CN107494517B - Serum-free frozen stock solution and application thereof in frozen mesenchymal stem cells - Google Patents

Abstract

A serum-free frozen stock solution comprises 8v/v% -15 v/v% DMSO, 85v/v% -92 v/v% DMEM basal medium and nutrient additives, wherein the nutrient additives contain fibroblast growth factors, insulin, growth hormone, transferrin, bone morphogenetic protein 4, glutamine, sodium pyruvate, beta-mercaptoethanol, human epidermal growth factors, sodium selenite, and various amino acids and vitamins. The frozen stock solution does not contain animal-derived serum, avoids pollution and allergen risks, and has higher clinical safety. The serum-free frozen stock solution is suitable for freezing and storing human placenta-derived, umbilical cord-derived and umbilical blood-derived mesenchymal stem cells, and compared with the conventional frozen stock solution with serum, the mesenchymal stem cells produced by freezing and storing the human placenta-derived, umbilical cord-derived and umbilical blood-derived mesenchymal stem cells in the serum-free frozen stock solution have high cell survival rate after recovery, good adherent growth state and good biological characteristics.

Description

Serum-free frozen stock solution and application thereof in frozen mesenchymal stem cells

Technical Field

The invention belongs to the field of cell biology, and particularly relates to a serum-free cryopreservation liquid for mesenchymal stem cells in a perinatal period and a cryopreservation method thereof.

Background

Mesenchymal Stem Cells (MSCs) are a type of Stem cell with self-proliferating ability and multi-directional differentiation potential, and can be differentiated into osteoblasts, chondroblasts, adipocytes, nerve Cells, muscle Cells, etc. under specific conditions. At first, MSCs were isolated from bone marrow, and then, it was found that a small amount of MSCs was present in various tissues such as liver, skin, muscle, cartilage, skin, and peripheral blood. The latest research shows that the MSCs have the functions of supporting hematopoiesis, regulating immunologic function, promoting damaged tissue repair, preventing and treating graft-versus-host disease and the like, and become important seed cells and potential treatment tools in the fields of regenerative medicine and tissue engineering. As the earliest source of MSCs, the amount of MSCs in bone marrow is very small (about 0.001% to 0.01% of the total number of nucleated cells), the number and differentiation potential of MSCs are reduced under aging and disease conditions, the stability of seed cell quality cannot be guaranteed, and bone marrow collection is a traumatic process with high acquisition cost. In contrast, perinatal Mesenchymal Stem Cells (P-MSCs) provide a new important source for scientific research and clinical application of MSCs.

The P-MSCs are mesenchymal stem cells which take birth-related tissues as sources, mainly comprise umbilical cord blood, umbilical cord and placenta-derived mesenchymal stem cells, have the common characteristics of MSCs, such as multidirectional differentiation potential, low immunogenicity, paracrine action and the like, and have the following advantages compared with spinal cords and other tissue sources: firstly, the cell content is rich, and the preparation method is simple; secondly, the acquisition process is non-invasive operation, no harm is caused to a donor, and meanwhile, the risk of bacterial and viral infection is lower than that of mesenchymal stem cells from adult tissues; moreover, because the cord blood, the umbilical cord and the placenta are originally biological wastes, the umbilical cord blood, the umbilical cord and the placenta are used as a mesenchymal stem cell source system, so that the mesenchymal stem cell source system is changed into valuables, and the moral ethical problem is not caused, and the umbilical cord blood, the umbilical cord and the placenta are particularly suitable for developing industrialized operation.

During 1999 to 2017, the proportion of research projects of MSCs is 4/5 in 6000 clinical studies of stem cells registered worldwide. In 13 stem cell clinical research projects which are recorded by Wei Ji Commission in China by 2016, 7 stem cell researches are carried out on MSCs, and 6 stem cell researches are carried out on P-MSCs, wherein the researches comprise the researches on the prevention and treatment effects of major diseases such as lupus nephritis, infantile cerebral palsy, spinal cord injury, acute myocardial infarction, heart failure and the like, and the suggestion that mesenchymal stem cells become a large research hotspot after embryonic stem cells and hematopoietic stem cells, has huge clinical value and wide application prospect. Therefore, in order to ensure effective development of clinical research on the MSCs in the middle and downstream and safely promote clinical application of the MSCs, the safety and quality of the MSCs seed cells become more important.

Cell cryopreservation is an important means for preserving cells, and the cells can be removed from a growth state within a certain period of time to preserve the cell characteristics, and can be revived when needed for experimental research or clinical application. Therefore, cell cryopreservation technology, especially cryopreservation solution, is important for maintaining the activity of cryopreserved cells and reducing cell damage. However, the existing MSCs frozen stock solution and method have many disadvantages, such as: contain a high proportion of Fetal Bovine Serum (FBS), which may increase the risk of foreign proteins causing rejection reactions in clinical studies and applications; in addition, the serum-free freezing method adopts serum substitutes, so that the reagent is expensive and the application range is limited. Such as: chinese patent application 201610936192.6 discloses a frozen stock solution of adipose mesenchymal stem cells, which consists of RPMI-1640 culture medium, FBS (2-5%, v/v), sulfolane, ethanol, ethylene glycol, propylene glycol, tween, glycerol, polyvinylpyrrolidone, agar powder, trehalose, penicillin, and cryoprotectant hydroxyethyl starch or dextran. Chinese patent application CN201510718538.0 discloses a cryopreservation solution and a cryopreservation method for placental amniotic mesenchymal stem cells, wherein the cryopreservation solution consists of DMSO, a basic culture medium and FBS (10-50%, v/v). The mesenchymal stem cell frozen stock solutions of the methods all contain FBS, and exogenous animal serum is introduced.

Disclosure of Invention

The invention aims to provide a frozen stock solution, which does not contain animal serum, has definite and easily obtained components, does not influence the dryness and activity of cells during freezing and can avoid potential clinical application risks caused by the animal serum in the frozen stock solution.

The invention also aims to provide a preparation method of the frozen stock solution.

Still another object of the present invention is to provide a use of a cryopreservation solution in cryopreservation of mesenchymal stem cells, such as: umbilical cord-derived, placenta-derived, and umbilical blood-derived mesenchymal stem cells, and the like.

The invention further aims to provide application of the cryopreservation liquid in cryopreservation of the mesenchymal stem cells. The cryopreservation method of the serum-free cryopreservation solution.

The frozen stock solution provided by the invention does not contain serum, and comprises 8v/v% -15 v/v% of DMSO, 85v/v% -92 v/v% of DMEM basic culture medium and nutrient additives.

The frozen stock solution provided by the invention further comprises: fibroblast Growth Factor (bFGF) 4.5-27 ng/mL, insulin 4.5-18 μ g/mL, growth hormone 2.7-3.5 ng/mL, transferrin (TF) 0.045-0.18 μ g/mL, bone Morphogenetic Protein 4 (Bone Morphogenetic Protein-4, BMP-4) 0.09-0.12 ng/mL, glutamine 150-400 mg/L, sodium pyruvate 45-70 mg/L, beta-mercaptoethanol 23-60 μ M, human Epidermal Growth Factor (Human Epidermal Growth Factor, hEGF) 10-20 ng/mL, sodium selenite 15-45 μ M, platelet Growth Factor (Growth Factor, human Factor) 4-18 mL. These components are used in the present invention either individually or in combination.

The frozen stock solution provided by the invention further comprises: vitamin B12.18 mg/L-0.9 mg/L, vitamin C0.1 mg/L-0.25 mg/L, vitamin B6 μ g/L-90 μ g/L, vitamin B2.2 mg/L-0.6 mg/L, glutamic acid 9.6 mg/L-16.8 mg/L, alanine 9.2 mg/L-21.3 mg/L, glycine 5.06 mg/L-7.2 mg/L, aspartic acid 8.6 mg/L-14.7 mg/L, proline 6.9 mg/L-13.2 mg/L and serine 6.0 mg/L-9.8 mg/L. These components are used in the present invention either individually or in combination.

In the frozen stock solution, the concentration of DMSO is preferably 9v/v% to 12v/v%, such as: but are not limited to 9v/v%, 10v/v%, 11v/v%, and 12v/v%.

In frozen stock solution, low-sugar DMEM basic culture medium (L-DMEM) is preferably selected, the concentration is 88v/v% -91 v/v%, for example: but are not limited to 88v/v%, 89v/v%, 90v/v%, and 91v/v%.

In the frozen stock solution, pH 7.0 to 8.5 is preferably selected.

The freezing medium provided by the invention comprises 10v/v% DMSO and 90v/v% DMEM basal medium.

The cryopreservation solution provided by the invention is suitable for long-term cryopreservation of umbilical cord-derived, placenta-derived and umbilical blood-derived mesenchymal stem cells, particularly for mesenchymal stem cells in peripheral production, and the density of the mesenchymal stem cells cryopreserved in per milliliter of the cryopreservation solution is 0.5-1 multiplied by 10 ⁷ And (4) storing the individual cells at-80 ℃ overnight, and transferring the cells into liquid nitrogen for long-term cryopreservation. The cell survival rate is high through the resuscitation verification after freezing storage.

A method for cryopreserving P-MSCs by using the cryopreservation liquid comprises the following steps:

firstly, digesting adherent cells with 0.25% trypsin preheated in a 37 ℃ water bath, terminating digestion with DMEM complete medium, gently blowing the cells (e.g., gently blowing the cells with a 3mL sterile dropper), collecting the cells in a container (e.g., a 15mL centrifuge tube), centrifuging (e.g., 1,500rpm,5 min), and discarding the supernatant;

then, the cell sediment is added with the serum-free freezing medium (the dosage is 0.5-1.0 multiplied by 10) ⁷ Adding 1mL of the frozen stock solution into each living cell), gently blowing and uniformly mixing, transferring into a sterile freezing tube, and sealing.

Finally, the cells were frozen overnight at-80 ℃ and then transferred to liquid nitrogen for long term cryopreservation.

The technical scheme of the invention has the following beneficial effects:

the cryopreservation liquid provided by the invention is a serum-free cryopreservation liquid, and is obtained by optimizing a cryopreservation protective agent, growth hormone, nutritional components and the using amount thereof, so that animal serum is not introduced in the cryopreservation process of mesenchymal stem cells (especially P-MSCs), and the defects of the conventional serum-containing cryopreservation liquid are avoided, such as: cytotoxic substances contained in animal serum, such as polyamine oxidase and the like, have toxic effects on cryopreserved cells; the quality of the frozen stock solution is unstable due to different individuals, producing areas, batch numbers and the like of the exogenous serum; exogenous animal proteins increase clinical allergen risk.

The cryopreservation solution provided by the invention has clear components, is lower in cost compared with the cryopreservation solution containing animal serum or a commercial serum substitute, can well keep the activity and surface antigen characteristics of mesenchymal stem cells, and is high in cell viability rate and good in wall adhesion effect after recovery.

The cryopreservation liquid provided by the invention is suitable for cryopreservation of mesenchymal stem cells (especially P-MSCs), and has the advantages of simple cryopreservation method and good cryopreservation effect.

Drawings

FIG. 1 is a morphological observation comparison of frozen cells in serum-free and serum-containing frozen stock solutions after recovery; wherein, the picture A1, the picture A2, the picture A3 and the picture A4 are respectively the morphological observation results of the culture 1 st, 2 nd, 3 rd and 4 th days after the recovery of the serum-free frozen medium group cells, and the picture B1, the picture B2, the picture B3 and the picture B4 are respectively the morphological observation results of the culture 1 st, 2 nd, 3 rd and 4 th days after the recovery of the serum-containing frozen medium group cells;

FIG. 2 is a flow chart of the surface labeled antigen of the thawed serum-free frozen cells of the present invention;

FIG. 3 is a surface labeled antigen obtained by flow detection after the recovery of frozen cells in a frozen stock solution containing serum.

Detailed Description

The technical scheme of the invention is described in detail in the following with reference to the accompanying drawings. Although the present invention has been described in detail with reference to the preferred embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the spirit and scope of the invention as defined in the appended claims.

Example 1 cryopreservation of umbilical cord mesenchymal stem cells in serum-free mesenchymal stem cell cryopreservation solution

The umbilical cord of the newborn is taken as a source of the mesenchymal stem cells, the preparation and primary culture of the umbilical cord mesenchymal stem cells are completed in 48h under the aseptic condition, and then subculture is carried out. Taking P3-generation umbilical cord MSCs, discarding culture solution after the cell fusion degree reaches 80-90%, adding 5mL PBS buffer solution to gently wash cells, discarding washing solution, digesting the cells with 0.25% trypsin preheated at 37 ℃, wherein the trypsin is used for just infiltrating the bottom of a culture container, such as:

when the cells are observed to be transparent spherical under a microscope and do not adhere to the wall any more, DMEM complete culture medium is added immediately, and the dosage is as follows: />

Then, gently blowing off cells by using an aseptic dropper, transferring the cell suspension into a 15mL centrifuge tube, adding 3 mL-5 mL of DMEM complete culture medium into an original culture container, washing once, combining washing solutions, adding into the centrifuge tube, centrifuging at 1,500rpm for 5min at room temperature, and discarding the supernatant; each (0.5-1.0) x 10 ⁷ Adding 1mL of serum-free cryopreservation liquid into each cell, gently blowing and uniformly mixing the cells by using a pipette, adding 1mL of cells into each cryopreservation tube, sealing the tube opening by using a sealing film, and marking cryopreservation information by using a tube body; the frozen tube was placed in a-80 ℃ freezer overnight and transferred to a liquid nitrogen tank for cryopreservation.

The P-MSCs serum-free freezing medium comprises the following components: in the form of a final concentration meter,

DMSO：12％(v/v)；

DMEM basal medium: 88% (v/v);

bFGF：10ng/mL；

insulin: 6.8 mu g/mL;

growth hormone: 2.7ng/mL;

TF：0.08μg/mL；

BMP-4：0.1ng/mL；

(ii) glutamine: 180mg/L;

sodium pyruvate: 55mg/L;

beta-mercaptoethanol: 30 mu M;

hEGF：12ng/mL；

sodium selenite: 20 mu M;

PGF：8.5ng/mL；

glutamic acid: 12.8mg/L, alanine: 16.8mg/L, glycine: 6.2mg/L, aspartic acid: 11.5mg/L, proline: 9.2mg/L, serine: 7.7mg/L;

vitamin B12:0.36mg/L, vitamin C:0.15mg/L, vitamin B6:35 μ g/L, vitamin B2:0.6mg/L.

Example 2 cryopreservation of umbilical cord mesenchymal stem cells with serum-free mesenchymal stem cell cryopreservation solution

MSCs isolated from umbilical cord were cryopreserved as described in example 1.

The serum-free freezing medium comprises the following components: in order to obtain the final concentration of the concentrated solution,

DMSO：9％(v/v)；

DMEM basal medium: 91% (v/v);

bFGF：25ng/mL；

insulin: 16.5. Mu.g/mL;

growth hormone: 3.0ng/mL;

TF：0.15μg/mL；

BMP-4：0.12ng/mL；

glutamine (b): 350mg/L;

sodium pyruvate: 65mg/L;

beta-mercaptoethanol: 50 mu M;

hEGF：15ng/mL；

sodium selenite: 42 mu M;

PGF：16ng/mL；

glutamic acid: 15.6mg/L, alanine: 10.5mg/L, glycine: 7.0mg/L, aspartic acid: 9.5mg/L, proline: 13.0mg/L, serine: 9.5mg/L;

vitamin B12:0.82mg/L, vitamin C:0.25mg/L, vitamin B6:65 μ g/L, vitamin B2:0.25mg/L.

Example 3 cryopreservation of umbilical cord mesenchymal stem cells with serum-free mesenchymal stem cell cryopreservation solution

Umbilical cord MSCs were cryopreserved as described in example 1.

The serum-free freezing medium comprises the following components: in the form of a final concentration meter,

DMSO：10％(v/v)；

DMEM basal medium: 90% (v/v);

bFGF：9ng/mL；

insulin: 9 mu g/mL;

growth hormone: 2.7ng/mL;

TF：0.09μg/mL；

BMP-4：0.09ng/mL；

(ii) glutamine: 270mg/L;

sodium pyruvate: 50mg/L;

beta-mercaptoethanol: 35 mu M;

hEGF：9ng/mL；

sodium selenite: 15 mu M;

PGF：10.6ng/mL；

glutamic acid: 13.2mg/L, alanine: 15.3mg/L, glycine: 6.1mg/L, aspartic acid: 11.7mg/L, proline: 10mg/L, serine: 7.9mg/L;

vitamin B12:0.54mg/L, vitamin C:0.18mg/L, vitamin B6:50 μ g/L, vitamin B2:0.4mg/L.

Example 4 cryopreservation of umbilical cord mesenchymal stem cells with serum-free mesenchymal stem cell cryopreservation solution

And (3) taking the placenta of the newborn as a source of the mesenchymal stem cells, completing the preparation and primary culture of the mesenchymal stem cells of the placenta under the aseptic condition within 48h, and then carrying out subculture. Cell cryopreservation was performed using P3-substituted placental MSCs as described in example 1.

The serum-free freezing medium used was the same as in example 1.

Example 5 cryopreservation of umbilical cord mesenchymal Stem cells with serum-free mesenchymal Stem cell cryopreservation solution

The preparation and primary culture of the placenta mesenchymal stem cells are finished in 48h under aseptic conditions by taking the placenta of the newborn as the source of the mesenchymal stem cells, and then subculture is carried out. Cell cryopreservation was performed using P3-substituted placental MSCs as described in example 1.

The serum-free freezing medium used was the same as in example 2.

Example 6 cryopreservation of umbilical cord mesenchymal stem cells in serum-free mesenchymal stem cell cryopreservation solution

And (3) taking the placenta of the newborn as a source of the mesenchymal stem cells, completing the preparation and primary culture of the mesenchymal stem cells of the placenta under the aseptic condition within 48h, and then carrying out subculture. Cell cryopreservation was performed using P3-substituted placental MSCs as described in example 1.

The serum-free freezing medium used was the same as in example 3.

Example 7 cryopreservation of umbilical cord mesenchymal Stem cells in serum-containing mesenchymal Stem cell cryopreservation solution

Umbilical cord MSCs were cryopreserved as described in example 1.

The used serum frozen stock solution comprises the following components: in order to obtain the final concentration of the concentrated solution,

DMSO：10％(v/v)；

DMEM basal medium: 60% (v/v);

FBS：30％(v/v)。

example 8 cryopreservation of placental mesenchymal Stem cells in serum-containing mesenchymal Stem cell cryopreservation solution

Placental MSCs were cryopreserved as described in example 4.

The formulation of all serum frozen stocks was the same as in example 7.

Example 9 recovery of mesenchymal Stem cells

After the mesenchymal stem cells of examples 1 to 8 were frozen for three months, the cells were taken out from the liquid nitrogen, shaken back and forth in a constant-temperature water bath at 37 ℃ and thawed within 1 min. Then, the outer wall of the cryopreservation tube was wiped with a 75% alcohol cotton ball, the cells were transferred to a 15mL centrifuge tube, 10mL of complete medium was added, the mixture was mixed, centrifuged at 1,500rpm for 5min, the supernatant was discarded, a proper amount of medium (1 mL to 3 mL) was added to the cell pellet, the mixture was gently blown down, mixed 1: 1 with trypan blue staining solution, and viable cells were counted at 5X 10 (see Table 1) ⁴ -1×10 ⁵ Per mL in

Plating, adding DMEM complete medium, such as: />

Put in CO ₂ In an incubator, 37 ℃ and 5% of CO ₂ And (5) performing conditioned culture. Biological safety with resuscitation operation in ten thousand grade sterile roomThe whole process is carried out in the whole cabinet.

Table 1 comparison of the resuscitative cell viability of frozen cells with serum-free and serum-containing freezing media.

Example 10 morphological Observation after recovery of mesenchymal Stem cells

Freezing and storing the placenta mesenchymal stem cells of the embodiments 4 and 8 for three months, recovering and culturing according to the method of the embodiment 9, replacing the total amount of the cells on the second day of recovery, replacing the total amount of the cells every 2 days, and putting the cells into CO ₂ In an incubator, 37 ℃ and 5% of CO ₂ The cells were cultured under the conditions of adherence and growth observed daily with a phase contrast microscope and photographed (see FIG. 1). The recovery and liquid change operations are carried out in a biological safety cabinet of a ten thousand-level sterile room.

Example 11 flow identification of surface-labeled antigens of mesenchymal Stem cells

After the umbilical cord mesenchymal stem cells of example 1 and example 7 are frozen and stored for three months, the umbilical cord mesenchymal stem cells are recovered and cultured according to the method of example 9, the next day is the total cell volume change solution, the cell healing degree reaches about 80-90%, 0.25% trypsin digestion is carried out, the digestion is stopped by DMEM complete culture medium, 1,500rpm centrifugation is carried out for 5min, the supernatant is discarded, and the cell concentration is adjusted to 1 × 10 by PBS ⁷ and/mL, taking 100 mu L of cells, adding into a flow tube, respectively adding mouse anti-human monoclonal labeled antibodies CD90-PerCP, CD105-PE, CD73-APC, CD14-/CD19-/CD34-/CD45-FITC, taking mouse anti-human IgGl-FITC, igG1-PE, igG1-APC and IgG1-PerCP as isotype controls, incubating for 20min at room temperature in a dark place, carrying out sample loading detection on a flow cytometer, and analyzing and determining results by FlowJo software (see figure 2 and figure 3).

In the examples, the formulation of DMEM complete medium was: based on DMEM basal medium, the medium also contains 10% (v/v) FBS, 100kU/L penicillin and 100mg/L streptomycin at final concentration.

In the examples, it will be appreciated by those skilled in the art that the cryopreservation solution used in the comparative examples is a classic serum cell cryopreservation solution formulation.

The sources of the main reagents of the above examples are as follows:

0.25% trypsin: the enzyme solution with the mass-to-volume ratio of 0.25% is prepared by 400mL PBS according to the specification of 1000mg of Gibco company in America;

DMEM medium, FBS: gibco, inc., 500mL Specification, USA;

DMSO, DMSO: sigma, USA, 10mL size.

Claims (3)

1. A method for freezing mesenchymal stem cells is characterized in that the mesenchymal stem cells are taken, trypsinized, centrifuged, supernatant is discarded, freezing stock solution is added, after being stored overnight at minus 80 ℃, the mesenchymal stem cells are transferred into liquid nitrogen for long-term freezing and stored, and the density of the mesenchymal stem cells frozen in each milliliter of the freezing stock solution is 0.5 to 1.0 multiplied by 10 ⁷ A cell;

the freezing solution comprises the following components: 8v/v% -15 v/v% DMSO, 85v/v% -92 v/v% DMEM, and

4.5-27 ng/mL fibroblast growth factor, 4.5-18 μ g/mL insulin, 2.7-3.5 ng/mL growth hormone, 0.045-0.18 μ g/mL transferrin, 0.09-0.12 ng/mL bone morphogenetic protein 4, 150-400 mg/L glutamine, 45-70 mg/L sodium pyruvate, 23-60 μ M beta-mercaptoethanol, 10-20 ng/mL human epidermal growth factor, 15-45 μ M sodium selenite, 4.5-18 ng/mL human platelet growth factor, 9.6-16.8 mg/L glutamic acid, 9.2-21.3 mg/L alanine, 5.06-7.2 mg/L glycine, 8.6-14.6 mg/L aspartic acid, 9.6-14.8 mg/L aspartic acid, 9-13 mg/L aspartic acid, and the concentrations of aspartic acid are all in the frozen solution of the concentrations of aspartic acid, i.6-13.13 mg/L, 6-13 mg/L aspartic acid, 6-13.6 mg/L aspartic acid, 6mg/L aspartic acid, 2-13.6 mg/L aspartic acid and 6mg/L aspartic acid;

also comprises one or more of 0.18 mg/L-0.9 mg/L vitamin B12, 0.1 mg/L-0.25 mg/L vitamin C, 10 mug/L-90 mug/L vitamin B6 and 0.2 mg/L-0.6 mg/L vitamin B2, and the concentration of each vitamin is the concentration in the frozen stock solution;

the DMEM culture medium is L-DMEM;

the pH value of the frozen stock solution is 7.0-8.5.

2. The method of cryopreserving mesenchymal stem cells according to claim 1, wherein the mesenchymal stem cells are perinatal mesenchymal stem cells.

3. The cryopreservation method of mesenchymal stem cells according to claim 1, characterized by comprising:

firstly, digesting adherent cells by 0.25% trypsin preheated by 37 ℃ water bath, stopping digestion by a DMEM complete culture medium, slightly blowing and beating the cells, collecting the cells in a container, centrifuging, and discarding supernatant;

then, the freezing medium is added into the cell sediment with the dosage of 0.5-1.0 multiplied by 10 ⁷ Adding 1mL of cryopreservation liquid into each living cell, lightly blowing and uniformly mixing, transferring into a sterile cryopreservation tube, and sealing;

finally, the cells were frozen overnight at-80 ℃ and then transferred to liquid nitrogen for long term cryopreservation.

Images