Cryopreservation solution suitable for fibroblasts as well as preparation method and application of cryopreservation solution

Abstract

The invention discloses a cryopreservation solution suitable for fibroblasts and a preparation method and application thereof, and relates to the technical field of cell cryopreservation solutions, the cryopreservation solution suitable for fibroblasts is prepared from the following components: a cryoprotectant, a cell nutrient, an antioxidant and a solvent, and does not contain animal-derived serum or dimethyl sulfoxide; the cryoprotectant comprises a propylene glycol solution and an ethylene glycol solution, trehalose is adopted as the cell nutrient; glutathione is adopted as the antioxidant; the volume ratio of the propylene glycol solution to the ethylene glycol solution to the trehalose to the glutathione to the compound electrolyte is 10: 10: 5: 1: 74; the fibroblast cryopreservation liquid is simple in component, free of DSMO and animal-derived serum, simple and convenient in preparation process, long in validity period, convenient in storage condition, free of programmed cooling and suitable for cryopreservation of fibroblasts; the recovery rate of the cryopreserved living cells is higher than 75.0%, the average recovery rate is up to 85%, the proliferation capacity of the cryopreserved and recovered cells is high, and the cell activity is kept stable.

Description

Technical Field

[0001] The present invention relates to the field of cell cryopreservation technology, in particular to a cryopreservation solution suitable for fibroblasts and a preparation method and application thereof. Background Art

[0002] During the subculture and daily maintenance of cell culture, a large amount of culture equipment, culture medium and various preparations are required. Once the cells leave the living body and begin primary culture, their various biological characteristics will gradually change and continue to change with the increase in the number of subcultures and changes in in vitro environmental conditions. Therefore, it is necessary to cryopreserve cells in a timely manner. Cells can be stored frozen in a -80℃ refrigerator for up to one year; cells stored in liquid nitrogen at a temperature of -196℃, theoretically, the storage time is unlimited and suitable for any type of cells.

[0003] Fibroblasts are the main cell components of loose connective tissue and have important application value in the fields of skin wound repair and tissue engineering. In order to preserve fibroblasts for a long time and maintain their biological activity, cell cryopreservation is usually required.

[0004] Currently, the commonly used cell freezing solution is mostly composed of dimethyl sulfoxide (DMSO) as the main component, and serum, culture medium, etc. are added. However, DMSO has certain cytotoxicity and may affect the activity and function of cells after recovery. In addition, serum components are complex, there is a potential risk of pathogen contamination, and the quality differences between different batches are large, which is not conducive to the standardization and quality control of freezing solutions.

[0005] Current technical deficiencies:

[0006] (1) Currently, there is no cryopreservation solution specifically for fibroblasts in the field of cell cryopreservation. Common stem cell cryopreservation solutions are usually used for cryopreservation.

[0007] (2) Commonly used cryopreservation fluids contain DMSO, which has certain cytotoxicity and may affect the activity and function of fibroblasts after recovery. Serum is also added, which has a complex composition and poses a potential risk of pathogen contamination. In addition, the quality of different batches varies greatly, which is not conducive to the standardization and quality control of cryopreservation fluids.

[0008] (3) Even if fibroblasts can survive after cryopreservation, their proliferation ability, differentiation potential, and secretory function may be affected, resulting in unsatisfactory performance of the cells in subsequent applications.

[0009] Currently, in the field of cell cryopreservation fluid, there is no cryopreservation fluid specifically for fibroblasts, and common stem cell cryopreservation fluid is usually used for cryopreservation; therefore, it is of great significance to develop a cryopreservation fluid suitable for fibroblasts that is efficient, low-toxic, has stable ingredients and is easy to standardize. Summary of the invention

[0010] In view of this, the present invention aims to address the deficiencies in the prior art, and its main purpose is to provide a freezing solution suitable for fibroblasts, a preparation method and an application thereof. The freezing solution has simple ingredients, does not contain DSMO, does not contain animal serum, has a simple preparation process, a long shelf life, convenient storage conditions, and does not require programmed cooling. After use, the freezing solution can be directly transferred to a liquid nitrogen tank for storage, and is suitable for the freezing of fibroblasts. The recovery rate of cryopreserved live cells is higher than 75.0%, with an average recovery rate of up to 85%. The cells have strong proliferation ability after freezing and recovery, and the cell activity remains stable.

[0011] To achieve the above purpose, the present invention adopts the following technical solutions:

[0012] A freezing solution suitable for fibroblasts is prepared from the following ingredients: a cryoprotectant, a cell nutrient, an antioxidant and a solvent, which does not contain animal serum and dimethyl sulfoxide; the cryoprotectant includes a propylene glycol solution and an ethylene glycol solution; the cell nutrient uses trehalose; the antioxidant uses glutathione; the solvent is a compound electrolyte solution; wherein, according to the volume ratio, propylene glycol solution: ethylene glycol solution: trehalose: glutathione: compound electrolyte solution = 10:10:5:1:74.

[0013] As a preferred solution: the concentration of the propylene glycol solution is 20%, the concentration of the ethylene glycol solution is 40%, the concentration of trehalose is 0.05-0.10 mol / L, and the concentration of glutathione is 0.1 mol / L.

[0014] As a preferred solution: the cell freezing density of the freezing solution is 5×10 5 cells / ml to 4×10 7 cells / ml.

[0015] As a preferred solution: after the cells are resuspended in the freezing solution, they are placed in a freezing box, cooled without a programmed cooling device or manual program, and stored at -80°C, and directly transferred to a liquid nitrogen tank for long-term storage.

[0016] As a preferred embodiment: the components of the composite electrolyte solution are sodium chloride, sodium gluconate, sodium acetate, potassium chloride and magnesium chloride.

[0017] The preparation method of the freezing solution suitable for fibroblasts comprises the following steps: propylene glycol solution, ethylene glycol solution, trehalose, glutathione and compound electrolyte solution are sequentially added and mixed in a volume ratio of 10:10:5:1:74, and the mixture is fully blown and sucked to form a mixed solution; the mixed solution is sterilized by filtering at 0.22 μm with a syringe to obtain the freezing solution suitable for fibroblasts; the freezing solution is stored at -20°C for a long term and at 4-10°C for a short term.

[0018] As a preferred solution, the specific steps of forming the mixed solution are: first, use a pipette to suck up propylene glycol solution, ethylene glycol solution, trehalose and glutathione in sequence, add them to a centrifuge tube in sequence, and mix them by suction and blowing; then use a pipette to suck up the compound electrolyte solution twice and add it to the centrifuge tube, and mix it by suction and blowing to form a mixed solution.

[0019] As a preferred solution, the specific steps of using a syringe to filter and sterilize the mixed solution at 0.22 μm are as follows: using a syringe to absorb the mixed cryopreservation solution, connecting a 0.22 μm disposable filter to the syringe, slowly pumping out the cryopreservation solution, and filtering and sterilizing.

[0020] As a preferred solution: the filtered and sterilized cryopreserved solution is transferred to a sterile bottle, sealed with a lid, and stored in a refrigerator at 4°C.

[0021] The application of the freezing solution suitable for fibroblasts is that the freezing solution is applied to the cryopreservation of fibroblasts.

[0022] Compared with the prior art, the present invention has obvious advantages and beneficial effects. Specifically, it can be seen from the above technical scheme that by providing a fibroblast cryopreservation solution, the composition is simple, does not contain DSMO, does not contain animal serum, has a simple preparation process, a long shelf life, convenient storage conditions, and no program cooling is required. After use, it can be directly transferred to a liquid nitrogen tank for storage, which is suitable for the cryopreservation of fibroblasts; the viability of cells frozen by the cryopreservation solution provided by the present application after recovery is as high as 95.0% or more, the recovery rate of frozen live cells is higher than 75.0%, the average recovery rate is as high as 85%, the cells after cryopreservation and recovery have strong proliferation ability, and the cell activity remains stable.

[0023] In order to more clearly illustrate the structural features and effects of the present invention, it is described in detail below in conjunction with the accompanying drawings and specific embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a schematic diagram of the number of cells cultured after recovery in Example 2 of the effect of the present invention;

[0025] Figure 2 This is a schematic diagram of the viability of cells cultured after recovery in Example 2 of the present invention. DETAILED DESCRIPTION

[0026] The present invention Figure 1 to Figure 2As shown, a freezing solution suitable for fibroblasts is prepared from the following ingredients: a cryoprotectant, a cell nutrient, an antioxidant and a solvent, which does not contain animal serum and dimethyl sulfoxide; the cryoprotectant includes a propylene glycol solution and an ethylene glycol solution; the cell nutrient is trehalose; the antioxidant is glutathione; the solvent is a compound electrolyte solution; wherein, according to the volume ratio, propylene glycol solution: ethylene glycol solution: trehalose: glutathione: compound electrolyte solution = 10:10:5:1:74.

[0027] The concentration of the propylene glycol solution is 20%, the concentration of the ethylene glycol solution is 40%, the concentration of trehalose is 0.05-0.10 mol / L, and the concentration of glutathione is 0.1 mol / L.

[0028] The cell freezing density of this freezing solution is 5×10 5 cells / ml to 4×10 7 cells / ml.

[0029] After the cells are resuspended in the freezing solution, they are placed in a freezing box and directly transferred to a liquid nitrogen tank for long-term storage without being cooled by a programmed cooling device or a manual program or temporarily stored at -80°C.

[0030] The components of the composite electrolyte solution are sodium chloride, sodium gluconate, sodium acetate, potassium chloride and magnesium chloride.

[0031] The preparation method of the cryopreservation solution suitable for fibroblasts comprises the following steps: adding propylene glycol solution, ethylene glycol solution, trehalose, glutathione and compound electrolyte solution in order according to a volume ratio of 10:10:5:1:74 and mixing them, and fully blowing and sucking to evenly form a mixed solution; using a syringe to filter and sterilize the mixed solution at 0.22 μm to obtain the cryopreservation solution suitable for fibroblasts; the cryopreservation solution is stored at -20°C for a long term and at 4-10°C for a short term; the cryopreservation solution can be stored for a long term of up to 12 months in a -20°C environment, and can be stored for a short term of 3 months in a 4-10°C environment.

[0032] The specific steps of forming the mixed solution are: first, using a pipette to sequentially absorb propylene glycol solution, ethylene glycol solution, trehalose and glutathione, and sequentially add them into a centrifuge tube by suction and blowing to mix; then, using a pipette to absorb the compound electrolyte solution twice and add it into the centrifuge tube, and suction and blowing to mix to form a mixed solution.

[0033] The specific steps of using a syringe to filter and sterilize the mixed solution at 0.22 μm are as follows: using a syringe to absorb the mixed cryopreservation solution, connecting a 0.22 μm disposable filter to the syringe, and slowly pumping out the cryopreservation solution for filtering and sterilization.

[0034] The filtered and sterilized cryopreserved solution was transferred to a sterile bottle, sealed with a lid, and stored in a 4°C refrigerator.

[0035] The application of the freezing solution suitable for fibroblasts is applied to the cryopreservation of fibroblasts.

[0036] Resuscitation Example

[0037] 1. Prepare reagents: propylene glycol solution, ethylene glycol solution, trehalose, glutathione, compound electrolyte solution, DPBS buffer, trypsin, DMEM basal medium. Prepare consumables: sterile square bottles, centrifuge tubes, pipettes, disposable filters, disposable syringes, cryopreservation tubes, cell culture bottles.

[0038] 2. Prepare fibroblast freezing solution: first use a 5mL pipette to take 5mL of propylene glycol solution, 5mL of ethylene glycol solution, 2.5mL of trehalose, and 0.5mL of glutathione in sequence, add them to a centrifuge tube in sequence, and mix them by suction and blowing. Then use a 25mL pipette to take 37mL of compound electrolyte solution in two times and add them to the above centrifuge tube, and mix them by suction and blowing.

[0039] 3. Filtration and sterilization: Remove the needle cap of the disposable syringe, insert the needle into the mixed liquid, pull the piston handle of the syringe, absorb the mixed cryopreservation liquid, cover the needle cap, remove the needle part, connect a 0.22um disposable filter to the needle mouth of the syringe, slowly push the piston handle to slowly eject the cryopreservation liquid for filtration and sterilization.

[0040] 4. Storage: Transfer the filtered and sterilized cryopreserved solution into a sterile square bottle, seal it with a lid, and store it in a refrigerator at 4°C.

[0041] 5. After the fibroblasts from the four groups of samples from different sources were cultured until the cell confluence reached 80-90%, the culture supernatant was discarded, 10 mL of PBS buffer was added to gently wash the cells, and the washing solution was discarded.

[0042] 6. Add 5 mL of 0.25% trypsin preheated at 37°C to digest the cells; then place in a 37°C incubator to digest for 3-5 minutes.

[0043] 7. Observe under the microscope that the adherent cells become round and fall off. Use a pipette to gently blow off the cells and transfer the cell suspension into a 50mL centrifuge tube.

[0044] 8. Mix the cell suspension by pipetting and blowing, and take a sample to count using an automatic cell counter.

[0045] Example 1: The first group of harvested fibroblasts were divided into two groups according to the number of viable cells, 9.99×10 6 100 living cells / group, one of which was resuspended and mixed with 2 mL of prepared fibroblast freezing solution, placed in a cryopreservation tube, sealed with a lid, and transferred to a liquid nitrogen tank for freezing.

[0046] Example 2: The second group of harvested fibroblasts were divided into two groups according to the number of viable cells, 9.99×10 6 100 living cells / group, one of which was resuspended and mixed with 2 mL of prepared fibroblast freezing solution, placed in a cryopreservation tube, sealed with a lid, and transferred to a liquid nitrogen tank for freezing.

[0047] Example 3: The fibroblasts harvested from the third group were divided into two groups according to the number of living cells, 10.00×10 6 100 living cells / group, one of which was resuspended and mixed with 2 mL of prepared fibroblast freezing solution, placed in a cryopreservation tube, sealed with a lid, and transferred to a liquid nitrogen tank for freezing.

[0048] Example 4: The fibroblasts harvested from the fourth group were divided into two groups according to the number of living cells, 10.00×10 6 100 living cells / group, one of which was resuspended and mixed with 2 mL of prepared fibroblast freezing solution, placed in a cryopreservation tube, sealed with a lid, and transferred to a liquid nitrogen tank for freezing.

[0049] Comparative Example 1: Another group of cells equally divided in Example 1 was resuspended and mixed with 2 mL of freezing solution containing 10% DMSO, placed in a freezing tube, sealed with a lid, transferred to a -80°C refrigerator overnight, and then transferred to a liquid nitrogen tank for freezing.

[0050] Comparative Example 2: Another group of cells equally divided in Example 2 was resuspended and mixed with 2 mL of freezing solution containing 10% DMSO, placed in a freezing tube, sealed with a lid, transferred to a -80°C refrigerator overnight, and then transferred to a liquid nitrogen tank for freezing.

[0051] Comparative Example 3: Another group of cells equally divided in Example 3 was resuspended and mixed with 2 mL of freezing solution containing 10% DMSO, placed in a freezing tube, sealed with a lid, transferred to a -80°C refrigerator overnight, and then transferred to a liquid nitrogen tank for freezing.

[0052] Comparative Example 4: Another group of cells equally divided in Example 4 was resuspended and mixed with 2 mL of freezing solution containing 10% DMSO, placed in a freezing tube, sealed with a lid, transferred to a -80°C refrigerator overnight, and then transferred to a liquid nitrogen tank for freezing.

[0053] Effect Example 1

[0054] After one month of freezing, the four groups of samples were taken out of the liquid nitrogen tank and revived in a 37.0℃ water bath. The revived cells were resuspended in DPBS buffer to a fixed volume, centrifuged and washed, the supernatant was discarded after washing, and then resuspended and mixed with DPBS buffer, and the samples were counted at a fixed volume. The counting data are shown in Table 1:

[0055] Table 1: Data of four groups of samples after cryopreservation and resuscitation

[0056]

[0057] The data of the above four groups of samples showed that there was no significant difference in the number and viability of cells after recovery in each group of samples under the protection of two different freezing solutions (p<0.05).

[0058] Effect Example 2

[0059] Post-thaw culture expansion

[0060] 1. In total, 8 groups of cells were recovered from the samples in Examples 1 to 4 and Comparative Examples 1 to 4. 5.25×10 6 Live cells were inoculated into three cell culture flasks using DMEM basal medium at a density of 10000×10 4 Pieces / cm 2 .

[0061] 2. When the confluence of the cultured cells reaches 80-90%, discard the culture supernatant, add 10 mL of DPBS buffer to gently wash the cells, and then discard the wash solution.

[0062] 3. Add 5 mL of 0.25% trypsin preheated at 37°C to digest the cells; then place in a 37°C incubator to digest for 3-5 minutes.

[0063] 4. Observe under the microscope that the adherent cells become round and fall off. Use a pipette to gently blow off the cells and transfer the cell suspension into a 50mL centrifuge tube.

[0064] 5. Mix the cell suspension by suction and blowing, take a sample and count it using an automatic cell counter to obtain the cell number and viability of Examples 1 to 4 and Comparative Examples 1 to 4.

[0065] 6. According to the counting results, press 10000×10 4 Pieces / cm 2 Continue to inoculate cell culture flasks at the same density and continue to culture and pass it down.

[0066] Follow the above process and culture until P7.

[0067] Table 2: Multiplication multiples data of Examples 1 to 4 and Comparative Examples 1 to 4

[0068] Multiplication times P4 P5 P6 P7 Example 1 2.80 2.71 2.68 2.62 Example 2 2.81 2.75 2.69 2.66 Example 3 2.79 2.73 2.66 2.60 Example 4 2.82 2.69 2.68 2.65 Comparative Example 1 2.55 2.45 2.39 2.34 Comparative Example 2 2.54 2.47 2.42 2.37 Comparative Example 3 2.47 2.45 2.38 2.37 Comparative Example 4 2.57 2.52 2.42 2.32

[0069] Table 3: Number of cells in Examples 1 to 4 and Comparative Examples 1 to 4

[0070]

[0071] Table 4: Cell viability of Examples 1 to 4 and Comparative Examples 1 to 4

[0072] Cell viability (%) P3 P4 P5 P6 P7 Example 1 98.90 97.80 97.70 98.00 98.10 Example 2 98.60 98.00 97.50 98.10 98.00 Example 3 98.80 98.10 97.60 98.30 98.10 Example 4 98.20 97.80 97.50 98.20 98.20 Comparative Example 1 98.40 95.00 94.90 95.50 95.70 Comparative Example 2 98.00 94.50 94.70 95.60 95.40 Comparative Example 3 98.40 94.30 94.30 95.30 95.70 Comparative Example 4 98.70 94.80 94.40 95.50 95.60

[0073] The number and viability of cells cultured and expanded by the samples of Examples 1 to 4 are better than those of the samples of Comparative Examples 1 and 4, and the cell proliferation ability and activity are maintained in a good state and are not affected by the freezing process; the use of the freezing solution is suitable for the freezing of fibroblasts, and the frozen cells can be directly transferred to a liquid nitrogen tank for long-term storage, and the cell viability after recovery is high and the proliferation ability is strong; the cell viability after recovery of the cells frozen by the freezing solution provided by the present application is as high as 95.0% or more, the recovery rate of frozen live cells is higher than 75.0%, and the average recovery rate is as high as 85%, the cell proliferation ability after freezing and recovery is strong, and the cell activity remains stable.

[0074] The design focus of the present invention is to provide a fibroblast cryopreservation solution, which has simple ingredients, does not contain DSMO, does not contain animal serum, has a simple preparation process, has a long shelf life, has convenient storage conditions, does not require programmed cooling, and can be directly transferred to a liquid nitrogen tank for storage after use, and is suitable for the cryopreservation of fibroblasts.

[0075] The above description is only a preferred embodiment of the present invention and does not limit the technical scope of the present invention. Therefore, any slight modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A freezing solution suitable for fibroblasts, characterized in that: The invention is prepared from the following ingredients: a cryoprotectant, a cell nutrient, an antioxidant and a solvent, and does not contain animal serum or dimethyl sulfoxide; the cryoprotectant includes a propylene glycol solution and an ethylene glycol solution; the cell nutrient is trehalose; the antioxidant is glutathione; and the solvent is a compound electrolyte solution; Among them, according to the volume ratio, propylene glycol solution: ethylene glycol solution: trehalose: glutathione: compound electrolyte solution = 10: 10：5：1：74。 2. The freezing solution for fibroblasts according to claim 1, characterized in that: The concentration of the propylene glycol solution is 20%, the concentration of the ethylene glycol solution is 40%, the concentration of trehalose is 0.05-0.10 mol / L, and the concentration of glutathione is 0.1 mol / L.

3. The freezing solution for fibroblasts according to claim 1, characterized in that: The cell freezing density of the freezing solution is 5×10 5 cells / ml to 4×10 7 cells / ml.

4. The freezing solution for fibroblasts according to claim 1, characterized in that: After the cells are resuspended in the freezing solution, they are placed in a freezing box, cooled without a programmed cooling device or manual program, and temporarily stored at -80°C, and directly transferred to a liquid nitrogen tank for long-term storage.

5. The freezing solution for fibroblasts according to claim 1, characterized in that: The components of the composite electrolyte solution are sodium chloride, sodium gluconate, sodium acetate, potassium chloride and magnesium chloride.

6. A method for preparing a freezing solution suitable for fibroblasts according to any one of claims 1 to 5, characterized in that: The method comprises the following steps: adding propylene glycol solution, ethylene glycol solution, trehalose, glutathione and compound electrolyte solution in a volume ratio of 10:10:5:1:74 in sequence and mixing, and fully blowing and sucking to form a mixed solution; using a syringe to filter the mixed solution through 0.22 μm for sterilization to obtain a cryopreservation solution suitable for fibroblasts; and storing the cryopreservation solution at -20°C for a long term and at 4-10°C for a short term.

7. The method for preparing a freezing solution suitable for fibroblasts according to any one of claim 6, characterized in that: The specific steps of forming the mixed solution are: first, using a pipette to sequentially absorb propylene glycol solution, ethylene glycol solution, trehalose and glutathione, and sequentially add them into a centrifuge tube by suction and blowing to mix; then, using a pipette to absorb the compound electrolyte solution twice, add it into the centrifuge tube, and suction and blow to mix to form a mixed solution.

8. The method for preparing a freezing solution suitable for fibroblasts according to any one of claim 6, characterized in that: The specific steps of using a syringe to filter and sterilize the mixed solution at 0.22 μm are as follows: using a syringe to absorb the mixed cryopreservation solution, connecting a 0.22 μm disposable filter to the syringe, and slowly pumping out the cryopreservation solution for filtering and sterilization.

9. The method for preparing a freezing solution suitable for fibroblasts according to any one of claim 8, characterized in that: The filtered and sterilized cryopreserved solution was transferred to a sterile bottle, sealed with a lid, and stored in a refrigerator at 4°C.

10. A use of the freezing solution for fibroblasts according to any one of claims 1 to 5, characterized in that: The freezing solution is used for the cryopreservation of fibroblasts.

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