s5, resuspending the stem cell cryopreservation agent of claim 1 or 2 into cells, and controlling the cell density at 1X 10⁵-5×10⁶/mL；

S7, subpackaging the cells resuspended in the S5 into a freezing tube, and directly placing the freezing tube in a refrigerator at the temperature of minus 80 ℃;

and (5) after 24 hours of S9, transferring the frozen cells in the S7 into liquid nitrogen for long-term storage.

4. The method of using a stem cell cryopreservation protectant according to any one of claims 1-3, wherein: the cryopreservation protective agent can be used for cryopreservation of all cells.

Technical Field

The invention relates to the technical field of stem cells, in particular to a stem cell cryopreservation protective agent with definite chemical components and a using method thereof.

Background

Stem cells have the ability to self-renew and differentiate into a variety of mature cells. Meanwhile, the biological active substances such as cell factors, growth factors and the like can be secreted, the damage repair or regeneration of tissues and organs is stimulated in a paracrine signal transduction mechanism mode, and the biological active substances are a seed cell source for cell transplantation treatment, drug screening or tissue engineering of tissue damage (such as liver damage and refractory wound surfaces). Some stem cells, such as mesenchymal stem cells, also have the function of immunoregulation, and can be used for treating autoimmune diseases, tumors and the like, and even can be used for carrying out allograft transplantation treatment. Research and application of stem cells have become one of the leading and hottest research hotspots in the biomedical field.

At present, the source of stem cells is mainly isolated from donor organs. The source and amount of the drug cannot meet the time and the requirement of clinical treatment. If the reserve of sufficient stem cells can be ensured, the time and the number of the stem cells required in the clinical treatment process can be solved at any time. The method for storing the stem cells is the long-term cryopreservation of the stem cells, however, during the cryopreservation process of the cells, ice crystals formed in the cells can cause irreversible damage to the cells, and the activity and the function of the cells are obviously and adversely affected. The use of the cell freezing protective agent can reduce the formation of ice crystals of cells in the process of rising temperature, and play a role in protecting the activity and the function of the cells.

At present, two types of commonly used cell cryopreservation protective agents are mainly used, and one type is a permeability protective agent, such as dimethyl sulfoxide (DMSO), glycerol, propylene glycol and ethylene glycol. Most of the protective agents are small molecular substances, are easy to penetrate cell membranes to enter cells, and reduce the concentration of electrolytes in unfrozen solution inside and outside the cells, so that the cells are protected from being damaged by high-concentration electrolytes. Meanwhile, the water in the cells cannot be excessively exosmosed, so that the cells are prevented from being excessively dehydrated and shrunk. Another class is impermeable protectants such as polyvinylpyrrolidone, sucrose, polyethylene glycol, dextran, albumin, and the like. The mechanisms by which they function may be: the non-permeable protective agent as a macromolecular substance can be preferentially combined with water molecules in the solution to reduce the content of free water in the solution, so that the freezing point is lowered and the formation of ice crystals is reduced; meanwhile, the concentration of electrolyte in the solution is reduced due to the large molecular weight of the compound, so that the damage of solute is reduced.

Dimethyl sulfoxide (DMSO) is the most commonly used cell freezing protective agent, but DMSO is a cytotoxic substance at normal temperature, and it is difficult to ensure that frozen cells can be eluted cleanly when being recovered in clinical use. The most common freezing technique currently used is to place the cells in a frozen stock solution containing 10% DMSO, then to incubate the cells in liquid nitrogen after a series of temperature steps. During the low-temperature cryopreservation process of cells, ROS (reactive oxygen species) and the like can be excessively generated and accumulated in the cells, so that oxidative stress injury can be formed on the cells during the recovery process, and a safe and reliable stem cell cryopreservation protective agent is urgently needed.

Disclosure of Invention

The invention aims to provide the stem cell cryopreservation protective agent which is definite in components, high in safety, low in price, convenient to use and good in cryopreservation effect and the preparation method thereof, so that the problems that the existing stem cell cryopreservation protective agent is high in cost and easily causes adverse reactions of patients are solved.

In order to achieve the aim, the invention provides a stem cell cryopreservation protective agent with definite chemical components, which comprises the following components in concentration:

glycerol: 15-30% (v/v),

sucrose: 0.25 to 0.5Mol of,

non-essential amino acid mixture: 1X of a first reaction solution and a second reaction solution,

N-acetyl-L cysteine: 1-5 mM.

Further, the solvent of the stem cell freezing protective agent is DMEM, DMEM/F12 and RPMI-1640 basic culture solution.

The invention provides a use method of a stem cell cryopreservation protective agent, which comprises the following steps:

s1, preparing the cells growing adherent to the skin into single cell suspension, and counting the number of the cells;

s3, centrifuging and collecting the single cell suspension in the S1, wherein the single cell suspension needs 500g and is centrifuged for 5 minutes, and removing a supernatant and keeping cell precipitates;

s5, the stem cell freezing protective agent is used for resuspending cells, and the cell density is controlled to be 1 x 10⁵-5×10⁶/mL；

S7, subpackaging the cells resuspended in the S5 into a freezing tube, and directly placing the freezing tube in a refrigerator at the temperature of minus 80 ℃;

and (5) after 24 hours of S9, transferring the frozen cells in the S7 into liquid nitrogen for long-term storage.

Further, the cryopreservation protective agent can be used for cryopreserving all cells.

Compared with the prior art, the beneficial effect of this scheme:

1. the scheme can ensure that the raw material preparation of the stem cell cryopreservation protective agent does not contain animal protein such as serum, albumin and the like or blood products, does not have the risk of disease transmission of human and livestock together, and does not have the risk of sensitization of foreign protein or antigen;

2. the stem cell cryopreservation protective agent provided by the scheme is definite in added raw material components, does not contain DMSO (dimethyl sulfoxide) which is toxic to a human body, and is high in safety;

3. the stem cell cryopreservation protective agent provided by the scheme can be directly placed in a refrigerator at the temperature of minus 80 ℃ in the cell cryopreservation process without using a program cooling device, so that the cell cryopreservation program is simplified.

4. The protective agent provided by the scheme has low manufacturing cost and high possibility of large-dose application, and the operation method for freezing and storing the cells is simple and convenient and is easy to popularize and apply.

Drawings

FIG. 1 is a scattergram of flow assays after PI staining in example 1;

FIG. 2 shows the cell survival rate in example 1

After the bone marrow mesenchymal stem cells are preserved in liquid nitrogen for 1 month, 3 months, 6 months and 12 months and are rapidly thawed and revived in a water bath at 37 ℃, PI staining is carried out, and the survival rate of the cells is detected by a flow cytometer. A, performing flow detection on a scatter diagram after PI dyeing; B. the statistical results of the proportion of PI staining negative viable cells at each time point suggest that there is no significant difference in cell survival rate between the time points.

FIG. 2: and (3) preserving the bone marrow mesenchymal stem cells in liquid nitrogen for 1 month, 3 months, 6 months and 12 months, rapidly thawing and recovering the cells in a water bath at 37 ℃, and culturing the cells for 24 hours to obtain a morphological map.

FIG. 3: CCK8 detects the proliferation capacity of bone marrow mesenchymal stem cells after being preserved in liquid nitrogen for 1 month, 3 months, 6 months and 12 months and then being thawed and revived quickly by water bath at 37 ℃.

FIG. 4: after the EpCAM positive liver stem cells are preserved in liquid nitrogen for 1 month, 6 months and 12 months and are quickly thawed and revived in a 37 ℃ water bath, PI staining is carried out, and the survival rate of the cells is detected by a flow cytometer. A, performing flow detection on a scatter diagram after PI dyeing; B. the statistical results of the proportion of PI staining negative viable cells at each time point suggest that there is no significant difference in cell survival rate between the time points.

FIG. 5: EpCAM positive liver stem cells are preserved in liquid nitrogen for 1 month, 6 months and 12 months, then are quickly thawed and revived in a 37 ℃ water bath, and form images are obtained after 24 hours of culture.

FIG. 6: CCK8 detects the proliferation capacity of EpCAM positive liver stem cells after being preserved in liquid nitrogen for 1 month, 6 months and 12 months and then being thawed and revived quickly by a water bath at 37 ℃.

FIG. 7: HT29 colon cancer cells are preserved in liquid nitrogen for 1 month, 6 months and 12 months, then are thawed and revived quickly in a water bath at 37 ℃, PI staining is carried out, and the survival rate of the cells is detected by a flow cytometer. A, performing flow detection on a scatter diagram after PI dyeing; B. the statistical results of the proportion of PI staining negative viable cells at each time point suggest that there is no significant difference in cell survival rate between the time points.

FIG. 8: HT29 colon cancer cell is preserved in liquid nitrogen for 1 month, 6 months and 12 months, then rapidly thawed and revived in 37 ℃ water bath, and cultured for 24 hours to obtain a morphological map.

FIG. 9: CCK8 was used to test the proliferation capacity of HT29 colon cancer cells after being stored in liquid nitrogen for 1 month, 6 months and 12 months and then thawed and revived rapidly in 37 ℃ water bath.

Detailed Description

The present invention will be described in further detail below by way of specific embodiments:

example 1:

a stem cell cryopreservation protective agent comprises the following components in concentration: 15% glycerol (v/v), 0.3Mol sucrose, 1 × mixture of non-essential amino acids and 1 mMN-acetyl-L cysteine in DMEM basal medium. The stem cell cryopreservation protective agent prepared by the method is used for cryopreserving the mesenchymal stem cells,

the specific implementation steps are as follows:

s1, digesting the bone marrow mesenchymal stem cells cultured to 80% fusion density with pancreatin to prepare single cell suspension, and counting the cells with a blood counting chamber;

s3, extracting the single cell suspension of S1, centrifuging 500g of the single cell suspension for 5 minutes by using a centrifuge, removing supernatant and retaining cell sediment;

s5, the stem cell freezing protective agent is used for resuspending cells, and the cell density is controlled to be 1 x 10⁵-5×10⁶/mL；

S7, adding 1ml of the cell frozen protective agent resuspended in the S5 into a 2ml frozen tube; directly placing the cells in a refrigerator at-80 deg.C;

and (5) after 24 hours of S9, transferring the frozen cells in the S7 to liquid nitrogen for long-term storage.

As shown in the attached figure 1, after the cells of S9 were stored in liquid nitrogen for 1 month, 3 months, 6 months and 12 months, the frozen cells were taken out and placed in a 37 ℃ water bath for rapid thawing, and then stained with PI, and the cell viability was measured by flow, which showed that the cell viability was maintained at 94% or more after 12 months of storage in liquid nitrogen, and no significant change was observed compared with the cells stored in a short time.

As shown in figure 2, after 24 hours of cell recovery, under-mirror observation showed no significant morphological differences in cells stored in liquid nitrogen for 1 month, 3 months, 6 months, and 12 months. As shown in FIG. 3, the results of the CCK8 test indicate that there was no significant difference in proliferation ability between cells stored in liquid nitrogen for 1 month, 3 months, 6 months, and 12 months.

Example 2:

a stem cell cryopreservation protective agent comprises the following components in concentration: 30% glycerol (v/v), 0.5Mol sucrose, 1 × mixture of non-essential amino acids and 5 mMN-acetyl-L cysteine in basal medium supplemented with DMEM/F12. The stem cell cryopreservation protective agent prepared in the above way is used for cryopreserving EpCAM positive liver stem cells,

the specific implementation steps are as follows:

s1, digesting EpCAM positive liver stem cells cultured to 80% fusion density with pancreatin to prepare single cell suspension, and counting the cells by using a blood counting chamber;

s3, extracting the single cell suspension of S1, wherein the single cell suspension needs 500g and is centrifuged for 5 minutes, and then the supernatant is removed and the cell sediment is reserved;

s5, the stem cell freezing protective agent is used for resuspending cells, and the cell density is controlled to be 1 x 10⁵-5×10⁶/mL；

S7, adding 1ml of the cell frozen protective agent resuspended in the S5 into a 2ml frozen tube; directly placing the cells in a refrigerator at-80 deg.C;

and (5) after 24 hours of S9, transferring the frozen cells in the S7 to liquid nitrogen for long-term storage.

As shown in FIG. 4, after the cells of S9 were stored in liquid nitrogen for 1 month, 6 months and 12 months, the frozen cells were taken out and placed in a 37 ℃ water bath for rapid thawing, and then stained with PI to detect the cell viability by flow, which revealed that the cell viability was maintained at 96% or more after 12 months of liquid nitrogen storage and was not significantly changed from that of the cells stored in a short time.

As shown in FIG. 5, after 24 hours of cell recovery, microscopic observation showed no significant morphological differences of the cells after 1, 6 and 12 months of storage in liquid nitrogen. As shown in FIG. 6, the results of the CCK8 test indicate that there was no significant difference in proliferation ability between cells stored in liquid nitrogen for 1 month, 3 months, 6 months, and 12 months.

Example 3:

a stem cell cryopreservation protective agent comprises the following components in concentration: 20% glycerol (v/v), 0.25Mol sucrose, 1 × non-essential amino acid mixture and 2.5 mMN-acetyl-L cysteine in basal medium added to RPMI-1640. The stem cell cryopreservation protective agent prepared in the above way is applied to HT29 colon cancer cells,

the specific implementation steps are as follows:

s1, digesting the HT29 colon cancer cells cultured to 70-80% fusion density by pancreatin to prepare single cell suspension, and counting the cells by using a blood counting chamber;

s3, extracting the single cell suspension of S1, wherein the single cell suspension needs 500g and is centrifuged for 5 minutes, and then the supernatant is removed and the cell sediment is reserved;

s5, the stem cell freezing protective agent is used for resuspending cells, and the cell density is controlled to be 1 x 10⁵-5×10⁶/mL；

S7, adding 1ml of the cell frozen protective agent resuspended in the S5 into a 2ml frozen tube; directly placing the cells in a refrigerator at-80 deg.C;

and (5) after 24 hours of S9, transferring the frozen cells in the S7 to liquid nitrogen for long-term storage.

As shown in FIG. 7, after the cells of S9 were stored in liquid nitrogen for 1 month, 6 months and 12 months, the frozen cells were taken out and placed in a 37 ℃ water bath for rapid thawing, and then stained with PI to detect the cell viability by flow, which revealed that the cell viability was maintained at 96% or more after 12 months of liquid nitrogen storage and was not significantly changed from that of the cells stored in a short time.

As shown in FIG. 8, after 24 hours of cell recovery, microscopic observation showed no significant morphological differences of the cells after 1, 6 and 12 months of storage in liquid nitrogen. As shown in FIG. 9, the results of the CCK8 test indicated that there was no significant difference in proliferation capacity between cells stored in liquid nitrogen for 1 month, 3 months, 6 months, and 12 months.

The foregoing are merely examples of the present invention and common general knowledge of known specific structures and/or features of the schemes has not been described herein in any greater detail. It should be noted that, for those skilled in the art, without departing from the structure of the present invention, several changes and modifications can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicability of the patent. The scope of the claims of the present application shall be determined by the contents of the claims, and the description of the embodiments and the like in the specification shall be used to explain the contents of the claims.