A cell freezing solution and a method for preparing the same

Abstract

The application discloses a kind of cell cryopreservation solution and preparation method thereof, the cell cryopreservation solution includes glycerol, platelet lysate, proline, L-glutamine and support material, basal medium.Compared with prior art, the cell cryopreservation solution prepared by the application can provide the nutrients and growth factors required by cells, help maintain the stability and survival rate of cells, reduce the damage to cells during freezing and thawing, and improve the survival rate and viability of cells.

Description

Technical Field

[0001] This invention relates to the field of cell cryopreservation technology, and in particular to a cell cryopreservation solution and its preparation method. Background Technology

[0002] Cell cryopreservation solution is a liquid preparation used for the long-term preservation of live cells. During cell cryopreservation, cells need to be rapidly frozen and stored at extremely low temperatures to prevent cell death and damage.

[0003] However, existing technologies still have some drawbacks. For example, cells are easily affected by temperature changes, ice crystals formed during freezing, chemicals, and mechanical stimulation during cryopreservation, leading to high cell mortality. The composition and formulation of cell cryopreservation solutions vary depending on the type of cell and the laboratory, lacking consistency and standardization. Cell contamination issues may arise during the preparation and use of cryopreservation solutions, such as contamination by bacteria, fungi, and viruses, which can affect the accuracy and reliability of experimental results. Different cell types have different adaptability to the composition and formulation of cryopreservation solutions, requiring adjustments and optimizations for different cell types. High storage conditions are required; cell cryopreservation solutions need to be stored at extremely low temperatures, such as liquid nitrogen or ultra-low temperature freezers, which places high demands on laboratory equipment and management.

[0004] To address these challenges, electrospinning technology has been widely applied in the manufacture of polymeric scaffold materials for the biomedical field, including drug delivery, tissue engineering, diagnostics, biosensors, and enzyme immobilization. In particular, electrospinning's unique ability to produce continuous fibers with diameters from nanometers to micrometers makes it a promising technology for constructing cocoon-like fibrous scaffolds. Rapid heating rates and uniform heat distribution during thawing can inhibit ice formation and growth, thereby improving cryopreservation effectiveness. However, most natural or synthetic polymers are poor thermal conductors. To overcome this obstacle, various conductive fillers, such as alumina, boron nitride, silicon carbide, and carbon nanotubes, have been added to polymers to increase the thermal conductivity of the support material; however, biocompatibility issues always exist, making it difficult to improve the activity and viability of cryopreserved cells.

[0005] Chinese patent CN112293409B discloses a cell cryopreservation solution comprising sodium, potassium, magnesium, chloride, acetate, gluconate, and mannitol, with water as the solvent. The cell cryopreservation solution and in vitro neutrophil storage method provided by this invention are not only simple and inexpensive, but also maintain neutrophil activity for extended periods. Compared to preservation methods using ordinary culture media, the cell cryopreservation solution provided by this invention better preserves neutrophil activity; frozen and thawed neutrophils still exhibit good phagocytic and bactericidal effects, improving the clinical application of neutrophils and thus showing promising industrialization prospects. However, the addition of various ions in the cell cryopreservation solution prepared by this invention requires precise control; otherwise, it may lead to adverse effects such as increased cell death rate or decreased metabolic function. Mannitol may also affect cell membrane permeability, impacting cell metabolism and growth.

[0006] Patent CN107027743B discloses a cell cryopreservation solution comprising basal culture medium, platelet lysate, bFGF, and L-glutamine. This cryopreservation solution is free of animal serum and DMSO, eliminating the possibility of introducing exogenous viruses from animal serum and the adverse effects of DMSO on adipose-derived stem cells. Furthermore, the invention provides a cell cryopreservation method that is simple to operate and requires no complex programmed cooling. After one year of cryopreservation of adipose-derived stem cells using the disclosed cell culture medium and cryopreservation method, the cell viability reaches 93% without affecting the differentiation capacity of the adipose-derived stem cells. However, the quality and concentration of platelet lysate and bFGF in the cell cryopreservation solution provided by this invention need to be strictly controlled; otherwise, it may affect cell growth and metabolism. This cell cryopreservation solution and method may not be suitable for all cell types, and the cryopreservation effect is unstable. Summary of the Invention

[0007] In view of the shortcomings of the poor cryopreservation and thawing effect of the existing cell cryopreservation solution, the technical problem to be solved by the present invention is to provide a cell cryopreservation solution with good cryopreservation and thawing effect and its preparation method.

[0008] To achieve the above-mentioned objectives, the present invention adopts the following technical solution:

[0009] A cell cryopreservation solution comprising the following components: glycerol, platelet lysate, proline, L-glutamine, scaffold material, and basal culture medium replenishment.

[0010] The glycerol content in the cell cryopreservation solution is 5-10 v / v.

[0011] The basal culture medium is one of RPMI-1640, MEM, high-glucose DMEM, low-glucose DMEM, or DMEM / F12.

[0012] The platelet lysate constitutes 3–8 v / v in the cell cryopreservation solution.

[0013] The proline content in the cell cryopreservation solution is 0.5–2 w / v.

[0014] The L-glutamine was present in the cell cryopreservation solution at a concentration of 0.4–0.6 w / v.

[0015] The scaffold material constitutes 0.5–2 w / v in the cell cryopreservation solution.

[0016] A method for preparing a cell cryopreservation solution is as follows:

[0017] Weigh each component according to volume or weight-volume ratio, and then mix each component by stirring at 5-20 rpm for 3-10 minutes to obtain the cell cryopreservation solution.

[0018] The preparation method of the scaffold material is as follows, in parts by weight:

[0019] S1. Add 1-3 parts of sodium alginate to 180-220 parts of water at 30-50℃, adjust the pH to 3-4 using 0.2-0.5 mol / L hydrochloric acid, then add 1-5 parts of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1.5-2 parts of N-hydroxysuccinimide, stir at 30-50℃ and 100-300 rpm for 3-8 hours, dialyze the reaction mixture using a dialysis tube with a molecular weight cutoff of 4000-6000 Da, and then freeze-dry to obtain the composite material;

[0020] S2. Add 0.5 to 2 parts of polyethylene oxide to 80 to 120 parts of 6 to 9 wt% silk protein aqueous solution, stir at 100 to 300 rpm for 1 to 5 hours to obtain a mixed solution; then, add 10 to 20 parts of polyamide-imide to the above mixed solution, stir at 40 to 50°C and 300 to 800 rpm for 8 to 15 hours to obtain spinning solution A;

[0021] S3. Add 3-8 parts of gelatin to 80-120 parts of 10-20 wt% polylactic acid solution and stir at 100-300 rpm for 10-50 min. Then add 3-8 parts of graphene oxide and stir at 300-800 rpm for 8-15 h to obtain spinning solution B. Transfer spinning solution A and spinning solution B prepared in step S2 to different syringes and perform electrospinning simultaneously. Add water to the composite material to prepare a 0.5-2 wt% aqueous solution for collecting the electrospun fibers from spinning solution A and spinning solution B. After collection, let it stand for 10-30 h, wash with water, and freeze dry to obtain the scaffold material.

[0022] The polylactic acid solution is prepared by mixing polylactic acid and hexaoxyisopropanol.

[0023] The positive voltage for electrospinning is 18–22 kV, and the flow rate is fixed at 0.4–0.6 mL / h.

[0024] The cryopreservation and thawing steps for the cells are as follows:

[0025] The adipocytes were added to the cell cryopreservation solution and stored at 1–5°C for 10–40 min, then at -10–-30°C for 1–3 h, and finally transferred to a -70–-90°C freezer for long-term storage. The thawing procedure for the adipocytes was to remove the frozen adipocytes from the -70–-90°C freezer and quickly transfer them to a 35–38°C water bath for gentle shaking to thaw.

[0026] Glycerol is a commonly used cryoprotectant that reduces cell damage during freezing by lowering intracellular water content. Platelet lysate is a mixture of growth factors and extracellular matrix released from platelets, providing growth factors and cell adhesion proteins necessary for cell growth and helping to maintain cell stability and viability.

[0027] Proline and L-glutamine are amino acids that play important roles in cellular metabolism. Proline can increase the resistance and survival ability of cells during cryopreservation, while L-glutamine can provide cells with the energy and metabolic substrates they need, helping to maintain cell growth and metabolism.

[0028] Scaffold materials are materials used to support and direct the growth of cells, and can be used in tissue engineering and biomedical applications. The scaffold material in this method is a composite material composed of various materials such as sodium alginate, silk fibroin, polylactic acid, and graphene oxide. It can provide support for cell attachment and a structure for directed growth, facilitating directed cell growth and the construction of tissue engineering projects.

[0029] Basal culture medium is a commonly used medium in cell culture, containing the nutrients and growth factors required by cells. It provides the necessary nutrients and growth factors to promote cell growth and proliferation. In this method, one of the commonly used basal culture media, such as RPMI-1640, MEM, high-glucose DMEM, low-glucose DMEM, or DMEM / F12, was selected to suit different cell types.

[0030] The preparation steps in this method mainly involve physicochemical and biotechnological methods, including weighing, stirring and mixing, dialysis, and electrospinning. Among them, electrospinning is an emerging biotechnological method that can prepare fibrous scaffold materials with nanoscale dimensions, high specific surface area, and large porosity, making it suitable for directed cell growth and tissue engineering.

[0031] In summary, the cell cryopreservation solution prepared by this method has advantages such as reasonable nutrient ratio, addition of scaffold materials, simple preparation, wide applicability, and strong adjustability. It can meet the needs of biomedical fields such as cell cryopreservation and tissue engineering, and has certain innovation and advancement in the preparation of cell cryopreservation solution and scaffold materials.

[0032] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0033] 1) The cell cryopreservation solution prepared by this invention has a rich composition and contains a variety of components, such as glycerol, platelet lysate, proline, L-glutamine, etc., which can provide the nutrients and growth factors required by cells and help maintain cell stability and survival rate.

[0034] 2) The components in the cell cryopreservation solution prepared by this invention can slow down the growth rate of cells, help reduce cell damage during freezing and thawing, and improve cell survival rate and viability.

[0035] 3) The cell cryopreservation solution prepared by this invention can enhance the cell's antifreeze ability and reduce cell damage during freezing and thawing.

[0036] 4) Inspired by the fiber structure and protective function of silkworm cocoons, this invention aims to improve the low-temperature preservation effect of cells. By combining electrospinning, in-situ surface functionalization and freeze-drying, a composite scaffold material of silk protein, polylactic acid and graphene oxide was developed. It has a three-dimensional fluffy structure, which can promote cell attachment and proliferation. It also has strong thermal conductivity, thereby providing rapid and uniform temperature transfer during freeze-thaw processes. Detailed Implementation

[0037] Main source of materials:

[0038] N-hydroxysuccinimide: Molecular weight: 3400 Da.

[0039] Polyethylene oxide: Hubei Hanwei Chemical Co., Ltd., molecular weight: 230, item number: HW050052.

[0040] Silk protein: Shanghai Yuanye Biotechnology Co., Ltd., Product No.: S26299.

[0041] Polyamide-imide: Shanghai Haoxilong Plastics Co., Ltd., Model: 4203L.

[0042] Polylactic acid: Shanghai Aladdin Biochemical Technology Co., Ltd., Product No.: P169115, CAS No.: 26100-51-6.

[0043] Graphene oxide: Jiangsu Xianfeng Nanomaterials Technology Co., Ltd., item number: 100602, sheet diameter: 0.5~5μm, thickness: 0.8~1.2nm, CAS number: 7440-44-0.

[0044] Platelet lysate: Beijing Nuowei Biotechnology Co., Ltd., catalog number: 902010.

[0045] Example 1

[0046] A cell cryopreservation solution comprising the following components: 8 v / v glycerol, 5 v / v % platelet lysate, 1 w / v % proline, 0.5 w / v % L-glutamine, and 1 w / v scaffold material, supplemented with DMEM / F12.

[0047] A method for preparing a cell cryopreservation solution is as follows:

[0048] Weigh each component according to volume or weight-volume ratio, then mix each component at 10 rpm for 5 minutes to obtain the cell cryopreservation solution.

[0049] The method for preparing the scaffold material is as follows:

[0050] S1. Add 2g of sodium alginate to 200g of water at 40℃, adjust the pH to 3.4 using 0.4mol / L hydrochloric acid, then add 3g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1.8g of N-hydroxysuccinimide, stir at 40℃ and 200rpm for 5h, dialyze the reaction mixture using a dialysis tube with a molecular weight cutoff of 5000Da, and then freeze-dry to obtain the composite material;

[0051] S2. Add 1g of polyethylene oxide to 100g of 7.5wt% silk protein aqueous solution and stir at 200rpm for 3h to obtain a mixed solution; then, add 15g of polyamide-imide to the above mixed solution and stir at 45℃ and 500rpm for 12h to obtain spinning solution A.

[0052] S3. Add 5g of gelatin to 100g of 15wt% polylactic acid solution and stir at 200rpm for 30min. Then add 5g of graphene oxide and stir at 500rpm for 12h. The polylactic acid solution is prepared by mixing polylactic acid and hexaoxide isopropanol to obtain spinning solution B. Transfer spinning solution A and spinning solution B prepared in step S2 to different syringes and perform electrospinning simultaneously. The positive voltage of electrospinning is 20kV and the flow rate is fixed at 0.5mL / h. Add water to the composite material prepared in step S1 to prepare a 1wt% aqueous solution for collecting the electrospun fibers of spinning solution A and spinning solution B. After collection, let it stand for 24h, wash with water, freeze dry, and obtain the scaffold material.

[0053] The cryopreservation and thawing steps for the cells are as follows:

[0054] Human leg fat cells were added to a cell cryopreservation solution, stored at 4°C for 30 minutes, at -20°C for 2 hours, and then transferred to a -80°C freezer for long-term storage. The thawing procedure for the human leg fat cells involved removing the frozen human leg fat cells from the -80°C freezer and quickly transferring them to a 37°C water bath for gentle shaking to thaw.

[0055] Example 2

[0056] A cell cryopreservation solution is basically the same as that in Example 1, the only difference being that the preparation method of the scaffold material is different.

[0057] The method for preparing the scaffold material is as follows:

[0058] S1. Adjust the pH of 200g of water at 40℃ to 3.4 using 0.4mol / L hydrochloric acid, then add 3g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1.8g of N-hydroxysuccinimide, and stir at 40℃ and 200rpm for 5h to obtain a composite solution.

[0059] S2. Add 1g of polyethylene oxide to 100g of 7.5wt% silk protein aqueous solution and stir at 200rpm for 3h to obtain a mixed solution; then, add 15g of polyamide-imide to the above mixed solution and stir at 45℃ and 500rpm for 12h to obtain spinning solution A.

[0060] S3. Add 5g of gelatin to 100g of 15wt% polylactic acid solution and stir at 200rpm for 30min. Then add 5g of graphene oxide and stir at 500rpm for 12h. The polylactic acid solution is prepared by mixing polylactic acid and hexaoxide isopropanol to obtain spinning solution B. Transfer spinning solution A and spinning solution B prepared in step S2 to different syringes and perform electrospinning simultaneously. The positive voltage for electrospinning is 20kV and the flow rate is fixed at 0.5mL / h. Add water to the composite solution prepared in step S1 to prepare a 1wt% aqueous solution for collecting the electrospun fibers of spinning solution A and spinning solution B. After collection, let it stand for 24h, wash with water 3 times, and freeze dry to obtain the scaffold material.

[0061] The preparation method of the cell cryopreservation solution is the same as that in Example 1.

[0062] The cryopreservation and thawing steps for the cells are the same as in Example 1.

[0063] Example 3

[0064] A cell cryopreservation solution is basically the same as that in Example 1, the only difference being that the preparation method of the scaffold material is different.

[0065] The method for preparing the scaffold material is as follows:

[0066] S1. Add 2g of sodium alginate to 200g of water at 40℃, adjust the pH to 3.4 using 0.4mol / L hydrochloric acid, then add 3g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1.8g of N-hydroxysuccinimide, stir at 40℃ and 200rpm for 5h, dialyze the reaction mixture using a dialysis tube with a molecular weight cutoff of 5000Da, and then freeze-dry to obtain the composite material;

[0067] S2. Add 5g of gelatin to 100g of 15wt% polylactic acid solution and stir at 200rpm for 30min. Then add 5g of graphene oxide and stir at 500rpm for 12h. The polylactic acid solution is prepared by mixing polylactic acid and hexaoxide isopropanol to obtain spinning solution B. Transfer spinning solution B to a syringe for electrospinning. The positive voltage for electrospinning is 20kV and the flow rate is fixed at 0.5mL / h. Add water to the composite material prepared in step S1 to prepare a 1wt% aqueous solution for collecting the electrospun fibers from spinning solution B. After collection, let it stand for 24h, wash it with water 3 times, and freeze dry to obtain the scaffold material.

[0068] The preparation method of the cell cryopreservation solution is the same as that in Example 1.

[0069] The cryopreservation and thawing steps for the cells are the same as in Example 1.

[0070] Example 4

[0071] A cell cryopreservation solution is basically the same as that in Example 1, the only difference being that the preparation method of the scaffold material is different.

[0072] The method for preparing the scaffold material is as follows:

[0073] S1. Add 2g of sodium alginate to 200g of water at 40℃, adjust the pH to 3.4 using 0.4mol / L hydrochloric acid, then add 3g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1.8g of N-hydroxysuccinimide, stir at 40℃ and 200rpm for 5h, dialyze the reaction mixture using a dialysis tube with a molecular weight cutoff of 5000Da, and then freeze-dry to obtain the composite material;

[0074] S2. Add 1g of polyethylene oxide to 100g of 7.5wt% silk protein aqueous solution and stir at 200rpm for 3h to obtain a mixed solution; then, add 15g of polyamide-imide to the above mixed solution and stir at 45℃ and 500rpm for 12h to obtain spinning solution A.

[0075] S3. Transfer the spinning solution A prepared in step S2 to a syringe for electrospinning. The positive voltage for electrospinning is 20kV, and the flow rate is fixed at 0.5mL / h. Add water to the composite material to prepare a 1wt% aqueous solution for collecting the electrospun fibers from the spinning solution A. After collection, let it stand for 24h, wash it three times with water, and freeze-dry it to obtain the scaffold material.

[0076] The preparation method of the cell cryopreservation solution is the same as that in Example 1.

[0077] The cryopreservation and thawing steps for the cells are the same as in Example 1.

[0078] Comparative Example 1

[0079] A cell cryopreservation solution is basically the same as that in Example 1, except that no scaffold material is added to the cell cryopreservation solution.

[0080] The preparation method of the cell cryopreservation solution is the same as that in Example 1.

[0081] The cryopreservation and thawing steps for the cells are the same as in Example 1.

[0082] Test Example 1

[0083] Activity detection experiment

[0084] The thawed adipocyte fluid from the examples and comparative examples was washed three times with PBS buffer to remove residual cryopreservation solution, and DMEM was added to obtain adipocyte fluid ready for use.

[0085] For trypan blue staining, take 1 mL of the adipocyte solution to be used, wash with PBS, add 3 mL of type I collagenase, and digest in a 37°C air bath shaker for 30 min at a shaking speed of 180 rpm. Then add physiological saline to stop digestion, filter through a 100-mesh copper mesh, and let stand for 4 min. After the filtrate separates into layers, take 0.1 mL of the adipocyte layer below the golden-yellow oil. Add 0.1 mL of 0.4% trypan blue staining solution, mix well, and stain for 3 min. Pipette approximately 10 μL of the mixture from the side onto a hemocytometer plate with a coverslip, and observe and count under an inverted microscope. Under the microscope, dead adipocytes are stained blue, while live cells are not stained. After trypan blue staining, count the adipocytes under a microscope and calculate the adipocyte rejection rate, i.e., the survival rate. The formula for calculating the adipocyte rejection rate is as follows.

[0086] Adipocyte rejection rate = (Number of live cells / (Number of live cells + Number of dead cells)) × 100%

[0087] The experiment was repeated three times. The average value was taken. The test results are shown in Table 1.

[0088] Table 1: Results of Activity Detection Experiment

[0089] Test Plan Survival rate / % Example 1 85.5 Example 2 81.8 Example 3 80.5 Example 4 81.2 Comparative Example 1 76.3 Comparative Example 2 72.8

[0090] Test Example 2

[0091] Cell viability assay

[0092] Cell viability was assessed using the CCK-8 assay. Cell plating was performed as follows: the control group used cell-free culture medium, while the experimental group used thawed adipocyte suspension from the examples and comparative studies. The assay time was 24 hours. Cells were collected, with 100 μL of cell suspension per well. Cells were cultured at 25°C and 5% CO2 until adherence. CCK-8 assay: 10 μL of CCK-8 solution was added to the culture medium in each well after cell adhesion, and the mixture was thoroughly mixed to ensure uniform color distribution. After 4 hours of further culture, the absorbance (D) at 450 nm was measured using a microplate reader. Relative cell viability was calculated using the following formula.

[0093] Relative cell viability = (D experimental group / D control group) × 100%

[0094] Each group was tested three times, and the average value was taken. The test results are shown in Table 2.

[0095] Table 2: Cell viability test results

[0096]

[0097]

[0098] The test results of Examples 1 and 2 show that Example 1 has the best overall performance. This may be because the cell cryopreservation solution of the present invention is composed of the following components: glycerol, platelet lysate, proline, L-glutamine, scaffold material, and DMEM / F12 supplementation. The scaffold material is prepared by adding sodium alginate to water, adjusting the pH to acidic using hydrochloric acid, then adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide, stirring and dialyzing, and freeze-drying to obtain a composite material. Polyethylene oxide is added to a silk protein aqueous solution and stirred to obtain a mixed solution. Then, polyamide imide is added to the mixed solution and stirred to obtain spinning solution A. Gelatin is added to a polylactic acid solution and stirred, and graphene oxide is added to the solution and stirred to obtain spinning solution B. Spinning solutions A and B are electrospun separately. The composite material is added to water to prepare an aqueous solution for collecting the electrospun fibers from spinning solutions A and B. The solution is allowed to stand for reaction, washed with water, and freeze-dried to obtain the scaffold material.

[0099] The ability to slow ice crystal growth and recrystallization is essential in the cryopreservation of cells and tissues to avoid damage associated with the physical and chemical reactions of freezing. Achieving rapid and uniform thawing is critical, as it reduces cryogenic damage to cells and enhances cell recovery.

[0100] The scaffold material of this invention has a three-dimensional structure, which not only provides a suitable microenvironment for cell growth and activity but also enhances the cells' resistance to cryoprotectants, thereby improving cell viability after thawing and achieving effective cryopreservation. Silk fibroin, polylactic acid, and graphene oxide provide mechanical support and protection. The protective function of silk fibroin stems from its delicate layered structure and unique composition; the combination of silk fibroin with the scaffold material improves the cryopreservation effect. The key lies in the good biocompatibility and biodegradability of silk fibroin attached to the scaffold. However, silk fibroin alone suffers from poor mechanical properties and low thermal conductivity. This invention prepares electrostatic fibers through electrostatic composite, which contain silk fibroin, polylactic acid, and graphene oxide, constructing a composite scaffold material. Using an aqueous solution of the composite material as the receiving material activates the carboxyl groups of silk fibroin in the fiber, reacting with the amino groups of polyamide-imide, and fixing the silk fibroin within the fiber, enhancing the fiber's hydrophilicity and improving cell adhesion. Sodium alginate solution can partially react with gelatin, fixing sodium alginate onto the fiber surface. Alginic acid solution can also act as an auxiliary solvent, spontaneously diffusing into the fiber network to increase the three-dimensional space between fibers, thereby generating a uniform and continuous three-dimensional fiber framework. This may improve spatial heat transfer during cell cryopreservation. Furthermore, sodium alginate modification can also enhance the hydrophilicity of the scaffold. The three-dimensional porous structure creates a natural growth environment for cells, significantly shortening thawing time and inhibiting ice crystal growth. This allows cryopreserved cells to maintain high cell viability after thawing and restore cell proliferation capacity during freeze-thaw cycles, thus achieving effective long-term cryopreservation.

[0101] In summary, Embodiment 1 of this invention draws inspiration from the fibrous structure and protective function of silkworm cocoons to improve the cryopreservation effect of cells. A composite scaffold material of silk protein, polylactic acid, and graphene oxide was developed using a combination of electrospinning, in-situ surface functionalization, and freeze-drying. This material possesses a three-dimensional, fluffy structure that promotes cell attachment and proliferation, and exhibits high thermal conductivity, thereby providing rapid and uniform temperature transfer during freeze-thaw cycles.

Claims

1. A cell cryopreservation solution, characterized in that, It contains the following components: glycerol, platelet lysate, proline, L-glutamine, scaffold material, and basal culture medium supplement; The preparation method of the scaffold material is as follows, in parts by weight: S1. Add 1-3 parts of sodium alginate to 180-220 parts of water at 30-50℃, adjust the pH to 3-4 using 0.2-0.5 mol / L hydrochloric acid, then add 1-5 parts of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1.5-2 parts of N-hydroxysuccinimide, stir at 30-50℃ and 100-300 rpm for 3-8 hours, dialyze the reaction mixture using a dialysis tube with a molecular weight cutoff of 4000-6000 Da, and then freeze-dry to obtain the composite material; S2. Add 0.5-2 parts of polyethylene oxide to 80-120 parts of 6-9 wt% silk protein aqueous solution, stir at 100-300 rpm for 1-5 h to obtain a mixed solution; then, add 10-20 parts of polyamide-imide to the above mixed solution, stir at 40-50℃ and 300-800 rpm for 8-15 h to obtain spinning solution A; S3. Add 3-8 parts of gelatin to 80-120 parts of 10-20 wt% polylactic acid solution and stir at 100-300 rpm for 10-50 min. Then add 3-8 parts of graphene oxide and stir at 300-800 rpm for 8-15 h to obtain spinning solution B. Transfer the spinning solution A and spinning solution B prepared in step S2 to different syringes and perform electrospinning simultaneously. Add water to the composite material to prepare a 0.5-2 wt% aqueous solution for collecting the electrospun fibers from spinning solution A and spinning solution B. After collection, let it stand for 10-30 h, wash with water, and freeze dry to obtain the scaffold material.

2. The cell cryopreservation solution as described in claim 1, characterized in that, The glycerol content in the cell cryopreservation solution is 5-10 v / v.

3. The cell cryopreservation solution as described in claim 1, characterized in that, The basal culture medium is one of RPMI-1640, MEM, high-glucose DMEM, low-glucose DMEM, or DMEM / F12.

4. The cell cryopreservation solution as described in claim 1, characterized in that, The platelet lysate constitutes 3-8 v / v in the cell cryopreservation solution.

5. The cell cryopreservation solution as described in claim 1, characterized in that, The proline content in the cell cryopreservation solution is 0.5~2 w / v.

6. The cell cryopreservation solution as described in claim 1, characterized in that, The L-glutamine was present in the cell cryopreservation solution at a concentration of 0.4–0.6 w / v.

7. The cell cryopreservation solution as described in claim 1, characterized in that, The scaffold material accounts for 0.5~2 w / v in the cell cryopreservation solution.

8. A method for preparing the cell cryopreservation solution according to any one of claims 1 to 7, characterized in that, The steps are as follows: Weigh each component according to the volume or weight-volume ratio, and then stir and mix each component at 5~20 rpm for 3~10 minutes to obtain the cell cryopreservation solution.

9. A method for cryopreserving and reviving cells using the cell cryopreservation solution as described in any one of claims 1 to 7, characterized in that, The steps are as follows: add the fat cells to the cell cryopreservation solution, store at 1~5℃ for 10~40 min, store at -10~-30℃ for 1~3 h, and then transfer to a -70~-90℃ freezer for long-term storage; the thawing step of the fat cells is to take the frozen fat cells out of the -70~-90℃ freezer, quickly transfer them to a 35~38℃ water bath and gently shake to thaw.

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