Cell freezing medium and application

Abstract

The invention belongs to the technical field of cell cryopreservation solutions, and particularly relates to a cell cryopreservation solution and application thereof.The cell cryopreservation solution is prepared from, by volume, 5%-30% of cell culture fluid, 45%-70% of calf serum, 10% of dimethyl sulfoxide, 2% of 3% glutamine, 3% of 7.5% sodium bicarbonate and 10% of prepared solution R. The cryopreservation solution is applied to liver cell cryopreservation and resuscitation, and the cryopreservation effect is good. According to the improved cell cryopreservation liquid, the cell survival rate can reach 90% or above, the cell adherence rate can reach 80% or above, the survival rate is remarkably improved compared with the conventional survival rate, the cell growth quality and biological characteristics are guaranteed, the proliferation cycle of cell resuscitation is shortened, resuscitated cells can be in time, and the cell resuscitation efficiency is improved. And quality and quantity can be guaranteed for biological experiment research and production.

Description

Technical Field

[0001] The invention belongs to the technical field of cell freezing solution, and in particular relates to a cell freezing solution and application thereof. Background Art

[0002] With the rapid development of biotechnology today, we are increasingly using tissue cell culture and reproduction technology in the research of biotechnology and related sciences. Tissue cells with research significance or application prospects are preserved at low temperatures for a long time. Once necessary, all cells can be resuscitated at any time to restore their complete morphological structure and biological characteristics for use in life science research. Cell freezing and resuscitation are key technologies that directly affect the success of cell proliferation.

[0003] Cryopreservation fluid is a necessary solution for cell cryopreservation. The function of the cryopreservation fluid is to disperse the cells evenly in the solution, provide the cells with an evenly distributed environment and space, and at the same time supply the nutrients necessary for the cell's life metabolism, preventing or reducing the damage of frozen ice crystals to the cells.

[0004] For a long time, a large number of domestic and foreign books on biological cells have introduced conventional cryopreservation solution formulas, and the cryopreservation solution formulas we have used for a long time are: cell culture medium 70-80%, bovine serum 10-20%, dimethyl sulfoxide 10%. Commonly used culture solutions such as MEM, DMEM, HyClone, CD293, HIPP-T009, etc. have been commercialized on the market.

[0005] Using the conventional freezing solution, the survival rate of frozen cells observed under an ordinary optical microscope after 12 hours of revival culture is generally around 60%. When the survival rate is less than 60%, the cells grow and reproduce slowly, the morphology is irregular, the cells are relatively small, the growth is relatively poor, and basically cannot survive. When the cell survival rate is above 60%, the cells are more evenly distributed, there are more living cells, the morphology is relatively normal, and it takes 6-8 days to grow a full monolayer.

[0006] Through a large number of actual biological cytology experiments and a large amount of statistical data, it is concluded that the survival rate of cells frozen with the existing freezing solution after thawing is generally 50-70%, which is relatively low, and the cell adhesion rate is 30-50%. Summary of the invention

[0007] In order to solve the problems in the prior art, the present invention provides a cell freezing solution and application thereof, so as to achieve the purpose of improving the survival rate of frozen cells, ensuring the quality of cell growth and shortening the time of cell proliferation.

[0008] The present invention solves the technical problem by adopting the following technical solutions:

[0009] The present invention aims to provide a cell freezing solution, comprising the following raw materials in volume percentage: 5-30% cell culture solution, 45-70% calf serum, 10% dimethyl sulfoxide, 2% 3% glutamine, 3% 7.5% sodium bicarbonate and 10% preparation solution R.

[0010] Further, the prepared solution R is composed of the following parts: 110-140 mg / mL D-glucose, 90-120 mg / mL hydroxyethyl starch, 70-100 mg / mL lactobionic acid, 30-60 mg / mL polyvinyl pyrrolidone, 30-60 mg / mL D-raffinose pentahydrate, 11-14 mg / mL potassium hydroxide, 7-10 mg / mL potassium dihydrogen phosphate, 2-5 mg / mL magnesium sulfate heptahydrate, 2-5 mg / mL adenosine, 60-90 μmol / L vitamin C and 15-45 μmol / L vitamin E.

[0011] Furthermore, the invention comprises the following raw materials in volume percentage: 5% cell culture medium, 70% calf serum, 10% dimethyl sulfoxide, 2% 3% glutamine, 3% 7.5% sodium bicarbonate and 10% prepared solution R; the prepared solution R comprises 110 mg / mL D-glucose, 90 mg / mL hydroxyethyl starch, 70 mg / mL lactobionic acid, 30 mg / mL polyvinyl pyrrolidone, 30 mg / mL D-raffinose pentahydrate, 11 mg / mL potassium hydroxide, 7 mg / mL potassium dihydrogen phosphate, 2 mg / mL magnesium sulfate heptahydrate, 2 mg / mL adenosine, 60 μmol / L vitamin C and 15 μmol / L vitamin E.

[0012] Furthermore, the invention comprises the following raw materials in volume percentage: 10% cell culture medium, 65% calf serum, 10% dimethyl sulfoxide, 2% 3% glutamine, 3% 7.5% sodium bicarbonate and 10% prepared solution R; the prepared solution R comprises 120 mg / mL D-glucose, 100 mg / mL hydroxyethyl starch, 80 mg / mL lactobionic acid, 40 mg / mL polyvinyl pyrrolidone, 40 mg / mL D-raffinose pentahydrate, 12 mg / mL potassium hydroxide, 8 mg / mL potassium dihydrogen phosphate, 3 mg / mL magnesium sulfate heptahydrate, 3 mg / mL adenosine, 70 μmol / L vitamin C and 25 μmol / L vitamin E.

[0013] Furthermore, the invention comprises the following raw materials in volume percentage: 20% cell culture medium, 55% calf serum, 10% dimethyl sulfoxide, 2% 3% glutamine, 3% 7.5% sodium bicarbonate and 10% prepared solution R; the prepared solution R comprises 130 mg / mL D-glucose, 110 mg / mL hydroxyethyl starch, 90 mg / mL lactobionic acid, 50 mg / mL polyvinyl pyrrolidone, 50 mg / mL D-raffinose pentahydrate, 13 mg / mL potassium hydroxide, 9 mg / mL potassium dihydrogen phosphate, 4 mg / mL magnesium sulfate heptahydrate, 4 mg / mL adenosine, 80 μmol / L vitamin C and 35 μmol / L vitamin E.

[0014] Furthermore, the invention comprises the following raw materials in volume percentage: 30% cell culture medium, 45% calf serum, 10% dimethyl sulfoxide, 2% 3% glutamine, 3% 7.5% sodium bicarbonate and 10% prepared solution R; the prepared solution R comprises 140 mg / mL D-glucose, 120 mg / mL hydroxyethyl starch, 100 mg / mL lactobionic acid, 60 mg / mL polyvinyl pyrrolidone, 60 mg / mL D-raffinose pentahydrate, 14 mg / mL potassium hydroxide, 10 mg / mL potassium dihydrogen phosphate, 5 mg / mL magnesium sulfate heptahydrate, 5 mg / mL adenosine, 90 μmol / L vitamin C and 45 μmol / L vitamin E.

[0015] A cell freezing solution is used in the freezing of hepatocytes. The freezing process of hepatocytes is as follows: culturing HL2, TG2, TG3, TGC4 cells grown into a monolayer, 1 bottle each → adding PBS with pH 7.0 to wash the cell surface once → digesting with 0.25% trypsin for 2 minutes → discarding the trypsin → adding 10 mL of culture solution, blowing and mixing the cell suspension → centrifuging at 1000 rpm / 5 minutes → discarding the supernatant → adding the improved freezing solution respectively, adjusting the cell concentration to (1±0.2)×10 7 viable cells / mL→dispense into 1mL / tube→put into programmed cooling box→-80℃ overnight→immerse in liquid nitrogen for long-term storage.

[0016] A cell freezing solution is used in liver cell recovery. The liver cell recovery process is as follows: take out HL2, TG2, TG3, and TGC4 cells frozen with each component of the freezing solution from liquid nitrogen, one tube each → place in a 37°C water bath to dissolve → centrifuge at 1000rpm / 3min → discard the supernatant → add 10mL of 20% calf serum culture solution → culture at 37°C.

[0017] The cell culture medium in the present invention can be any one of the following: MEM, DMEM, HyClone, CD293, HIPP-T009.

[0018] The cryopreservation solution of the present invention is applied to the freezing and recovery of liver cells, and adopts cell culture fluid, calf serum, dimethyl sulfoxide, glutamine, sodium bicarbonate, and solution R. The solution R is composed of the following parts: D-glucose, hydroxyethyl starch, lactobionic acid, polyvinyl pyrrolidone, D-raffinose pentahydrate, potassium hydroxide, potassium dihydrogen phosphate, magnesium sulfate heptahydrate, adenosine, vitamin C, and vitamin E. All of them are prepared in a certain proportion. The survival rate of frozen cells can be increased to more than 90%, and the cell adhesion rate can reach more than 80%, thereby ensuring the quality of cell growth, shortening the proliferation cycle of cell recovery, and saving time cost.

[0019] Compared with the prior art, the beneficial technical effects of the present invention are:

[0020] The improved cell freezing solution of the present invention can achieve a cell survival rate of more than 90% and a cell adhesion rate of more than 80%, which is significantly improved compared with the conventional survival rate, ensures the quality and biological characteristics of cell growth, shortens the proliferation cycle of cell recovery, and enables the recovered cells to be provided to biological experimental research and production use in a timely manner with guaranteed quality and quantity.

[0021] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention, it can be implemented according to the contents of the specification. In order to make the above contents of the present invention and its objectives, features and advantages more obvious and easy to understand, the specific implementation methods of the present invention are listed below. DETAILED DESCRIPTION

[0022] The technical solution of the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments 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.

[0023] In addition, unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0024] Example 1

[0025] The cell freezing solution for improving the survival rate of frozen cells has the following volume ratios of the components: 5% cell culture medium, 70% calf serum, 10% dimethyl sulfoxide, 2% 3% glutamine, 3% 7.5% sodium bicarbonate, and 10% preparation solution R. The preparation solution R components are 110 mg / mL D-glucose, 90 mg / mL hydroxyethyl starch, 70 mg / mL lactobionic acid, 30 mg / mL polyvinyl pyrrolidone, 30 mg / mL D-raffinose pentahydrate, 11 mg / mL potassium hydroxide, 7 mg / mL potassium dihydrogen phosphate, 2 mg / mL magnesium sulfate heptahydrate, 2 mg / mL adenosine, 60 μmol / L vitamin C, and 15 μmol / L vitamin E.

[0026] Example 2

[0027] The cell freezing solution for improving the survival rate of frozen cells has the following volume ratios of the components: 10% cell culture medium, 65% calf serum, 10% dimethyl sulfoxide, 2% 3% glutamine, 3% 7.5% sodium bicarbonate, and 10% preparation solution R. The preparation solution R comprises 120 mg / mL D-glucose, 100 mg / mL hydroxyethyl starch, 80 mg / mL lactobionic acid, 40 mg / mL polyvinyl pyrrolidone, 40 mg / mL D-raffinose pentahydrate, 12 mg / mL potassium hydroxide, 8 mg / mL potassium dihydrogen phosphate, 3 mg / mL magnesium sulfate heptahydrate, 3 mg / mL adenosine, 70 μmol / L vitamin C, and 25 μmol / L vitamin E.

[0028] Example 3

[0029] The cell freezing solution for improving the survival rate of frozen cells has the following volume ratios: 20% cell culture medium, 55% calf serum, 10% dimethyl sulfoxide, 2% 3% glutamine, 3% 7.5% sodium bicarbonate, and 10% preparation solution R. The preparation solution R comprises 130 mg / mL D-glucose, 110 mg / mL hydroxyethyl starch, 90 mg / mL lactobionic acid, 50 mg / mL polyvinyl pyrrolidone, 50 mg / mL D-raffinose pentahydrate, 13 mg / mL potassium hydroxide, 9 mg / mL potassium dihydrogen phosphate, 4 mg / mL magnesium sulfate heptahydrate, 4 mg / mL adenosine, 80 μmol / L vitamin C, and 35 μmol / L vitamin E.

[0030] Example 4

[0031] The cell freezing solution for improving the recovery rate of frozen cells has the following volume ratios: 30% cell culture medium, 45% calf serum, 10% dimethyl sulfoxide, 2% glutamine, 3% sodium bicarbonate, and 10% preparation solution R. The preparation solution R comprises 140 mg / mL D-glucose, 120 mg / mL hydroxyethyl starch, 100 mg / mL lactobionic acid, 60 mg / mL polyvinyl pyrrolidone, 60 mg / mL D-raffinose pentahydrate, 14 mg / mL potassium hydroxide, 10 mg / mL potassium dihydrogen phosphate, 5 mg / mL magnesium sulfate heptahydrate, 5 mg / mL adenosine, 90 μmol / L vitamin C, and 45 μmol / L vitamin E.

[0032] Effects of cryopreservation and thawing of cells using improved cryopreservation solution

[0033] Cell freezing solution consists of the following volume ratios

[0034]

[0035]

[0036] Mix the above components evenly.

[0037] During the cell freezing process, culture and grow monolayer HL2, TG2, TG3, and TGC4 cells, one bottle each → add PBS with pH 7.0 to wash the cell surface once → digest with 0.25% trypsin for 2 minutes → discard the trypsin → add 10 mL of culture medium, pipette and mix the cell suspension → centrifuge at 1000 rpm / 5 minutes → discard the supernatant → add the improved freezing solution respectively, and adjust the cell concentration to (1±0.2)×10 7 viable cells / mL→dispense into 1mL / tube→put into programmed cooling box→-80℃ overnight→immerse in liquid nitrogen for long-term storage.

[0038] Cell recovery process and effect: Take out 1 HL2, TG2, TG3, TGC4 cells frozen with each component of freezing solution and 1 HL2 cell frozen in conventional manner from liquid nitrogen as control group → Place in 37℃ water bath to dissolve → Centrifuge at 1000rpm / 3min → Discard supernatant → Add 10mL 20% calf serum culture medium to calculate cell viability → Place in 37℃ CO2 incubator. Observe after 12h of culture → It can be seen that about 70% of cells are attached to the wall, the cell morphology is good, the cells are relatively plump, there are many live cells, and the size is uniform → Replace with 10% calf serum culture medium and continue to culture → Grow into a monolayer of cells for 2-3 days and then subculture.

[0039] The recovery results and cell viability statistics are shown in the following table:

[0040] Cryopreservation solution components HL2 survival rate % TG2 activity % TG3 activity% TGC4 activity % The survival rate of the control group % Example 1 92 93 95 95 51 Example 2 93 94 92 94 53 Example 3 90 92 93 93 48 Example 4 93 95 93 91 61

[0041] The recovery results and cell adhesion rate are shown in the following table:

[0042]

[0043] After 72 hours of culture, the cells multiplied into a monolayer of adherent cells and could be subcultured.

[0044] The serial numbers of the above embodiments of the present invention are only for description and do not represent the advantages or disadvantages of the embodiments.

[0045] The embodiments of the present invention are described above, but the present invention is not limited to the above-mentioned specific implementation modes. The above-mentioned specific implementation modes are merely illustrative and not restrictive. Under the guidance of the present invention, ordinary technicians in this field can also make many forms without departing from the scope of protection of the purpose of the present invention and the claims, which are all within the protection of the present invention.

Claims

1. A cell freezing solution, characterized in that: The invention comprises the following raw materials in volume percentage: 5-30% of cell culture fluid, 45-70% of calf serum, 10% of dimethyl sulfoxide, 2% of 3% glutamine, 3% of 7.5% sodium bicarbonate and 10% of preparation solution R.

2. A cell freezing solution as claimed in claim 1, characterized in that: Solution R is prepared and composed of the following parts: 110-140 mg / mL D-glucose, 90-120 mg / mL hydroxyethyl starch, 70-100 mg / mL lactobionic acid, 30-60 mg / mL polyvinylpyrrolidone, 30-60 mg / mL D-raffinose pentahydrate, 11-14 mg / mL potassium hydroxide, 7-10 mg / mL potassium dihydrogen phosphate, 2-5 mg / mL magnesium sulfate heptahydrate, 2-5 mg / mL adenosine, 60-90 μmol / L vitamin C and 15-45 μmol / L vitamin E.

3. A cell freezing solution as claimed in claim 2, characterized in that: The invention comprises the following raw materials in volume percentage: 5% cell culture medium, 70% calf serum, 10% dimethyl sulfoxide, 2% 3% glutamine, 3% 7.5% sodium bicarbonate and 10% prepared solution R; the prepared solution R comprises 110 mg / mL D-glucose, 90 mg / mL hydroxyethyl starch, 70 mg / mL lactobionic acid, 30 mg / mL polyvinyl pyrrolidone, 30 mg / mL D-raffinose pentahydrate, 11 mg / mL potassium hydroxide, 7 mg / mL potassium dihydrogen phosphate, 2 mg / mL magnesium sulfate heptahydrate, 2 mg / mL adenosine, 60 μmol / L vitamin C and 15 μmol / L vitamin E.

4. A cell freezing solution as claimed in claim 2, characterized in that: The invention comprises the following raw materials in volume percentage: 10% cell culture medium, 65% calf serum, 10% dimethyl sulfoxide, 2% 3% glutamine, 3% 7.5% sodium bicarbonate and 10% prepared solution R; the prepared solution R comprises 120mg / mL D-glucose, 100mg / mL hydroxyethyl starch, 80mg / mL lactobionic acid, 40mg / mL polyvinyl pyrrolidone, 40mg / mL D-raffinose pentahydrate, 12mg / mL potassium hydroxide, 8mg / mL potassium dihydrogen phosphate, 3mg / mL magnesium sulfate heptahydrate, 3mg / mL adenosine, 70μmol / L vitamin C and 25μmol / L vitamin E.

5. A cell freezing solution as claimed in claim 2, characterized in that: The invention comprises the following raw materials in volume percentage: 20% cell culture medium, 55% calf serum, 10% dimethyl sulfoxide, 2% 3% glutamine, 3% 7.5% sodium bicarbonate and 10% prepared solution R; the prepared solution R comprises 130mg / mL D-glucose, 110mg / mL hydroxyethyl starch, 90mg / mL lactobionic acid, 50mg / mL polyvinyl pyrrolidone, 50mg / mL D-raffinose pentahydrate, 13mg / mL potassium hydroxide, 9mg / mL potassium dihydrogen phosphate, 4mg / mL magnesium sulfate heptahydrate, 4mg / mL adenosine, 80μmol / L vitamin C and 35μmol / L vitamin E.

6. A cell freezing solution as claimed in claim 2, characterized in that: The invention comprises the following raw materials in volume percentage: 30% cell culture medium, 45% calf serum, 10% dimethyl sulfoxide, 2% 3% glutamine, 3% 7.5% sodium bicarbonate and 10% prepared solution R; the prepared solution R comprises 140 mg / mL D-glucose, 120 mg / mL hydroxyethyl starch, 100 mg / mL lactobionic acid, 60 mg / mL polyvinyl pyrrolidone, 60 mg / mL D-raffinose pentahydrate, 14 mg / mL potassium hydroxide, 10 mg / mL potassium dihydrogen phosphate, 5 mg / mL magnesium sulfate heptahydrate, 5 mg / mL adenosine, 90 μmol / L vitamin C and 45 μmol / L vitamin E.

7. Use of a cell freezing solution according to any one of claims 1 to 6 in the freezing of hepatocytes, characterized in that: Hepatocyte cryopreservation process: culture hepatocytes grown into a monolayer → add PBS with pH 7.0 to wash the cell surface once → digest with 0.25% trypsin for 2 minutes → discard trypsin → add 10 mL of culture medium, pipette and mix the cell suspension → centrifuge at 1000 rpm / 5 minutes → discard the supernatant → add cell cryopreservation solution and adjust the cell concentration to (1±0.2)×10 7 viable cells / mL→dispense into 1mL / tube→put into programmed cooling box→-80℃ overnight→immerse in liquid nitrogen for long-term storage.

8. Use of a cell freezing solution according to any one of claims 1 to 6 in liver cell resuscitation, characterized in that: The recovery process of hepatocytes: take out the hepatocytes frozen with freezing solution from liquid nitrogen → place in 37℃ water bath to dissolve → centrifuge at 1000rpm / 3min → discard the supernatant → add 10mL of 20% calf serum culture medium → culture at 37℃.

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