CN118318828A - Cryopreservation liquid based on liquid-liquid condensed phase and preparation method and application thereof - Google Patents

Abstract

The invention discloses a cryopreservation liquid based on liquid-liquid condensed phase and a preparation method and application thereof. The cryopreservation liquid of the invention does not contain DMSO or DMF, does not contain serum, and maintains good cryopreservation effect. It has good biological safety and does not destroy the connection structure between cells in the tissue. Is suitable for freezing and preserving various cells, and can also preserve simple tissues and small organs.

Description

A cryopreservation solution based on liquid-liquid condensed phase and its preparation method and application

Technical Field

The invention belongs to the technical field of biomedical materials, and specifically relates to a cryopreservation solution and a preparation method and application thereof.

Background technique

As a major advance in life science and medicine in the 21st century, cell therapy and organ transplantation technologies have saved the lives of countless patients. When cell products are cultured in vitro, they will age, mutate, and die during the process of passage. Cryopreservation is an effective way to preserve cells for a long time and maintain their vitality. The current gold standard for clinical use is to achieve cell cryopreservation by adding serum, culture medium, dimethyl sulfoxide (DMSO), glycerol (GCL), dimethylformamide (DMF), etc., through programmed cooling or vitrification.

Regarding DMSO, DMF, etc. contained in the cryopreservation solution, it has been found that they have adverse effects such as causing intracellular protein aggregation, causing different degrees of DNA methylation or demethylation, affecting DNA replication and expression, inducing changes in germ cell genes, causing disorder or loss of cell spindle microtubules, obvious damage to the cytoskeleton, and obvious damage to the intercellular connections of monolayer cells. Therefore, the development of cryopreservation materials that do not contain DMSO and other cytotoxic organic small molecules, have good biocompatibility, are non-toxic, and do not damage tissue structure is an inevitable trend in the development of biological sample cryopreservation.

At the same time, traditional cell freezing fluid contains serum, but the source of serum is unstable, individual differences are large, and the composition is complex and unclear. Therefore, the quality between batches is often unstable, and the state of cell culture is also greatly affected. The development of serum-free freezing preservation fluid can reduce batch differences, ensure the stability of the state of growing cells, and reduce the possibility of unknown effects of unknown components of biological origin on cells.

Summary of the invention

In order to improve the above-mentioned defects of the prior art, the present invention provides a cryopreservation solution, which comprises the following components: an amphiphilic macromolecule (a), a salting-out agent (b) and a basic solution.

According to an embodiment of the present invention, the cryopreservation solution further comprises a protective agent (c).

According to an embodiment of the present invention, the cryopreservation solution is prepared by mixing and layering the amphiphilic macromolecule (a), the salting-out agent (b) and the basic solution.

According to an embodiment of the present invention, the freezing preservation solution is prepared by adding amphiphilic macromolecules (a) and salting-out agent (b) to a base solution to obtain a mixed solution, separating the mixed solution into layers, and then taking the layer rich in amphiphilic macromolecules (a) and adding protective agent (c).

According to an embodiment of the present invention, in the mixed solution, the mass ratio of the amphiphilic macromolecule (a) to the salting-out agent (b) is (10-40): (10-40), preferably (15-30): (15-30), and further (20-30): (20-30).

According to an embodiment of the present invention, the volume ratio of the mixed solution to the layer rich in amphiphilic macromolecules (a) is 100:(50-99), preferably 100:(80-99) or 100:(85-99), for example 100:80 or 100:95.

According to an embodiment of the present invention, the volume ratio of the protective agent (c) to the layer rich in amphiphilic macromolecules (a) is (1-50):(50-99), preferably (5-20):(80-95), for example 20:80 or 5:95.

According to an embodiment of the present invention, the mass volume ratio of the amphiphilic macromolecule (a) and the protective agent (c) is (10-40) g: (2-20) mL, preferably (15-30) g: (5-18) mL, further (20-30) g: (5-15) mL or (20-30) g: (5-10) mL.

Those skilled in the art will appreciate that the layer rich in amphiphilic macromolecules (a) refers to a layer enriched in amphiphilic macromolecules (a) obtained by two-phase separation, and the layer may contain more than 50 wt % of amphiphilic macromolecules (a), preferably more than 60 wt %, and more preferably 60-99 wt %.

According to an embodiment of the present invention, the cryopreservation solution is prepared by the following method:

(1) adding an amphiphilic macromolecule (a) and a salting-out agent (b) to a base solution and mixing to obtain a mixed solution;

(2) After the mixed solution is separated into layers, the layer rich in amphiphilic macromolecules (a) is taken and a protective agent (c) is added.

According to an embodiment of the present invention, the amphiphilic macromolecule (a) comprises at least one of amphiphilic proteins having coexisting hydrophilic/hydrophobic micro-regions and amphiphilic macromolecules having hydrophilic and hydrophobic regions.

Preferably, the amphiphilic proteins in which the hydrophilic/hydrophobic microdomains coexist include at least one of bovine serum albumin, human serum albumin, and the like.

Preferably, the amphiphilic macromolecule having hydrophilic and hydrophobic regions includes at least one of polyethylene glycol (PEG), chondroitin sulfate, hyaluronic acid, chitosan and the like.

According to an embodiment of the present invention, the degree of polymerization of the amphiphilic macromolecule (a) is in the range of 600 to 20,000, preferably 5,000-20,000, more preferably 6,000-20,000, further preferably 6,000-10,000, and most preferably 6,000.

According to an embodiment of the present invention, the amphiphilic macromolecule (a) can be used alone in the cryopreservation solution, or two or more types can be used in combination, but it is preferably used alone.

According to a preferred embodiment of the present invention, in the cryopreservative solution, the amphiphilic macromolecule (a) is selected from at least one of bovine serum albumin (BSA), polyethylene glycol (PEG), and chondroitin sulfate. Preferably, the amphiphilic macromolecule (a) only comprises bovine serum albumin. Most preferably, the amphiphilic macromolecule (a) only comprises polyethylene glycol.

According to an embodiment of the present invention, each 100 mL of the mixed solution contains 10 g to 40 g of the amphiphilic macromolecule (a), preferably 15 g to 30 g, and more preferably 20 g to 30 g.

According to an embodiment of the present invention, the salting-out agent (b) is selected from at least one of sodium citrate, potassium citrate, sodium phosphate, sodium sulfate, sodium chloride, sodium bromide and other salts having a salting-out effect. Preferably, the salting-out agent (b) is selected from at least one of sodium citrate, potassium citrate or sodium chloride.

According to an embodiment of the present invention, every 100 mL of the mixed solution contains 10 g to 40 g of the salting-out agent (b), preferably 15 g to 30 g, and more preferably 20 g to 30 g.

According to an embodiment of the present invention, the basic solution is selected from at least one of the following: an aqueous solution, a PBS buffer or a basic culture medium. Preferably, the basic solution can be a basic culture medium known in the art, such as MEM culture medium.

According to an embodiment of the present invention, the protective agent (c) is selected from polyhydroxy or polyamino substances.

Preferably, the polyhydroxy or polyamino substance is selected from at least one of ethylene glycol (EG), glycerol (GCL), urea, glucose, propylene glycol, 1,3-propylene glycol, isoprene glycol, betaine, etc. Preferably, the polyhydroxy or polyamino substance is selected from at least one of ethylene glycol, urea, glycerol, glucose, betaine; more preferably, selected from at least one of ethylene glycol, urea, glycerol, betaine; most preferably, selected from one of glycerol, betaine or ethylene glycol.

According to an embodiment of the present invention, the cryopreservation solution may further contain other ingredients. Preferably, the other ingredients are selected from one, two or more of antioxidants, buffer solutions, structural stabilizers, humectants, excipients, preservatives, antibiotics, etc. Further, the other ingredients are preferably antioxidants, such as ascorbic acid.

According to an embodiment of the present invention, the pH value of the cryopreservation solution is in the range of 6.0-7.5, preferably 6.5-7.0.

According to an embodiment of the present invention, the cryopreservation solution does not contain at least one of dimethyl sulfoxide (DMSO), dimethylformamide (DMF) and serum.

According to an exemplary embodiment of the present invention, the cryopreservation solution is obtained by the following preparation method:

(1) adding an amphiphilic macromolecule (a), a salting-out agent (b) and a basic solution to obtain a mixed solution, wherein, per 100 mL of the mixed solution, the content of the amphiphilic macromolecule (a) is 10 g to 40 g, the content of the salting-out agent (b) is 10 g to 40 g, and the balance is the basic solution;

(2) After the mixed solution is separated into layers, the layer rich in amphiphilic macromolecules (a) is taken and the protective agent (c) is added to make the volume up to 100 mL.

Preferably, in the mixed solution, the mass ratio of the amphiphilic macromolecule (a) to the salting-out agent (b) is (15-30):(15-30), further (20-30):(20-30).

Preferably, the volume ratio of the mixed solution to the layer rich in amphiphilic macromolecules (a) is 100:(80-99) or 100:(85-99), for example, 100:80 or 100:95.

Preferably, the volume ratio of the protective agent (c) to the layer rich in amphiphilic macromolecules (a) is (5-20):(80-95), for example, 20:80 or 5:95.

Preferably, the mass volume ratio of the amphiphilic macromolecule (a) and the protective agent (c) is (15-30) g: (5-18) mL, further (20-30) g: (5-15) mL or (20-30) g: (5-10) mL.

The present invention also provides a method for preparing the above-mentioned cryopreservation solution, and the preparation method is as follows:

The first step is to add the amphiphilic macromolecule (a), the salting-out agent (b) and the base solution to obtain a mixed solution;

In the second step, after the mixed solution is separated into layers, the layer rich in the amphiphilic macromolecule (a) obtained after separation is taken, and a protective agent (c) is added to obtain the cryopreservation solution.

According to an embodiment of the present invention, the preparation method of the cryopreservation solution is as follows:

The first step is to add the amphiphilic macromolecule (a), the salting-out agent (b) and the base solution to obtain a mixed solution;

In the second step, after the mixed solution is layered, the upper layer is a condensed phase solution, which contains a layer rich in the amphiphilic macromolecule (a), and the lower layer is a condensed phase solution. The upper condensed phase solution is taken and a protective agent (c) is added to obtain the cryopreservation solution.

According to an embodiment of the present invention, the dilute phase solution refers to a solution in which the content of the amphiphilic macromolecule (a) is less than the content of the amphiphilic macromolecule (a) in the condensed phase solution.

According to an embodiment of the present invention, in the first step, per 100 mL of the mixed solution, 10 g to 40 g of the amphiphilic macromolecule (a) is contained, preferably 15 g to 30 g, and more preferably 20 g to 30 g.

According to an embodiment of the present invention, in the first step, per 100 mL of the mixed solution, 10 g to 40 g of the salting-out agent (b) is contained, preferably 15 g to 30 g, and more preferably 20 g to 30 g.

According to an embodiment of the present invention, in the second step, the volume ratio of the condensed phase solution to the protective agent (c) is (80-95):(5-20), for example, 80:20, 95:5.

The present invention also provides a method for freezing cells using the above-mentioned cryopreservation solution, the method comprising freezing cells using the above-mentioned cryopreservation solution.

The present invention also provides the use of the above-mentioned cryopreservation solution, preferably for cryopreservation of cells, tissues and/or organs.

According to an embodiment of the present invention, the cells are selected from cells suitable for slow freezing, preferably, the cells for slow freezing are selected from suspension cells and/or adherent cells of mammals. Further, the adherent cells can be selected from cells in vivo cultured in vitro known in the art, selected from but not limited to normal cells and tumor cells, such as fibroblasts, myocardial and smooth muscle, liver, lung, kidney, breast skin glial cells, endocrine cells, melanocytes and various tumor cells. Further, the suspension cells can be selected from a type of cells known in the art that do not need to rely on supports and can grow in a suspended state in a culture medium, such as lymphocytes and most blood system derived cells, such as T cells, B cells, NK cells, mouse leukemia cells WEHI-3, human leukemia cells K-562, HL-60, etc.

Exemplarily, the cells for slow freezing are selected from at least one of red blood cells, stem cells, T cells, NK cells, and corneal endothelial cells. The stem cells include at least one of embryonic mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, and adipose-derived mesenchymal stem cells.

According to an embodiment of the present invention, the tissue is, for example, at least one selected from corneal monolayer tissue and ovarian tissue.

According to the embodiment of the present invention, the source of the cells in the present invention is not particularly limited, and can be insects, animals, plants, preferably animal cells. Among them, the source of the animal cells includes but is not limited to rats, mice, sheep, cattle, horses, pigs, dogs, cats, rabbits, monkeys, humans, etc.

Beneficial Effects

The present invention provides a cryopreservation solution, which does not contain DMSO, DMF, etc., and does not contain serum, and maintains a good cryopreservation effect. It has good biosafety and does not destroy the connection structure between cells in the tissue. It is suitable for the cryopreservation of various cells and can also preserve simple tissues and small organs.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Schematic diagram of the liquid-liquid phase separation system composed of PEG 6000 and SC system.

Figure 2. Proliferation activity of stem cells after cryopreservation in cryopreservation solution 31 in Experiment 7.

Figure 3. Osteogenic and adipogenic differentiation activities of stem cells after cryopreservation in cryopreservation solution 31 in experiment 8.

Figure 4. The osteogenic and adipogenic differentiation activities of stem cells after cryopreservation in the commercial cryopreservation solution CS10 were tested in Experiment 8.

FIG. 5 . Morphology maintenance of corneal endothelial cells in Example 3.

Figure 6. Metabolism of corneal endothelial cells in Example 3.

Figure 7. Morphology of HE staining of ovarian tissue after cryopreservation in cryopreservation solution 38.

Figure 8. Morphology of HE staining of ovarian tissue after cryopreservation in cryopreservation solution 39.

Figure 9. Morphology of HE staining of ovarian tissue after cryopreservation in cryopreservation solution 40.

Figure 10. Ovarian tissue comparison, traditional cryopreservation group, morphology of HE staining of ovarian tissue after cryopreservation.

Note: The magnification of the images in Figures 7-10 is 50 times.

Detailed ways

DETAILED DESCRIPTION OF THE INVENTION

In this specification, unless otherwise specified, the term has the common meaning in this field. For example, the freezing solution in the present invention refers to the cryopreservation solution unless otherwise specified.

The preparation method of the present invention will be described in further detail below in conjunction with specific examples. It should be understood that the following examples are only exemplary illustrations and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope that the present invention is intended to protect.

Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the reagents, materials, etc. used in the following examples, unless otherwise specified, can be obtained from commercial channels.

[Example]

Example 1. Cryopreservation of stem cells

1. Materials

<Cell Cryopreservation Solution>

The acquisition of the cell cryopreservation solution is divided into two steps:

In the first step, the following components are added per 100 mL volume: 10 g-40 g of amphiphilic macromolecule (a); 10 g-40 g of salting-out agent (b); the remainder is a base solution (MEM solution in Example 1), and the solution is allowed to stand for stratification, usually the upper layer is a coacervate phase (rich in amphiphilic molecules) and the lower layer is a dilute phase (less amphiphilic molecules).

In the second step, 80-95 mL of the upper condensed phase solution obtained in the first step is taken, 5-20 mL of a polyhydroxy or polyamino protective agent (c) having cell membrane permeability is added, and the solution is filtered using a 0.22 μm filter to obtain the cryopreservation solution.

The sources of the above ingredients are as follows:

Polyethylene glycol (PEG): CAS No.: 25322-68-3, purchased from J&K Company, molecular weight 600-20,000, 6000 was used in the following examples.

Sodium citrate (SC), CAS No. 6132-04-3, potassium citrate CAS No. 6100-05-6, sodium phosphate CAS No. 10049-21-5, sodium sulfate CAS No. 7757-82-6, sodium chloride CAS No. 7647-14-5 and sodium bromide CAS No. 7647-15-6, with a purity of more than 98%, were from J&K Company.

Ethylene glycol (EG): CAS No. 107-21-1 from J&K Company.

Glycerol: CAS No. 56-81-5 from TCI.

Glucose: CAS No. 50-99-7 from TCI.

Urea: CAS No. 57-13-6 from J&K Company.

Ascorbic acid: CAS No. 134-03-2 from Sigma-Aldrich.

CS10 cryopreservation medium: purchased from STEMCELL Technologies Inc., Lot#19167.

Basic solution: The basic solution used for stem cell cryopreservation is MEM medium from Gibco.

Cells: The stem cells used in the experiment were primary mesenchymal stem cells (h-MSC) from human embryos and mesenchymal stem cells (BMSC) from human bone marrow, both purchased from Solebol.

2. Methods

In the embodiments of the present invention, the cryopreservation method for all cells mainly adopts a traditional programmed cooling slow freezing scheme, and the specific cryopreservation steps are as follows:

(1) Select cells that are growing vigorously, in the logarithmic phase, and between P3 and P7. Digest with 0.25% Tripsin at 37°C for 1 min. After observing that the cells are rounded and detached under a microscope, use 4 times the volume of α-MEM medium containing 10% FBS to neutralize the trypsin and pipette until the cells detach from the culture dish.

(2) Transfer the cell suspension to a 15 mL centrifuge tube and centrifuge at 250 g for 5 min.

(3) Resuspend the cells in 1.0 mL of PBS and mix thoroughly by pipetting.

(4) Add 10 μL of AO/PI fluorescent dye to a 500 μL centrifuge tube. Mix 10.0 μL of cell suspension with 10 μL of fluorescent dye. Add 20.0 μL of the mixture to the round hole of the cell counting plate.

(5) Use a cell counter to count cells and detect cell viability.

(6) Calculate the cell count and cell density: For cells that grow normally, the survival rate should be above 90%. If the cell count result is 2.5×10 ⁶ and the volume is 1.0 mL, the cell density is 2.5×10 ⁶ cells/mL. The total number of cells is 2.5×10 ⁶ .

(7) Mix the cell suspension by pipetting and then divide it into each cryopreservation tube according to the proportion. The sample size is 5×10 ⁵ cells/cryopreservation tube, and the cryopreservation volume is 500 μL, which is the total volume of the cell suspension and the cryopreservation solution. The volume ratio of cell suspension to cryopreservation solution is 1:1.

(8) Add cryopreservation solution to each cryopreservation tube and mix thoroughly by pipetting to ensure that the cryopreservation solution is in full contact with the cells. Tighten the cryopreservation tube cap, mark the tube, and place it in a programmed cooling box.

(9) Place the programmed cooling box in a -80°C freezer overnight, and then transfer to liquid nitrogen for storage the next day.

<Cell freezing and resuscitation plan-immediate resuscitation rate and proliferation activity detection>

Theoretically, cells in liquid nitrogen can be stored indefinitely. However, in order to shorten the experimental period, cells can be revived and subjected to subsequent functional testing experiments after being stored in liquid nitrogen for more than 24 hours. The specific steps for cell resuscitation are as follows:

(1) Remove the cryovial from liquid nitrogen and transfer it to a 37°C water bath. Shake the cryovial to allow the contents to melt within 1 minute.

(2) Transfer the cell suspension to 5.0 mL of MEM. Centrifuge at 250 g for 5 minutes, discard the supernatant, and resuspend the cells in 1.0 mL of MEM.

(3) Trypsin was used to digest the normally proliferating MSCs in culture. After about 1 minute, the trypsin was neutralized with MEM to terminate the digestion. The supernatant was discarded after centrifugation. This was used as the Fresh group.

(4) Take out 10.0 μL of cell suspension, mix it thoroughly with 10.0 μL of AO/PI fluorescent dye in an EP tube, and add it dropwise into the round hole of the cell counting plate. The front and back protective films of the cell counting plate need to be torn off.

(5) Use a cell counter to detect and calculate cell count and cell viability.

(6) Calculate the cell amount and cell density: Dilute the cell suspension of each sample, including the Fresh group, according to the proportion and add them to a 96-well plate. Add 5,000 cells to each well. It should be noted that the amount of cells added to each well must be determined in a preliminary experiment to avoid squeezing out too many cells after 72 hours of culture.

(7) Cell proliferation was continuously observed for 72 h, and CCK-8 detection was performed at 24 h, 48 h, and 72 h, i.e., the Cell Counting Kit-8 (CCK-8) method was used for determination.

<Cell Differentiation Protocol after Cryopreservation>

Directed differentiation of stem cells into osteoblasts and adipocytes (the culture medium components are shown in Table 1-2). The commercial stem cell cryopreservation solution CS10 was used as the control group in the experiment. The experimental results were analyzed and the cryopreservation effect of the cryopreservation solution was evaluated.

[Table 1]. Osteogenic differentiation medium

Components concentration Manufacturer Fetal bovine serum (FBS) 10% Gibco Dexamethasone 0.1μmol/L Sigma Ascorbyl phosphate 0.2mmol/L Sigma β-Glycerol phosphate 10mmol/L Sigma

Note: The basal culture medium is DMEM low glucose medium, purchased from Solebio Biotechnology Co., Ltd.

[Table 2]. Adipogenic differentiation medium

Note: The basal culture medium is DMEM low glucose medium, purchased from Solebio Biotechnology Co., Ltd.

3. Description of test methods and test results

(1) Recovery rate: refers to the ratio of the absolute viability after cryopreservation (i.e., number of live cells/total number of cells) to the absolute viability of the fresh group of cells before cryopreservation (i.e., number of live cells/total number of cells). The absolute viability is measured by a cell counter after AO/PI staining. The method is as follows: Add 10 μL of AO/PI fluorescent dye to a 500 μL centrifuge tube. Mix 10.0 μL of cell suspension with 10.0 μL of fluorescent dye. Add 20.0 μL of cell suspension to the round hole of the cell counting plate. Use a cell counter to count cells and detect the absolute cell viability (i.e., number of live cells/total number of cells).

(2) Proliferation activity: refers to the increase in cells, that is, the increase in cell number through mitosis. The determination method uses the Cell Counting Kit-8 (CCK-8) method. The principle is to use a kit containing WST-8 [chemical name: 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonic acid benzene)-2H-tetrazole monosodium salt] to stain the cells. WST-8 is reduced by the dehydrogenase in the cells to a highly water-soluble yellow formazan product (Formazan dye). The amount of formazan generated is proportional to the number of living cells. The specific operation is as follows:

1. Inoculate the cell suspension (100 μL/well) in a 96-well plate. Pre-incubate the plate in a humidified incubator (e.g., at 37°C, 5% volume concentration _CO2 );

2. Add 10 μL of CCK-8 solution to each well of the plate;

3. Incubate the culture plate in the incubator for 1.5 hours;

4. Measure the absorbance at 450 nm using a microplate reader.

(3) Osteogenic and adipogenic differentiation ability: refers to the ability of stem cells to differentiate into osteocytes or adipocytes in the presence of certain induction factors. It can be described by qualitative results such as "excellent", "good" or "poor".

4. Test and results

The inventors conducted the following experiments 1-9:

The cryopreservative solution in the following experiments 1-9, the cryopreservative solution of fetal bovine serum (FBS)/DMSO (9:1) (denoted as FDM), and the CS10 cryopreservative solution were subjected to the slow freezing protocol, cell cryo-thawing protocol, and differentiation protocol after cryo-thawing described in the above 2. method, respectively, and the results were compared.

(1) Experiment 1: Effect of PEG concentration.

The cell cryopreservation solution in Experiment 1 is obtained in two steps: the first step is to add the following components per 100 mL volume: 3g-40g of polyethylene glycol 6000 (PEG6000); 20g of sodium citrate (SC); the remainder is the base solution (MEM solution in Example 1), and the solution is separated into layers after standing, the upper layer is the coacervate phase (rich in PEG6000), and the lower layer is the dilution phase (less PEG6000 content). The second step is to take 90mL of the upper coacervate phase solution obtained in the first step, add 10mL EG, and 100mL of cryopreservation solution can be obtained. According to the different amounts of PEG6000 used, the cryopreservation solution is divided into cryopreservation solutions 1-5, as shown in Table 3 below.

[Table 3] Effect of PEG concentration

In Experiment 1, liquid-liquid phase separation occurred in the cryopreservation solution 1-5, and the solution was divided into two incompatible phases. The upper condensed phase was taken and 10 mL EG was added as the cryopreservation solution 1-5. Figure 1 shows a diagram of the liquid-liquid phase separation system. Bone marrow-derived mesenchymal stem cells (BMSCs) were used as cryopreservation objects, and the immediate recovery rate after the cells were cryopreserved in liquid nitrogen for 24 hours by various cryopreservation solutions was measured. The results of the recovery rate are shown in Table 4.

[Table 4] Effect of PEG concentration on resuscitation rate

Cryopreservation fluid number 1 2 3 4 5 CS10 FDM Recovery rate (%) 90.5 92.0 95.0 96.5 91.5 93.5 95.5 variance(%) 3.1 4.2 2.8 0.7 1.3 2.1 0.7

As shown in Table 4, in cryopreservation solutions 1-5, that is, when the content of PEG6000 per 100 mL of liquid-liquid phase separation solution was 10-40 g (cryopreservation solutions 1-5), the recovery rate was relatively high, reaching a recovery rate of more than 90%. Among them, 15-30 g (cryopreservation solutions 2-4) was better, and 20-30 g (cryopreservation solutions 2-4) was the best. In particular, cryopreservation solution 4 showed a survival rate of up to 96%, which was better than or comparable to the cryopreservation recovery rate of the traditional FDM group and the CS10 group.

Experiment 2: Effect of salting-out agent concentration.

The cell cryopreservation solution in Experiment 2 was obtained in two steps: the first step was to add the following components per 100 mL volume: 20 g of polyethylene glycol 6000 (PEG6000); 10-40 g of sodium citrate (SC); the remainder was MEM solution, which was separated into layers after standing, with the upper layer being the coacervate phase (rich in PEG6000) and the lower layer being the dilution phase (containing less PEG6000). The second step was to take 90 mL of the upper coacervate phase solution obtained in the first step, add 10 mL of EG, and 100 mL of cryopreservation solution was obtained. According to the different amounts of SC used, the cryopreservation solution was divided into cryopreservation solutions 6-10, as shown in Table 5 below.

[Table 5] Effect of salting-out agent concentration

In Experiment 2, bone marrow-derived mesenchymal stem cells (BMSCs) were used as cryopreservation objects, and the immediate recovery rates after the cells were cryopreserved in liquid nitrogen for 24 hours using various cryopreservation solutions were measured. The results of the recovery rates are shown in Table 6.

[Table 6] Effect of salting-out agent concentration on recovery rate

Cryopreservation fluid number 6 7 8 9 10 CS10 FDM Recovery rate 89.95 91.5 95 94.5 91.0 93.5 95.5 variance 1.5 3.5 2.8 2.7 2.8 2.1 0.7

As shown in Table 6, for the cryopreservation solution containing PEG6000, SC and EG, that is, when the concentration of the salting-out agent SC for each 100 mL of liquid-liquid phase separation solution is 10g-40g (cryopreservation solution 6-10), there is a better recovery rate, which can be close to or higher than 90%, and the recovery rate is higher. When the concentration of SC is 15g-30g (cryopreservation solution 7-9), the recovery rate is higher, and when the concentration of SC is 20g-30g (cryopreservation solution 8-9), the recovery rate is the highest. It is better than or equivalent to the cryopreservation recovery rate of the traditional FDM group and the CS10 group.

Experiment 3. Effect of the content of cell membrane permeability protectant

The cell cryopreservation solution in Experiment 3 was obtained in two steps: the first step was to add the following components per 100 mL volume: 20 g of polyethylene glycol 6000 (PEG6000); 20 g of sodium citrate (SC); the remainder was MEM solution, which was separated into layers after standing, with the upper layer being the coacervate phase (rich in PEG6000) and the lower layer being the dilution phase (containing less PEG6000). The second step was to take 95-80 mL of the upper coacervate phase solution obtained in the first step, add 5-20 mL of EG, and 100 mL of cryopreservation solution was obtained. According to the different amounts of EG used, the cryopreservation solution was divided into cryopreservation solutions 11-15, as shown in Table 7 below.

Bone marrow-derived mesenchymal stem cells (BMSCs) were cryopreserved. The immediate recovery rates of cells stored in liquid nitrogen for 24 hours after being cryopreserved in various cryopreservation solutions using standard procedures were measured. The results of the recovery rates are shown in Table 8.

[Table 7] Effect of the content of cell membrane permeability protectant

【data】

[Table 8] Effect of cell membrane permeability protective agent content on recovery rate

Cryopreservation fluid number 11 12 13 14 15 CS10 FDM Recovery rate 93.8 95.0 92.5 91.5 83.0 93.5 95.5 variance 1.4 2.8 0.7 3.3 8.5 2.1 0.7

As shown in Table 8, the groups containing 5-20 mL EG (cryopreservatives 11-15) all showed a recovery rate of more than 80%, among which the group containing 5-18 mL EG (cryopreservatives 11-14) was better, the group containing 5-15 mL EG (cryopreservatives 11-13) was further better, and the group containing 5-10 mL EG (cryopreservatives 11-12) was the best, and its recovery rate was comparable to or better than that of the traditional FDM group and the CS10 group.

Experiment 4. Effect of the type of cell membrane permeability protectant

The cell cryopreservation solution in Experiment 4 was obtained in two steps: the first step was to add the following components per 100 mL volume: 20 g of polyethylene glycol 6000 (PEG6000); 20 g of sodium citrate (SC); the remainder was MEM solution, which was separated into layers after standing, with the upper layer being the coacervate phase (rich in PEG6000) and the lower layer being the dilution phase (containing less PEG6000). The second step was to take 90 mL of the upper coacervate phase solution obtained in the first step, add 10 mL of a cell membrane permeable molecule, 10 mL of EG, 10 mL of glycerol GCL, respectively, to obtain cryopreservation solutions 16 and 17, and take 100 mL of the upper coacervate phase solution and add 10 g of glucose Glu or 10 g of urea Urea or 10 g of betaine Betaine to obtain cryopreservation solutions 18-20. As shown in Table 9 below.

Bone marrow-derived mesenchymal stem cells (BMSCs) were cryopreserved. The immediate recovery rates of various cryopreservation solutions after the cells were cryopreserved in liquid nitrogen for 24 hours were determined, and the results of the recovery rates are shown in Table 10. 20% PEG and 20% SC were mixed and separated, and the upper condensed phase was taken, and 10% of different types of cell membrane permeability small molecule protective agents were added to serve as cryopreservation solutions 16-20.

[Table 9] Effect of the type of cell membrane permeability protectant

【data】

[Table 10] Effect of cell membrane permeability protectant types on recovery rate

Cryopreservation fluid number 16 17 18 19 20 CS10 FDM Recovery rate 95 97.0 50.5 73.0 92.0 93.5 95.5 variance 2.8 1.4 7.8 4.3 1.8 2.1 0.7

As shown in Table 10, the recovery rates of cryopreservation groups 16, 17, and 20, namely EG, GCL, and Betaine, were relatively good. Among them, the EG and GCL groups were the best, and their recovery rates were comparable to those of the traditional FDM cryopreservation group.

Test 5. Effect of salting-out agent type

The cell cryopreservation solution in Experiment 5 was obtained in two steps: the first step was to add the following components per 100 mL volume: 20 g of polyethylene glycol 6000 (PEG6000); 20 g of sodium citrate (SC), potassium citrate SCK, one of sodium phosphate, sodium sulfate, sodium chloride, and sodium bromide; the remainder was MEM solution, which was separated after standing, with the upper layer being the coacervate phase (rich in PEG6000) and the lower layer being the dilution phase (containing less PEG6000). The second step was to take 90 mL of the upper coacervate phase solution obtained in the first step, add 10 mL of a cell membrane permeable molecule, 10 mL of EG, respectively, and obtain cryopreservation solutions 21-26 according to the type of salting-out agent. As shown in Table 11 below.

[Table 11] Effect of salting-out agent type

Experiment 5 was conducted using the cell cryopreservation solutions in Table 11 to cryopreserve bone marrow-derived mesenchymal stem cells (BMSCs). The immediate recovery rates of cells stored in liquid nitrogen for 24 hours after being cryopreserved using various cryopreservation solutions in a standard procedure were measured. The results of the recovery rates are shown in Table 12.

[Table 12] Effect of salting-out agent type on recovery rate

Cryopreservation fluid number twenty one twenty two twenty three twenty four 25 26 CS10 FDM Recovery rate 95 94.5 93.0 92.5 98.0 94.0 93.5 95.5 variance 2.8 1.1 1.4 2.7 4.0 5.7 2.1 0.7

As shown in Table 12, cryopreservation groups 21-26 all showed good recovery rates. Among them, sodium citrate (cryopreservation solution 21), potassium citrate (cryopreservation solution 22), and sodium chloride group (cryopreservation solution 25) were better, and the recovery rates of sodium citrate (cryopreservation solution 21) and sodium chloride group (cryopreservation solution 25) were the best, which was comparable to the recovery rate of the traditional FDM cryopreservation group.

Experiment 6. Effect of PEG polymerization degree

The cell cryopreservation solution in Experiment 6 was obtained in two steps: the first step was to add the following components per 100 mL volume: 20 g of polyethylene glycol, whose molecular weight increased from 600 to 20,000 (PEG600-PEG20000); 20 g of sodium citrate (SC); the remainder was MEM solution, which was separated after standing, with the upper layer being the coacervate phase (rich in PEG6000) and the lower layer being the dilution phase (containing less PEG6000). The second step was to take 90 mL of the upper coacervate phase solution obtained in the first step, add 10 mL of a cell membrane permeable molecule, 10 mL of EG, respectively, and obtain cryopreservation solution 27-33 according to the different PEG molecular weights. As shown in Table 13 below.

[Table 13] Effect of PEG polymerization degree

Experiment 6 was conducted using the cell cryopreservation solutions in Table 13. Bone marrow-derived mesenchymal stem cells (BMSCs) were cryopreserved. The immediate recovery rates of cells stored in liquid nitrogen for 24 hours after being cryopreserved using various cryopreservation solutions in a standard procedure were measured. The results of the recovery rates are shown in Table 14.

[Table 14] Effect of PEG polymerization degree on recovery rate

Cryopreservation fluid number 27 28 29 30 31 32 33 CS10 FDM Recovery rate 3.5 4.5 30.5 91.5 95.0 92.0 91.5 93.5 95.5 variance 1.1 2.4 6.4 3.5 2.8 1.7 1.9 2.1 0.7

As shown in Table 14, cryopreservation groups 30-33 all showed good recovery rates. Among them, cryopreservation solutions 31-33 were better, among which cryopreservation solutions 31-32 were better, and among which cryopreservation solution 31 (PEG6000) was the best, with a recovery rate comparable to that of the traditional FDM cryopreservation group and the CS10 group.

Experiment 7. Testing the proliferation activity of frozen cells

In Experiment 7, cryopreservation solution 31 was used to perform standard procedure procedures for bone marrow-derived mesenchymal stem cells (BMSCs), and they were stored in liquid nitrogen for 24 hours before revival. After revival, they were seeded in a 96-well plate at a density of 8,000 cells/plate, and the traditional FDM cryopreservation group and the fresh unfrozen group were used as controls to measure their proliferation at 24 hours, 48 hours, and 72 hours. The statistical data is shown in Figure 2, where the cryopreservation group using cryopreservation solution 31, the cryopreservation group using CS10, the traditional FDM cryopreservation group, and the fresh unfrozen group are recorded as 31, CS10, FDM, and fresh, respectively.

As shown in Figure 2, the 72h proliferation capacity of mesenchymal stem cells after cryopreservation and resuscitation in cryopreservation solution 31 was comparable to the recovery rate of the traditional FDM cryopreservation group, and there was no significant difference in their proliferation capacity after cryopreservation compared with the fresh group.

Experiment 8. Testing the osteogenic and adipogenic differentiation abilities of cryopreserved cells

Experiment 8 was conducted using cryopreservation solution 31. Bone marrow-derived mesenchymal stem cells (BMSCs) were cryopreserved in a standard procedure, stored in liquid nitrogen for 24 hours, and then revived. After revivification, they were cultured for 48 hours, and then induced to differentiate into osteoblasts and adipocytes. The fresh unfrozen group was used as a control, and the results are shown in Figures 3 and 4.

As shown in Figures 3 and 4, the mesenchymal stem cells after cryopreservation and resuscitation in cryopreservation solution 31 maintained good osteogenic and adipogenic differentiation capabilities, which were rated "excellent", comparable to those of the traditional FDM cryopreservation group, and had no significant difference from the traditional CS10 cryopreservation group.

Experiment 9. Testing the protective ability of liquid-liquid coagulants composed of different types of amphiphilic macromolecules on stem cells

The PEG6000 in the cryopreservation solution 31 was replaced with 20 g of bovine serum albumin, chondroitin sulfate, or a mixture of hyaluronic acid and chitosan, respectively, to obtain cryopreservation solutions 34-36.

The above-mentioned cryopreservation solutions 34-36 were used for cryopreservation of bone marrow-derived mesenchymal stem cells, and the immediate recovery rates of the cells after being cryopreserved in liquid nitrogen for 24 hours using various cryopreservation solutions were measured. The results of the recovery rates are shown in Table 15.

[Table 15] Liquid-liquid condensed phases composed of different types of amphiphilic macromolecules

As shown in Table 15, cryopreservation groups 34-36 all showed good recovery rates, among which cryopreservation solution 34 was the best, and its immediate recovery rate was comparable to that of the CS10 cryopreservation group.

Through the above experiments, it can be known that the present invention provides a cell cryopreservation solution, which does not contain DMSO and serum. This cryopreservation solution can be used for the cryopreservation of various stem cells (including bone marrow stem cells derived from umbilical cord blood, etc.). The frozen stem cells maintain a good immediate recovery rate, proliferation activity and differentiation ability, and there is no significant difference between the traditional CS10 cryopreservation group and the cryopreservation group.

Example 2. Cryopreservation of immune cells (including T cells and NK cells, etc.)

1. Materials

<Cell Cryopreservation Solution>

The aforementioned freezing solution 31 can be used in cell freezing experiments only after being filtered through a 0.22 μm filter.

The preparation of cryopreservation solution 31 is divided into two steps: the first step is to add the following components per 100 mL volume: 20 g of PEG6000, 20 g of salting-out agent SC, and the balance is the base solution. The solution is allowed to stand and then separated into layers, usually the upper layer is the coacervate phase (rich in amphiphilic molecules) and the lower layer is the dilution phase (less amphiphilic molecules). The second step is to take 90 mL of the upper coacervate phase solution obtained in the first step, add 10 Ml EG (c), and 100 mL of cryopreservation solution 31 can be obtained.

The preservation solution used for immune cryopreservation includes the following components:

PEG6000: from Sigma-Aldrich.

Sodium citrate: from J&K Company.

Base solution:

The basic solution for storing NK92 cells, H9 cells (αT cell line), and γT cells may include: PBS buffer: from TCI.

MEM medium: from Gibco.

cell:

The γδT cells used in the experiment were donated by Xi'an Jiaotong University;

NK92 cells and H9 cells (αT cell line) were purchased from Wuhan Pronocell Life Science Technology Co., Ltd.

2. Methods

Cryopreservation

Resuspend to freezing solution at 2x10 ⁷ /mL, place in a program cooling box at -80℃ for more than 4 hours, and then transfer to liquid nitrogen for storage.

recovery

T cells or NK cells frozen in liquid nitrogen for 24 hours were revived in a 37-degree water bath, and all ice crystals were melted within 1 minute. After resuspending in 1 mL of PBS, AO/PI was used to count and determine the viability.

Experiment 13. Testing the cryopreservation of immune cells

Experiment 13 was carried out using cryopreservation solution 31. The cryopreservation solution was used to cool and freeze NK cells, T cells (αT cell line), γδT cells, etc. according to standard procedures, and then stored in liquid nitrogen for 24 hours before reviving. The commercial formula CS10 was used as the control group. The results are recorded in Table 16.

3. Results

[Table 16]

Immediate recovery rate H9 cells NK92 cells γδ T cells CS10 95.3±2.3 75.4±3.6 82.3±4.34 Cryopreservation medium 31 96.5±1.3 80.7±3.9 95.43±3.6

The results of the recovery rate are shown in Table 16. NK92 cells, H9 cells (αT cell line), γδT cells, etc. cryopreserved in cryopreservation solution 31 all maintained a high recovery rate, which was better than the commercial CS10.

Example 3. Cryopreservation of corneal endothelial cell layer

1. Materials

<Cell Cryopreservation Solution>

The cryopreservation of corneal endothelial cells was performed using a cell cryopreservation solution of the following composition. The cell cryopreservation solution in Example 3 was obtained in two steps: the first step was to add the following components per 100 mL volume: 20 g of polyethylene glycol 6000 (PEG6000), 20 g of sodium citrate (SC); the remainder was a MEM solution, which was separated into layers after standing, with the upper layer being a coacervate phase (rich in PEG6000) and the lower layer being a dilution phase (containing less PEG6000). The second step was to take 100 mL of the upper coacervate phase solution obtained in the first step, add 3 g of betaine, and obtain a cryopreservation solution 37, which could be used for cell cryopreservation experiments after being filtered through a 0.22 μm filter.

The preservation solution used for cryopreservation of corneal endothelial cells includes the following components:

Polyethylene glycol (PEG): from J&K.

Sodium citrate: from J&K Company.

Betaine: From J&K Company.

The base solution for corneal endothelial cell preservation contains:

MEM basal culture medium: from Gibco.

Commercial corneal preservation solution JTSD: from Gibco.

The cryopreservation solution used for corneal cryopreservation is CS10.

cell:

The cells used in the experiment were human corneal endothelial cell lines (purchased from Solebol).

2. Methods

Cryopreservation of corneal endothelial cells

(1) The cells in a 10 cm culture dish were digested with 0.25% Tripsin at 37°C for 1 min. After the cells were observed to be rounded and detached under a microscope, 4-5 times the volume of α-MEM medium containing 10% FBS (hereinafter referred to as M10) was used to neutralize the trypsin and pipette until the cells detached from the culture dish.

(2) Transfer the cell suspension to a 15 mL centrifuge tube and centrifuge at 250 g (1200-1600 rpm) for 5 min. Collect the supernatant.

(3) Resuspend the cells in 1 mL of PBS and mix by pipetting.

(4) Use a cell counter to count cells and detect cell viability.

(5) The cell suspension was divided into 96-well plates according to the proportion, culture medium was added, and cultured in a cell culture incubator at 37°C for 24 h.

(6) Aspirate the culture medium, add preservation solution, and freeze it in a -40°C refrigerator for 7 days, 14 days, and 21 days.

(7) After the cornea is removed, the freezing solution is aspirated out, and the cornea is rinsed twice with complete culture medium. After adding complete culture medium, the cornea is placed in a 37°C cell culture incubator for culture.

(8) The CCK-8 value of the cells was measured at 24h, 48h and 72h, respectively. The proliferation of the cells after cryopreservation was observed. The cryopreservation of the adherent corneal endothelial cell line by the liquid-liquid coacervate phase cryopreservation medium was tested.

3. Results

The results are shown in Figures 5 and 6. In Figure 5, the fresh group, experimental group, commercial cryopreservation agent and control group represent unfrozen cells, cells frozen with cryopreservation solution 37 and CS10 solution, respectively. The fresh group experimental group includes the morphology before (upper figure) and 1 day after (lower figure); the commercial cryopreservation agent and control group include the morphology before (upper figure), and after 7 days of freezing (middle figure) and 21 days (lower figure). The ordinate in Figure 6 represents the proliferation rate of corneal cells after 24h, 48h and 72h of culture, with 24h as the cell number as the benchmark. That is, Proliferation = (number of cells at the moment) / (number of cells at 24h), and the horizontal axis represents the proliferation multiples of different groups, namely the fresh unfrozen group, and the cells frozen in No. 37 cryopreservation solution for 7 days, 14 days and 21 days, and the cells frozen in CS10 cryopreservation solution for 7 days, 14 days and 21 days, at 24h, 48, and 72h. Cryopreservation solution 37 cryopreserves the corneal endothelial cell line that grows on the wall. At 7 days and 21 days of freezing, the cells maintain a good morphology. The CCK-8 results show that the cell metabolism is normal and there is no significant difference with the commercial cryopreservation solution. Its metabolic rate is lower than that of the fresh group, and is basically the same as that of the fresh group after 48h of culture. After 21 days of freezing, the CCK-8 result of cryopreservation solution 37 is significantly lower than that of the fresh group, but better than that of the commercial group.

Example 4. Cryopreservation of ovarian tissue

1. Materials

<Ovarian tissue cryopreservation solution>

The cryopreservation of ovarian tissue is performed using the cryopreservation solution of the following composition. The cryopreservation process of ovarian tissue includes two steps, balancing and freezing, involving balancing solution and freezing solution, and the configurations thereof are as follows:

Cryopreserved tissue:

Ovarian tissue was obtained from SPF female severe combined immunodeficient NPG mice, 10 weeks old, weighing 25-35 g, purchased from Beijing Weitongda Biotechnology Co., Ltd. (Animal Production License No.: SCXK (Beijing) 2019-0002). Quarantine and environmental adaptation for 4 days (including the day of receipt) were carried out in accordance with the "Subscription, Receipt and Quarantine of Laboratory Animals" (SOPT001).

2. Freezing and thawing:

1) Cryopreservation steps

(1) Transport ovarian tissue to the laboratory at low temperature;

(2) Wash the ovarian tissue three times with DPBS;

(3) Cut the ovarian tissue into 5×5×1 mm size;

(4) Equilibrate in the corresponding equilibrium solution at room temperature for 15 min;

(5) Further dehydration in cryopreservation agent at room temperature for 5 min;

(6) Place the ovarian tissue on a single layer of gauze and quickly place it in liquid nitrogen;

(7) Under liquid nitrogen environment, transfer the ovarian tissue into a 1.8 mL cryotube;

(8) Transfer to liquid nitrogen for storage.

2) Thawing steps

Thawing liquid Element T1 1M sucrose + HTK solution T2 0.5M sucrose + HTK solution T3 0.25M sucrose + HTK solution T4 HTK Liquid G

(1) Remove ovarian tissue from liquid nitrogen and let stand at room temperature for 10 seconds;

(2) Transfer the ovarian tissue in the cryopreserved tube to T1 and incubate at 37°C for 5 min.

(3) At room temperature, treat in T2, T3, and T4 thawing solutions for 5 min each.

3. Characterization of cryopreservation effect:

1) HE staining (Hematoxylin-eosin staining) steps Experimental steps:

(1) Dewaxing of paraffin sections to water:

Put the slices into xylene I for 10 min - xylene II for 10 min - anhydrous ethanol I for 5 min - anhydrous ethanol II for 5 min - 95% alcohol for 5 min - 90% alcohol for 5 min - 80% alcohol for 5 min - 70% alcohol for 5 min - and wash with distilled water.

(2) Hematoxylin staining of cell nuclei:

The sections were stained with Harris hematoxylin for 3-8 minutes, washed with tap water, differentiated with 1% hydrochloric acid alcohol for a few seconds, rinsed with tap water, blued with 0.6% ammonia water, and rinsed with running water.

(3) Eosin staining of cytoplasm:

The sections were stained in eosin solution for 1-3 min.

(4) Dehydration and sealing:

Put the slices in 95% alcohol I for 5 min-95% alcohol II for 5 min-anhydrous ethanol I for 5 min-anhydrous ethanol II for 5 min, xylene I for 5 min-xylene II for 5 min to dehydrate and make them transparent. Take the slices out of xylene, dry them slightly, and seal them with neutral gum.

(5) Microscope examination, image acquisition and analysis.

2) The number of normal follicles is counted based on the morphological results of frozen sections of the ovarian cortex. The morphologically normal standard must meet the following criteria at the same time:

a. Granular cells are intact;

b. The oocyte is intact;

c. The nucleolus is clear;

d. The basement membrane is intact.

The calculation formula for normal follicles is as follows: normal rate = number of normal follicles/total number of follicles*100%.

4. Results

As shown in Figures 7-10, HE staining results show that after the above four groups of ovarian tissues in this embodiment were frozen and thawed, each group of ovarian tissues contained normal follicles and some abnormal follicles. After using a microscope to observe each group of ovarian tissues and obtain images of at least 100 follicles respectively, image analysis showed that the normal follicle rates of ovarian tissues frozen with cryopreservation solutions 38, 39 and 40 (i.e., cryopreservation solution 38 group, cryopreservation solution 39 group, and cryopreservation solution 40 group) were 68.7%, 82.4% and 76.6% respectively, while the normal follicle rate of the control group was 68.1%. The normal rates of cryopreservation solution 38 group, cryopreservation solution 39 group and cryopreservation solution 40 group were not lower than that of the control group, among which cryopreservation solutions 38 and 40 were better, and cryopreservation solution 39 was the best.

The above is an explanation of the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims (10)

1. A cryopreservation solution, characterized in that the cryopreservation solution comprises the following components: an amphiphilic macromolecule (a), a salting-out agent (b) and a basic solution. 2. The cryopreservation solution according to claim 1, characterized in that the cryopreservation solution further comprises a protective agent (c). Preferably, the cryopreservation solution is prepared by mixing and layering the amphiphilic macromolecule (a), the salting-out agent (b) and the basic solution. Preferably, in the mixed solution, the mass ratio of the amphiphilic macromolecule (a) to the salting-out agent (b) is (10-40): (10-40). Preferably, the volume ratio of the mixed solution to the layer rich in amphiphilic macromolecules (a) is 100:(50-99). Preferably, the volume ratio of the protective agent (c) to the layer rich in amphiphilic macromolecules (a) is (1-50):(50-99). Preferably, the mass volume ratio of the amphiphilic macromolecule (a) and the protective agent (c) is (10-40) g: (2-20) mL. 3. The cryopreservative solution according to claim 1 or 2, characterized in that the amphiphilic macromolecule (a) comprises at least one of amphiphilic proteins with coexisting hydrophilic/hydrophobic microregions and amphiphilic macromolecules with hydrophilic and hydrophobic regions. Preferably, the amphiphilic proteins in which the hydrophilic/hydrophobic microdomains coexist include at least one of bovine serum albumin and human serum albumin. Preferably, the amphiphilic macromolecule having hydrophilic and hydrophobic regions includes at least one of polyethylene glycol (PEG), chondroitin sulfate, hyaluronic acid, and chitosan. Preferably, the degree of polymerization of the amphiphilic macromolecule (a) is between 600 and 20,000. Preferably, the amphiphilic macromolecule (a) is used alone or as a mixture of two or more types in the cryopreservation solution. Preferably, the amphiphilic macromolecule (a) is at least one of bovine serum albumin (BSA), polyethylene glycol (PEG), and chondroitin sulfate. Preferably, every 100 mL of the cryopreservation solution contains 10 g to 40 g of the amphiphilic macromolecule (a). Preferably, the salting-out agent (b) is selected from at least one of sodium citrate, potassium citrate, sodium phosphate, sodium sulfate, sodium chloride, and sodium bromide salts having a salting-out effect. Preferably, the salting-out agent (b) is selected from one of sodium citrate, potassium citrate, or sodium chloride. Preferably, each 100 mL of the mixed solution contains 10 g to 40 g of the salting-out agent (b). Preferably, the basic solution is selected from at least one of the following: an aqueous solution, a PBS buffer or a basic culture medium. 4. The cryopreservation solution according to any one of claims 1 to 3, characterized in that the protective agent (c) is selected from polyhydroxy or polyamino substances. Preferably, the polyhydroxy or polyamino substance is selected from at least one of ethylene glycol, glycerol, urea, glucose, propylene glycol, 1,3-propylene glycol, isoprene glycol and betaine. 5. The cryopreservation solution according to any one of claims 1 to 4, characterized in that the cryopreservation solution may further contain other ingredients. Preferably, the other ingredients are selected from at least one of antioxidants, buffer solutions, structural stabilizers, humectants, excipients, preservatives, and antibiotics. Preferably, the pH value of the cryopreservation solution is between 6.0 and 7.5. The freezing preservation solution does not contain at least one of dimethyl sulfoxide (DMSO), dimethylformamide (DMF) and serum. 6. The method for preparing the cryopreservation solution according to any one of claims 1 to 5, characterized in that the preparation method is as follows: The first step is to add the amphiphilic macromolecule (a), the salting-out agent (b) and the basic solution, and mix them to obtain a mixed solution; the second step is to separate the mixed solution into layers, take the layer rich in the amphiphilic macromolecule (a) obtained after the layers are separated, and add the protective agent (c) to obtain the cryopreservation solution. Preferably, the preparation method of the cryopreservation solution is as follows: The first step is to add the amphiphilic macromolecule (a), the salting-out agent (b) and the base solution to obtain a mixed solution; In the second step, after the mixed solution is layered, the upper layer is a condensed phase solution, which contains a layer rich in the amphiphilic macromolecule (a), and the lower layer is a condensed phase solution. The upper condensed phase solution is taken and a protective agent (c) is added to obtain the cryopreservation solution. 7. The preparation method according to claim 6, characterized in that in the first step, 10 g to 40 g of the amphiphilic macromolecule (a) is contained per 100 mL of the mixed solution. Preferably, in the first step, 10 g to 40 g of the salting-out agent (b) is contained per 100 mL of the mixed solution. Preferably, in the second step, the volume ratio of the condensed phase solution to the protective agent (c) is (80-95):(5-20). 8. A method for freezing cells using the cryopreservation solution according to any one of claims 1 to 5. 9. Use of the cryopreservation solution according to any one of claims 1 to 5, characterized in that the cryopreservation solution according to any one of claims 1 to 5 is used for cryopreservation of cells, tissues and/or organs. Preferably, the cells are selected from cells suitable for slow freezing, preferably, the cells for slow freezing are selected from mammalian suspension cells and/or adherent cells. Exemplarily, the cells used for slow freezing are selected from at least one of red blood cells, stem cells, T cells, NK cells, and corneal endothelial cells. Further, the stem cells include embryonic mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, and adipose-derived mesenchymal stem cells. Preferably, the tissue comprises corneal monolayer tissue and ovarian tissue. 10. The use according to claim 9, characterized in that the cells are derived from insects, animals or plants.