Cell cryopreservation protection solution and cell cryopreservation method

Abstract

The invention provides a cell cryopreservation protection solution and a cell cryopreservation method, and relates to the technical field of biology. The cell cryopreservation protective solution contains 2-5% v / v of dimethyl sulfoxide and 200-100 [mu] M of gastrodin. According to the cell cryopreservation protection liquid, the content of DMSO is reduced by adding gastrodin, meanwhile, the use of human serum albumin (HSA) is replaced, the effect of protecting the cell state is achieved, good cell viability and dry function can be maintained after cell resuscitation, and important reference value is provided for subsequent cell research and cell product application.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a cell cryopreservation protection solution and a cell cryopreservation method. Background Art

[0002] Cell cryopreservation and thawing are key links in the application of umbilical cord blood hematopoietic stem cells, which directly affect the viability and function of cells after cryopreservation and thawing, and determine the effectiveness of subsequent scientific research or clinical application.

[0003] At present, "slow freezing and fast thawing" is still the basic principle followed by cell cryopreservation and resuscitation. Researchers have added a variety of protective ingredients to the cryopreservation protective solution to lower the freezing point, increase the stability of cell osmotic pressure and reduce cell stress damage, thereby protecting cells during the cryopreservation and resuscitation process. However, due to the different components contained in each cell cryopreservation protective solution, there are certain differences in the effects of cryopreservation and resuscitation, and the clinical application restrictions are also different. Therefore, screening out cryopreservation protective solutions suitable for umbilical cord blood hematopoietic stem cells is crucial for the scientific research and clinical application of hematopoietic stem cells.

[0004] Dimethyl sulfoxide (DMSO) is a commonly used cell cryoprotectant and the main active ingredient in cryoprotectant solutions used in cell therapy products that have been marketed so far. As an important osmotic cell protectant, DMSO can reduce mechanical damage to cells by reducing the formation of ice crystals inside and outside cells during cooling, but its cytotoxicity can also affect cell activity. At the same time, cryoprotectant solutions containing DMSO may also cause some complications after being infused into patients, such as nausea, vomiting, diarrhea, neurological symptoms, cardiac toxicity, etc.

[0005] Therefore, there are many products on the market or under development for cryopreservation protection solutions with low or no DMSO. Among them, human serum albumin (HSA) is currently a commonly used cryoprotectant, which protects cells and maintains the cell state during the cryopreservation and resuscitation process. However, in clinical applications, cell products are directly returned without cleaning, and HSA (blood products) also has the risk of causing adverse reactions such as allergies to patients. In addition, HSA has factors such as large batch differences, long or unstable supply cycles, etc. Therefore, improved cryopreservation reagents with low DMSO content are currently needed in the market.

[0006] In view of this, the present invention is proposed. Summary of the invention

[0007] The purpose of the present invention is to provide the use of gastrodin in the preparation of cell cryopreservation reagents, and the cell cryopreservation protection solution prepared by using gastrodin, so as to improve the defects of the existing low-content DMSO cryopreservation reagents.

[0008] In order to solve the above technical problems, the present invention particularly adopts the following technical solutions:

[0009] In a first aspect, a cell cryopreservation protection solution is provided, wherein the cell cryopreservation protection solution contains 2-5% v / v dimethyl sulfoxide and 200 nM-100 μM gastrodin.

[0010] In the second aspect, the application of gastrodin in the preparation of cell cryopreservation reagents is provided.

[0011] In a third aspect, a cell freezing method is also provided, comprising freezing cells using the cell freezing protection solution of the first aspect.

[0012] Compared with the prior art, the present invention has the following beneficial effects:

[0013] The cell cryopreservation protection solution provided by the present invention contains 2-5% v / v dimethyl sulfoxide (DMSO) and 200nM-100μM gastrodin, which has the following advantages:

[0014] (1) The DMSO content used in the cryopreservation protection solution provided by the present invention is 2-5% v / v, which can reduce the side effects caused by DMSO during clinical application.

[0015] (2) The present invention uses a new method of using gastrodin, a monomer component of traditional Chinese medicine, to replace HSA during cell cryopreservation, thereby avoiding inflammatory reactions such as allergies that may be caused by HSA, and at the same time avoiding the impact of large batch differences and unstable delivery time.

[0016] (3) The Chinese medicine monomer components added to the cell cryopreservation protection solution provided by the present invention can protect cells, improve cell viability and recovery rate, and have clinical anti-inflammatory, sedative, and hypnotic effects. As the main active ingredient in the gastrodin series preparations, gastrodin has obvious clinical effects and no obvious side effects, so this solution can also be applied to subsequent clinical applications.

[0017] In summary, the present invention reduces the DMSO content in the cell cryopreservation protection solution and adds the Chinese medicine monomer component gastrodin to replace HSA, thereby avoiding the allergic reaction caused by HSA, as well as the differences between HSA commodity batches and the supply cycle. At the same time, it achieves the same cryopreservation and resuscitation effect as the classic 10% v / v DMSO cryopreservation protection solution, reducing the possible impact of DMSO in subsequent applications. The present invention provides a cell cryopreservation protection solution and a cryopreservation and resuscitation method with reduced DMSO content through the new use of the Chinese medicine monomer component gastrodin, thereby achieving the purpose of preserving cells, and is particularly suitable for preserving hematopoietic stem cells, providing more reference value for subsequent scientific research and clinical applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0019] Figure 1 Comparison of cryopreservation and resuscitation effects of different concentrations of DMSO in effect example 1, data are expressed as mean ± standard deviation, n = 3, * indicates p < 0.05, the difference is statistically significant;

[0020] Figure 2 The comparison results of the cryopreservation and resuscitation effects of the cryopreservation protection solutions of Comparative Examples 1 to 5 in Effect Example 2;

[0021] Figure 3 The comparison results of the cryopreservation and resuscitation effects of the cryopreservation protection solutions provided in Examples 1 to 5 and Comparative Examples 3 and 5 in Effect Example 3;

[0022] Figure 4 Comparison results of the cryopreservation and resuscitation effects of the cryopreservation protection solutions provided in Examples 6 to 10 and Comparative Examples 1, 2 and 4 in Effect Example 4;

[0023] Figure 5 It is a comparison result of the CFU formation ability after the cells frozen and revived in Comparative Example 5, Example 1, Example 5, Comparative Example 4, Example 6, Example 10 and Comparative Example 1 in Effect Example 5, wherein BFU-E is erythrocyte forming unit, CFU-GM is granulocyte-macrophage colony forming unit, and CFU-GEMM is granulocyte-erythroid-macrophage-megakaryocyte mixed colony forming unit. DETAILED DESCRIPTION

[0024] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0025] As used herein, "prepared from" is synonymous with "comprising". As used herein, the terms "comprising", "including", "having", "containing" or any other variation thereof, are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus that comprises the listed elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.

[0026] As used herein, the conjunction "consisting of excludes any unspecified elements, steps or components. If used in a claim, this phrase will render the claim closed such that it excludes materials other than those described, except for normal impurities associated therewith.

[0027] Herein, when an amount, concentration, or other value or parameter is expressed as a range, a preferred range, or a range defined by a series of upper preferred values ​​and lower preferred values, this should be understood to specifically disclose all ranges formed by any pairing of any range upper limit or preferred value with any range lower limit or preferred value, regardless of whether the range is disclosed separately. For example, when a range of "1 to 5" is disclosed, the described range should be interpreted as including ranges "1 to 4", "1 to 3", "1 to 2", "1 to 2 and 4 to 5", "1 to 3 and 5", etc. When a numerical range is described herein, unless otherwise stated, the range is intended to include its end values ​​and all integers and fractions within the range.

[0028] As used herein, "and / or" is used to indicate that one or both of the described situations may occur, for example, A and / or B includes (A and B) and (A or B).

[0029] In a first aspect, a cell cryopreservation protection solution is provided, which contains 2-5% v / v dimethyl sulfoxide (DMSO) and 200nM-100μM gastrodin.

[0030] The content of DMSO may be, for example but not limited to, 2, 2.5, 3, 3.5, 4, 4.5 or 5% v / v, preferably 5% v / v.

[0031] The content of gastrodin can be, for example but not limited to, 200, 300, 500 or 800 nM, or 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 μM, preferably 200 nM to 5 μM.

[0032] In an optional embodiment, the cell cryopreservation protection solution contains 5% v / v dimethyl sulfoxide and 1 μM gastrodin. The cryopreservation protection solution provided by this embodiment has the same effect as the classic 10% v / v DMSO cryopreservation protection solution.

[0033] The DMSO content in the cell cryopreservation protection solution provided by the present invention does not exceed 5% v / v, and the Chinese medicine monomer component gastrodin is added on this basis. Gastrodin, also known as gastrodin glycoside, has a chemical name of 4-hydroxyphenyl-β-D-pyranoglucoside hemihydrate. It is the main component of the Chinese herbal medicine gastrodia elata, and its structural formula is shown in formula (I). It has anti-inflammatory effects. Chinese aliases: Tianliao, gastrodin, Tianxuanqing, natural gastrodia extract. Gastrodin preparations are widely used in the clinical treatment of dizziness, headaches, cardiovascular and cerebrovascular diseases, etc., and are recommended by multiple guidelines and expert consensus. It can be used as a single therapeutic drug or in combination with other drugs, and has few adverse reactions, good safety, and high clinical application value.

[0034]

[0035] The inventors found that adding 200nM to 100μM of gastrodin to the cell cryopreservation protection solution can make the frozen cells have better CFU formation ability after thawing; and / or, perform well in one or more aspects of maintaining cell viability and improving total nucleated cell recovery rate and CD34+ cell recovery rate. The cell cryopreservation protection solution prepared by the present invention reduces the DMSO content in the cryopreservation protection solution while ensuring the viability and function of the cells after cryopreservation and thawing.

[0036] The present invention does not limit the types of cells that can be cryopreserved in the cell cryopreservation protection solution. In an optional embodiment, the cell cryopreservation protection solution can be suitable for hematopoietic stem cells, such as umbilical cord blood hematopoietic stem cells.

[0037] The matrix of the cell cryopreservation protection solution provided by the present invention can be selected from any matrix for cell cryopreservation in the art, including but not limited to one or more of PBS, serum, physiological saline, compound electrolyte injection, compound electrolyte injection (II) and cell culture medium.

[0038] The cell culture medium may be any culture medium acceptable in the art, including but not limited to DMEM, DMEM / F12, RPMI1640, Iscove modified Dulbeco medium (IMDM), αEagle minimal essential medium (αMEM), and one or more of pre-made nutrient supplements (such as transferrin and insulin, etc.) added alone or mixed.

[0039] In an optional embodiment, the matrix is ​​a compound electrolyte injection, which contains 5.26 g of sodium chloride, 5.02 g of sodium gluconate, 3.68 g of sodium acetate, 0.37 g of potassium chloride and 0.30 g of magnesium chloride per 1000 mL.

[0040] Based on the cell cryopreservation protection solution provided in the first aspect, it can be confirmed that gastrodin can replace human serum albumin in traditional cell cryopreservation protection solution, indicating that gastrodin helps maintain cell viability during cell cryopreservation. Based on this discovery, the second aspect also provides the use of gastrodin in the preparation of cell cryopreservation reagents.

[0041] The cell freezing reagent in this application may also contain optional components with cell freezing protection in the art, including but not limited to DMSO, serum (such as fetal bovine serum), sugars (such as but not limited to glucose, glucose polymers such as dextran, raffinose and trehalose), alcohols (such as but not limited to mannitol, polyethylene glycol and 1,2-propylene glycol), amino acids, cytokines and one or more of salts.

[0042] In an optional embodiment, the working concentration of the gastrodin in the cell freezing reagent is 200nM to 100μM, for example, but not limited to 200, 300, 500 or 800nM, or 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100μM.

[0043] In an optional embodiment, the cell freezing reagent is used to freeze hematopoietic stem cells.

[0044] In an optional embodiment, the hematopoietic stem cells include umbilical cord blood hematopoietic stem cells.

[0045] In a third aspect, a cell cryopreservation method is also provided, the cell cryopreservation method comprising freezing cells using the cell cryopreservation protection solution of the first aspect. The freezing procedure of the freezing method can select a general freezing procedure in the art, for example, freezing according to the basic principle of "slow freezing and fast thawing".

[0046] In an optional embodiment, the freezing method includes precooling the cell freezing protection solution at 2-8°C before use.

[0047] In an optional embodiment, the freezing method includes placing the cells to be frozen from which the culture medium has been removed in a pre-cooled cell cryopreservation protection solution, and then using a programmed cooling box to perform programmed cooling in an environment of -80°C for 12 to 24 hours and transferring the cells to a liquid nitrogen tank for long-term liquid phase storage.

[0048] In an alternative embodiment, the cells to be cryopreserved include hematopoietic stem cells.

[0049] In an optional embodiment, the hematopoietic stem cells include umbilical cord blood hematopoietic stem cells.

[0050] The present invention is further described below by means of specific examples. However, it should be understood that these examples are only used for more detailed description and should not be construed as limiting the present invention in any form.

[0051] The methods for preparing reagents and freezing and thawing cells in the following embodiments and effect examples are as follows:

[0052] 1. Main reagents:

[0053] Compound electrolyte injection: Shanghai Baxter Medical Products Co., Ltd.;

[0054] DMSO (Dimethyl sulfoxide, DMSO): Sigma, D4540-100ML;

[0055] Gastrodin: MedChemExpress, HY-N0115.

[0056] 2. Cell cryopreservation: According to the counting results of the harvested cells and the expected freezing density and volume, collect the required cells, centrifuge at 400g for 10 minutes, increase the speed to 9 and decrease the speed to 3, and remove as much original culture medium as possible; pre-cool the program cooling box at 2-8℃ in advance, pre-warm the cryopreservation protection solution at 2-8℃ for use, resuspend the cells in the cryopreservation protection solution and transfer them to a 2mL cryopreservation tube, and mix gently; before freezing, place the cell suspension at 2-8℃ for 10 minutes, then place it in a program cooling box, freeze it at -80℃ overnight, and then transfer it to a liquid nitrogen tank for long-term liquid phase storage.

[0057] 3. Cell recovery: Precool the programmed cooling box in a -80℃ refrigerator half a day in advance, preheat the water bath to 37℃, take out the cells from the liquid nitrogen, and transfer the cells in the cooling box. Thaw the cells quickly in the water bath until only tiny ice cubes (about 1mm in diameter) are visible in the cryopreservation tube, then immediately take out the cryopreservation tube (the temperature should not exceed 4℃), and disinfect with 75% alcohol. Gently mix the cells in the safety cabinet, add all the cells to about 10 times the volume of the culture medium, dilute and mix, count and use for subsequent detection.

[0058] Example 1

[0059] This embodiment provides a cell cryopreservation protection solution, which uses a compound electrolyte injection solution as a matrix, has a DMSO content of 2% v / v, and is added with 200 nM gastrodin to prepare the required cell cryopreservation protection solution, which is prepared on the spot and stored at 4° C. before use.

[0060] Example 2

[0061] This embodiment provides a cell cryopreservation protection solution, which uses a compound electrolyte injection solution as a matrix, has a DMSO content of 2% v / v, and is added with 1 μM gastrodin to prepare the required cell cryopreservation protection solution, which is prepared on the spot and stored at 4° C. before use.

[0062] Example 3

[0063] This embodiment provides a cell cryopreservation protection solution, which uses a compound electrolyte injection solution as a matrix, has a DMSO content of 2% v / v, and is added with 5 μM gastrodin to prepare the required cell cryopreservation protection solution, which is prepared on the spot and stored at 4° C. before use.

[0064] Example 4

[0065] This embodiment provides a cell cryopreservation protection solution, which uses a compound electrolyte injection solution as a matrix, has a DMSO content of 2% v / v, and is added with 20 μM gastrodin to prepare the required cell cryopreservation protection solution, which is prepared on the spot and stored at 4° C. before use.

[0066] Example 5

[0067] This embodiment provides a cell cryopreservation protection solution, which uses a compound electrolyte injection solution as a matrix, has a DMSO content of 2% v / v, and is added with 100 μM gastrodin to prepare the required cell cryopreservation protection solution, which is prepared on the spot and stored at 4° C. before use.

[0068] Example 6

[0069] This embodiment provides a cell cryopreservation protection solution, which uses a compound electrolyte injection solution as a matrix, has a DMSO content of 5% v / v, and is added with 200 nM gastrodin to prepare the required cell cryopreservation protection solution, which is prepared on the spot and stored at 4° C. before use.

[0070] Example 7

[0071] This embodiment provides a cell cryopreservation protection solution, which uses a compound electrolyte injection solution as a matrix, has a DMSO content of 5% v / v, and is added with 1 μM gastrodin to prepare the required cell cryopreservation protection solution, which is prepared on the spot and stored at 4° C. before use.

[0072] Example 8

[0073] This embodiment provides a cell cryopreservation protection solution, which uses a compound electrolyte injection solution as a matrix, has a DMSO content of 5% v / v, and is added with 5 μM gastrodin to prepare the required cell cryopreservation protection solution, which is prepared on the spot and stored at 4° C. before use.

[0074] Example 9

[0075] This embodiment provides a cell cryopreservation protection solution, which uses a compound electrolyte injection solution as a matrix, has a DMSO content of 5% v / v, and is added with 20 μM gastrodin to prepare the required cell cryopreservation protection solution, which is prepared on the spot and stored at 4° C. before use.

[0076] Example 10

[0077] This embodiment provides a cell cryopreservation protection solution, which uses a compound electrolyte injection solution as a matrix, has a DMSO content of 5% v / v, and is added with 100 μM gastrodin to prepare the required cell cryopreservation protection solution, which is prepared on the spot and stored at 4° C. before use.

[0078] Comparative Example 1

[0079] This comparative example provides a cell cryopreservation protection solution, which uses the compound electrolyte injection solution as a matrix and a DMSO content of 10% v / v to prepare the required cell cryopreservation protection solution, which is prepared on the spot and stored at 4° C. before use.

[0080] Comparative Example 2

[0081] This comparative example provides a cell cryopreservation protection solution, which uses the compound electrolyte injection solution as a matrix and is prepared with a DMSO content of 5% v / v to prepare the required cell cryopreservation protection solution, which is prepared on the spot and stored at 4° C. before use.

[0082] Comparative Example 3

[0083] This comparative example provides a cell cryopreservation protection solution, which uses a compound electrolyte injection solution as a matrix and a DMSO content of 2% v / v to prepare the required cell cryopreservation protection solution, which is prepared on the spot and stored at 4° C. before use.

[0084] Comparative Example 4

[0085] This comparative example provides a cell cryopreservation protection solution, which uses a compound electrolyte injection solution as a matrix, has a DMSO content of 5% v / v, and is added with 5% w / v HSA to prepare the required cell cryopreservation protection solution, which is prepared on the spot and stored at 4° C. before use.

[0086] Comparative Example 5

[0087] This comparative example provides a cell cryopreservation protection solution, which uses a compound electrolyte injection solution as a matrix, has a DMSO content of 2% v / v, and is added with 5% w / v HSA to prepare the required cell cryopreservation protection solution, which is prepared on the spot and stored at 4° C. before use.

[0088] Effect Example 1

[0089] Comparing the cryopreservation effects of cell cryopreservation protection solutions with different DMSO concentrations (Comparative Examples 1 to 3), it was found that the viability of cells cryopreserved with 10% v / v DMSO after recovery was higher than that of cells cryopreserved with 2% v / v and 5% v / v DMSO, while there was no significant difference in the purity of CD34+ cells. Although there was no statistical difference in CFU formation ability, the CFU formation ability showed a downward trend as the DMSO concentration decreased. The experimental results are as follows Figure 1 shown.

[0090] Effect Example 2

[0091] In order to reduce the content of DMSO in the cryopreservation protection solution, other protective ingredients can be added to maintain the vitality and function of the cells. The effects of different DMSO concentrations on the cryopreservation and resuscitation effects were compared (Comparative Examples 1 to 3). The results showed that compared with 5% v / v DMSO (Comparative Example 2), the cryopreservation and resuscitation effect of 2% v / v DMSO (Comparative Example 3) was poor, and the cell viability and recovery rate (total nucleated cells and CD34+ cells) were lower than 5% v / v DMSO (Comparative Example 2); but after adding 5% v / v HSA (Comparative Examples 4 and 5), the cell viability and recovery rate (total nucleated cells and CD34+ cells) were significantly improved ( Figure 2 ). This indicates that the cell protection and cryopreservation recovery effects can be further enhanced by adding 5% v / v HSA.

[0092] Effect Example 3

[0093] Gastrodin is the main active ingredient of the traditional Chinese medicine Gastrodia elata, has anti-inflammatory effects, and is often used in the research and treatment of dizziness, epilepsy, stroke and dementia. Comparison of the cryopreservation protection solutions provided in Examples 1 to 5, Comparative Example 3 and Comparative Example 5 revealed that the cryopreservation and resuscitation effect can be improved by adding gastrodin under the premise of reducing the DMSO content. The results showed that the viability was improved after adding gastrodin to 2% v / v DMSO; after adding 200nM gastrodin under the condition of 2% v / v DMSO (Comparative Example 3) (Example 1), the viability and recovery rate (total nucleated cell recovery rate and CD34+ cell recovery rate) of cells after cryopreservation and resuscitation were significantly improved, the purity of CD34+ cells did not change significantly, and the level of cell apoptosis was also significantly reduced ( Figure 3 Although the recovery rate is slightly lower than that of the cryopreservation protection solution of 2% v / v DMSO plus 5% v / v HSA (Comparative Example 5), it also shows the protective effect of gastrodin in the cryopreservation and recovery of umbilical cord blood hematopoietic stem cells.

[0094] Effect Example 4

[0095] Since HSA is a blood product, it has the defects of large batch differences, unstable delivery time, and possible allergic and other inflammatory reactions. We continued to compare the cryopreservation effects of adding different concentrations of gastrodin (Examples 6-10) to replace HSA under 5% v / v DMSO. The results showed that when 1 μM gastrodin was added to 5% v / v DMSO instead of HSA, the cell viability and recovery rate after cryopreservation and recovery were significantly improved, and the level of cell apoptosis was significantly reduced. The effects were comparable to or even better than those of the classic 10% DMSO (Comparative Example 1), and there was no significant change in the purity of CD34+ cells ( Figure 4). The protective effect of gastrodin in the cryopreservation and resuscitation of umbilical cord blood hematopoietic stem cells was further demonstrated. The amount of DMSO can be reduced by adding gastrodin at an appropriate concentration, and HSA can be replaced when the DMSO concentration is 5% v / v.

[0096] Effect Example 5

[0097] The colony-forming capacity (CFU) of hematopoietic stem cells is the gold standard for in vitro functional testing. This protocol further compares the CFU formation capacity of cells after recovery by adding cryopreservation protection solutions prepared with different concentrations of gastrodin under different DMSO concentrations (Comparative Example 3, Comparative Example 5, Example 1, Example 5, Comparative Example 2, Comparative Example 4, Example 6, Example 10 and Comparative Example 1). The results show that ( Figure 5 ) Under 5% v / v DMSO conditions, the CFU formation ability of hematopoietic stem cells in cryopreservation protection solutions with different concentrations of gastrodin was not significantly different from that in the classic 10% v / v DMSO cryopreservation protection solution. Under 2% v / v DMSO conditions, there was no significant difference in the CFU formation ability of each group of cryopreservation protection solutions with different concentrations of gastrodin. In summary, it can be shown that 2%-5% v / v DMSO with the addition of a specific concentration of gastrodin can maintain the state and differentiation potential of hematopoietic stem cells well. Gastrodin can replace HSA and reduce the amount of DMSO used.

[0098] The detection method of the cell freezing effect in the above effect example is as follows:

[0099] (1) Cell counting: Count the cells before and after freezing. Mix the cell suspension to be counted with AOPI staining reagent (CountStar, catalog number: RE010213) in a 1:1 ratio and add to the cell counting plate. Count the cells using a CountStar cell counter to obtain the cell viability and number before and after freezing.

[0100] (2) Cell apoptosis detection: Cell apoptosis detection was performed using the PE-coupled Annexin-V apoptosis detection kit (BD, catalog number: 559763). Take 0.1M of cells to be detected and wash the cells with 1mL of 4-degree pre-cooled cell staining buffer (Biolegend, catalog number: 420201). Centrifuge at 300g for 5min, with an increase speed of 9 and a decrease speed of 3. After discarding the supernatant, resuspend and mix the cells with 100μL of 1x binding buffer according to the instructions, add AnnexinⅤ and 7-AAD reagents respectively, incubate at room temperature in the dark for 15min, add 400μL of 1x binding buffer and mix the cells for detection on a flow cytometer (BD FACS Celesta).

[0101] (3) Cell phenotype detection: Cell phenotype detection is performed by incubating labeled cells with cell surface flow cytometry antibodies and then using a flow cytometer to identify and phenotype the corresponding cells. Take 0.1M cells to be detected (volume about 100μL) and incubate with antibodies CD34 (BD, catalog number: 555821) or CD3 (Beijing Kuangbo, catalog number: Z6410048-100T), CD56 (Beijing Kuangbo, catalog number: A6803), CD16 (Beijing Kuangbo, catalog number: A6322) according to the antibody instructions. After incubation, wash the cells with 1mL PBS, centrifuge at 300g for 5min to remove the supernatant, and resuspend the cells in 100μL PBS for flow cytometer detection.

[0102] (4) CFU culture: Perform CFU detection culture on CD34+ cells before and after cryopreservation (Day 14). Prepare the cell suspension required for CFU according to the cell count, add the required cell suspension to methylcellulose culture medium according to the instructions (R&D, CAS: HSC003), inoculate in a six-well plate at a seeding density of 2000 cells / well, and set up 2 replicate wells. Add physiological saline to the blank wells, seal the six-well plate with medical tape, maintain the humidity in the well plate, and place it in an incubator. Take pictures and count after 10 to 14 days of culture.

[0103] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A cell cryopreservation protection solution, characterized in that: Contains 2-5% v / v dimethyl sulfoxide and 200nM-100μM gastrodin.

2. The cell cryopreservation protection solution according to claim 1, characterized in that: Contains 200nM~5μM gastrodin.

3. The cell cryopreservation protection solution according to claim 1 or 2, characterized in that: Contains 5% v / v dimethyl sulfoxide.

4. The cell cryopreservation protection solution according to claim 3, characterized in that: Contains 1 μM gastrodin.

5. The cell cryopreservation protection solution according to any one of claims 1 to 4, characterized in that: The matrix of the cell cryopreservation protection solution includes one or more of PBS, serum, physiological saline, compound electrolyte injection, compound electrolyte injection (II) and cell culture medium.

6. The cell cryopreservation protection solution according to claim 5, characterized in that: The matrix is ​​a compound electrolyte injection.

7. Application of Gastrodin in the preparation of cell cryopreservation reagents.

8. The use according to claim 7, characterized in that: The working concentration of the gastrodin in the cell freezing reagent is 200nM to 1μM.

9. The use according to claim 7, characterized in that: The cell freezing reagent is used for freezing hematopoietic stem cells; Optionally, the hematopoietic stem cells include umbilical cord blood hematopoietic stem cells.

10. A cell freezing method, characterized in that: The method comprises freezing cells using the cell cryopreservation protection solution according to any one of claims 1 to 6; Optionally, the cells include hematopoietic stem cells; Optionally, the hematopoietic stem cells include umbilical cord blood hematopoietic stem cells.

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