CN115777689A - Serum-free and protein-free non-programmed cell cryopreservation solution - Google Patents
Serum-free and protein-free non-programmed cell cryopreservation solution Download PDFInfo
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Abstract
The invention discloses a serum-free and protein-free non-programmed cell frozen stock solution, which contains DMSO, a PBS buffer solution, hydroxyethyl starch, glucose, trehalose, L-glutamine dipeptide, sodium pyruvate, ascorbic acid and taurine, does not contain components such as serum, protein and the like, and avoids the problems of potential animal virus risk of serum, batch-to-batch difference and the like. The stability of the cell freezing solution among different batches is improved. The intracellular protective agent and the extracellular protective agent have double protection effects, and can achieve good cryopreservation effect without gradient transfer or program cooling cryopreservation of cells by using a program cryopreservation box, thereby reducing 'ice crystal damage' and 'solute damage' during cell cryopreservation. The cell cryopreservation solution disclosed by the invention has a good cryopreservation effect on various cells, has a remarkable effect on sensitive cells such as SP2/0 and NIH3T3, and effectively improves the average cell viability and the convenience of cryopreserving the cells.
Description
Technical Field
The invention relates to the technical field of cell cryopreservation, in particular to a serum-free and protein-free non-procedural cell cryopreservation solution.
Background
Cells are the basic structure and functional unit of organisms, and have an important position in the fields of fundamental mechanism research, medical research and the like in modern biological research. The cell freezing is to put the cells into a low-temperature environment to reduce the basic metabolism of the cells and enable the cells to enter a temporary hibernation state, thereby achieving the purposes of long-term storage or transportation and the like. And when the cell lysate is used, the cryopreserved cells can be recovered only by placing the cryopreserved cells at 37 ℃ for rapid dissolution.
In the traditional cell freezing and storing technology, the traditional cell freezing and storing liquid generally comprises components such as a cell basic culture medium, fetal calf serum, DMSO (dimethyl sulfoxide) and the like, and in the cell freezing and storing process, the 'ice crystal damage' caused by liquid ice crystallization during freezing and the 'solute damage' caused by osmotic pressure change can be reduced or prevented. However, the fetal bovine serum is of animal origin and has a potential risk of animal viruses. The fetal calf serum has complex and unknown components, and fetal calf serum in different batches can have great difference, so that experimental results of different freezing batches can be influenced. The traditional cell cryopreservation method still needs to adopt a program cell cryopreservation mode, namely, the cells are subjected to program cooling cryopreservation by gradient transfer at 4-20 ℃ and-80 ℃ or assisted by a program cryopreservation box, so that not only is the time consumption long, the operation is troublesome, and the treatment capacity is limited, but also the large-scale cell cryopreservation requirement cannot be met.
Disclosure of Invention
The invention aims to overcome the defects of the existing cell cryopreservation solution technology and provides a serum-free protein-free non-procedural cell cryopreservation solution. The cell frozen stock solution does not contain serum components in the traditional cell frozen stock solution, does not use natural or recombinant protein and the like, and ensures that the cell frozen stock solution has stable effect among different batches. And the intracellular and extracellular protective agents are matched for use, so that the programmed cooling cryopreservation of the cells is changed into non-programmed cooling, the 'ice crystal damage' and 'solute damage' during the cell cryopreservation are reduced, and the survivability and the use convenience of the cells are effectively improved. The non-programmed cell freezing medium is serum-free and protein-free and is stable in effect, convenient to use and suitable for most cells.
It is a first object of the present invention to provide a composition.
The second purpose of the invention is to provide the application of the composition in preparing cell freezing medium.
The third purpose of the invention is to provide a cell freezing medium.
The fourth purpose of the invention is to provide the application of the cell freezing solution in the preparation of a kit for freezing and storing cells.
The fifth purpose of the invention is to provide a kit for freezing and preserving cells.
It is a sixth object of the present invention to provide a method for cryopreserving cells.
In order to achieve the purpose, the invention is realized by the following scheme:
a composition comprising DMSO, PBS buffer, hydroxyethyl starch, glucose, trehalose, L-glutamine dipeptide, sodium pyruvate, ascorbic acid, and taurine.
Preferably, the composition is a mixed solution of DMSO containing 50-150 g/L hydroxyethyl starch, 24.9-25.1 g/L glucose, 0-40 g/L trehalose, 9.9-10.1 g/L L-glutamine, 2.9-3.1 g/L sodium pyruvate, 2.9-3.1 g/L ascorbic acid, 0-0.125 g/L taurine and PBS buffer, the volume ratio of the DMSO to the PBS buffer is 1-2: 18 to 19.
More preferably, the composition is a mixed solution of DMSO containing 100g/L hydroxyethyl starch, 25g/L glucose, 25g/L trehalose, 10g/L L-glutamine dipeptide, 3g/L sodium pyruvate, 3g/L ascorbic acid, 0.125g/L taurine and PBS buffer, wherein the volume ratio of the DMSO to the PBS buffer is 1:9.
the composition is applied to preparation of cell cryopreservation liquid.
A cell cryopreservation solution contains the composition.
Preferably, the cell is an animal cell.
More preferably, the cell is a mammalian cell.
More preferably, the cell is a Hela cell, a NIH3T3 cell, a MG-63 cell, a mesenchymal cell, a cranial cell, a myofibroblast, a SP2/0 cell, a Hek293 cell, a 143b cell, and/or a HGC27 cell.
The application of the cell cryopreservation liquid in the preparation of the kit for cryopreserving cells.
Preferably, the cell is an animal cell.
More preferably, the cell is a mammalian cell.
More preferably, the cell is a Hela cell, a NIH3T3 cell, a MG-63 cell, a mesenchymal cell, a cranial cell, a myofiber cell, a SP2/0 cell, a Hek293 cell, a 143b cell and/or a HGC27 cell.
A kit for cryopreserving cells contains the cell cryopreservation solution.
The cell freezing solution is used for resuspending cells, and the cells are directly frozen without gradient cooling.
Preferably, the cell is an animal cell.
More preferably, the cell is a mammalian cell.
More preferably, the cell is a Hela cell, a NIH3T3 cell, a MG-63 cell, a mesenchymal cell, a cranial cell, a myofibroblast, a SP2/0 cell, a Hek293 cell, a 143b cell, and/or a HGC27 cell.
Compared with the prior art, the invention has the following beneficial effects:
the invention discloses a serum-free and protein-free non-programmed cell frozen stock solution, which contains DMSO, a PBS buffer solution, hydroxyethyl starch, glucose, trehalose, L-glutamine dipeptide, sodium pyruvate, ascorbic acid and taurine, does not contain components such as serum, protein and the like, and avoids the problems of potential animal virus risk of serum, batch-to-batch difference and the like. The stability of the cell frozen stock solution among different batches is improved. The intracellular protective agent (DMSO) and the extracellular protective agent (hydroxyethyl starch, taurine and trehalose) have double protection effects, and a good cryopreservation effect can be achieved without gradient transfer or programmed cooling cryopreservation of cells by using a programmed cryopreservation box, so that ice crystal damage and solute damage during cell cryopreservation are reduced. The cell cryopreservation solution disclosed by the invention has a good cryopreservation effect on various cells, has a remarkable effect on sensitive cells such as SP2/0 and NIH3T3, and effectively improves the average cell viability and the convenience of cryopreserving the cells.
Drawings
FIG. 1 shows the cell state of different types of cells cryopreserved with the cell cryopreserving solution of Experimental group 2. Wherein A is Hela cell, B is NIH3T3 cell, C is MG-63 cell, D is SP2/0 cell, and E is Hek293 cell.
FIG. 2 shows the cell state of different types of cells cryopreserved with the cell cryopreserving solution of the conventional control group. Wherein A is Hela cell, B is NIH3T3 cell, C is MG-63 cell, D is SP2/0 cell, and E is Hek293 cell.
FIG. 3 shows the state of cells after different types of cells were frozen in the cell freezing medium of A. Wherein A is Hela cell, B is NIH3T3 cell, C is MG-63 cell, D is SP2/0 cell, and E is Hek293 cell.
FIG. 4 shows the state of cells after different types of cells were frozen in the cell frozen stock solution from the company B. Wherein A is Hela cell, B is NIH3T3 cell, C is MG-63 cell, D is SP2/0 cell, and E is Hek293 cell.
Detailed Description
The present invention will be described in further detail with reference to the drawings and specific examples, which are provided for illustration only and are not intended to limit the scope of the present invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.
Example 1 Effect of extracellular protectant on programmed and non-programmed cell cryopreservation
The extracellular protectant is a non-permeable protectant, mostly macromolecular substances, has certain viscosity and can not enter cells. The main function is to reduce the content of free water outside the bag, lower the freezing point and reduce the generation of ice crystals by maintaining the stability of cell membranes and combining with water molecules in the solution. The extracellular protectant is hydroxyethyl starch, taurine, trehalose and the like, the nutritional energy substance is L-glutamine, sodium pyruvate, glucose and the like, the antioxidant is ascorbic acid, reduced glutathione and the like, and the intracellular protectant is DMSO and the like.
1. Preparing a cell freezing solution (the following percentages are by mass):
experimental group 1:25g trehalose, 25g glucose, 50g hydroxyethyl starch, 10g L-glutamine dipeptide, 3g sodium pyruvate, 3g ascorbic acid and 0.125g taurine.
Experimental group 2:25g trehalose, 25g glucose, 100g hydroxyethyl starch, 10g L-glutamine propyl dipeptide, 3g sodium pyruvate, 3g ascorbic acid and 0.125g taurine.
Experimental group 3:25g trehalose, 25g glucose, 150g hydroxyethyl starch, 10g L-glutamine propyl dipeptide, 3g sodium pyruvate, 3g ascorbic acid and 0.125g taurine.
Experimental group 4:25g glucose, 100g hydroxyethyl starch, 10g L-glutamine dipeptide, 3g sodium pyruvate, 3g ascorbic acid and 0.125g taurine.
Experimental group 5:10g trehalose, 25g glucose, 100g hydroxyethyl starch, 10g L-glutamine propyl dipeptide, 3g sodium pyruvate, 3g ascorbic acid and 0.125g taurine.
Experimental group 6:40g of trehalose, 25g of glucose, 100g of hydroxyethyl starch, 10g of L-glutamine dipeptide, 3g of sodium pyruvate, 3g of ascorbic acid and 0.125g of taurine.
Experimental group 7:25g trehalose, 25g glucose, 100g hydroxyethyl starch, 10g L-glutamine dipeptide, 3g sodium pyruvate and 3g ascorbic acid.
Experimental group 8:25g glucose, 100g hydroxyethyl starch, 10g L-glutamine dipeptide, 3g sodium pyruvate and 3g ascorbic acid.
Control group 1:25g trehalose, 25g glucose, 10g L-glutamine dipeptide, 3g sodium pyruvate, 3g ascorbic acid and 0.125g taurine.
Traditional control group: mixing 70% by volume DMEM high carbohydrate cell culture medium, 20% fetal bovine serum and 10% DMSO in a clean bench.
Preparing a solution composed of 10% DMSO and 90% PBS by volume, accurately weighing each component of the above experimental groups 1-8 or control group 1, stirring and dissolving each component with the solution, and fixing the volume to 1L with the solution; adjusting the pH value to 7.2-7.6, and fixing the volume; filtering and sterilizing by using a 0.22 mu m filter to obtain the cell frozen stock solution.
2. Experimental methods
(1) Hela cells are adherent cells, the Hela cells with good state are selected, washed for 1 to 2 times by PBS buffer solution, the PBS buffer solution is removed, and the cells are digested by adding pancreatin greenhouse (20 to 30 ℃); when the visual field of most (50-70% at most) cells becomes round under a microscope, adding a cell culture medium containing serum (in the embodiment, DMEM (DMEM) is used for Hela cells) to stop digestion, and gently blowing and beating the cells to shed the cells;
treatment of suspension cell conditions: centrifuging at 800-1200 rpm/min for 3-5 min and eliminating supernatant; washing the cells for 1-2 times by using PBS buffer solution, and then carrying out the treatment of the subsequent step (2);
(2) Collecting the cells treated in the step (1), centrifuging at 800-1200 rpm/min for 3-5 min; removing supernatant, respectively resuspending cells with cell cryopreservation solution prepared from the experimental group 1-8, the control group 1 and the conventional control group to ensure cell density of 8 × 10 5 ～5×10 6 one/mL, preferably 3X 10 6 Obtaining cell suspension per mL;
(3) Non-programmed freezing: transferring the cell suspension (except the traditional control group) obtained in the step (2) into a cell freezing tube with the volume of 1 mL/cell in triplicate; the direction of the cell freezing tube is upward, and the cell freezing tube is directly put into a refrigerator at minus 80 ℃ for overnight (10 to 12 hours, if no special description is provided, the same is true below); the next day, according to the actual use condition, the mixture is transferred into liquid nitrogen for long-term storage or short-term storage at minus 80 ℃.
(4) And (4) program freezing: transferring the traditional control group cell suspension obtained in the step (2) into a cell cryopreservation tube with 1mL per cell in triplicate; freezing the cells in a tube, standing for 30min at the temperature of 2-8 ℃, standing for 2h at the temperature of-20 ℃, and standing overnight at the temperature of-80 ℃; the next day, according to the actual use condition, the mixture is transferred into liquid nitrogen for long-term storage or stored at minus 80 ℃ for short-term storage.
(5) Cell recovery: taking the cell freezing tube obtained in the steps (3) and (4) out of liquid nitrogen, placing the tube on a floating plate, placing the tube in a 37 ℃ water bath kettle, and slightly shaking to accelerate melting; the frozen cell suspension is transferred to DMEM medium or PBS buffer solution (HBSS or physiological saline can be used, the purpose is to wash cells, the osmotic pressure is equivalent to that of the cells, and the cells cannot be damaged) with the volume 10 times of the original volume, the cells are gently mixed, centrifuged, the supernatant is removed, and the cells are suspended by using 1mL of DMEM medium to obtain the revived cells.
(5) The cells recovered in step (4) were stained with trypan blue, and subjected to viability and density detection using a cell counter, and the detection was repeated 2 times each. After the cells are recovered, partial cells are fragmented or lost, the cell counting instrument can analyze the cells abnormally, although the cell survival rate is high, the cell amount actually used for continuous culture is small, the false positive phenomenon is generated, and the false positive phenomenon of the cell survival rate is avoided by comparing the density change before and after the freezing storage.
3. Results of the experiment
The survival rate and recovery rate of Hela cells after being thawed after being frozen for 1 month are shown in Table 1.
The traditional cell freezing solution (traditional control group) has a certain difference between the effects of non-programmed freezing and programmed freezing, and the cell state of programmed freezing is superior to that of non-programmed freezing.
According to results of a control group 1 and experimental groups 1-8, hydroxyethyl starch plays an important role in cell cryopreservation protection, and has a good protection effect on cells within a certain range of use concentration; the trehalose effectively protects cells from the invariable inactivation of cell membranes and protein molecules under special environment; taurine has the extensive cell protection functions of resisting oxidation, stabilizing cell membranes and the like.
From example 2 and experimental groups 4 to 7, trehalose and taurine can enhance the tolerance of cells and influence the survival rate of cells after cryopreservation recovery. Under the condition that both trehalose and taurine are deficient (experimental group 8), the cell viability is reduced by 7-10%, and the influence of non-programmed freezing is more obvious; lack of taurine (experiment group 7) causes the survival rate of the revived cells to be reduced by about 2 percent; the optimal value of the trehalose is 25g/L, the trehalose is removed (experiment group 4), and the cell viability is reduced by 4-5%; and the cell survival rate is positively increased along with the increase of the trehalose proportion; however, as the trehalose concentration was further increased, the effect on the cells gradually saturated. The cell preservation solution has a synergistic effect on the maintenance of the survival rate of Hela cells.
TABLE 1 Effect of extracellular protectants on programmed and non-programmed cryopreservation of cells
Example 2 Effect of intracellular protectant classes and ratios on cell program cryopreservation
The intracellular protective agent is a permeability protective agent, is mostly a small molecular substance, can enter the inside of cells, reduces the water molecule content in the cells, and has a certain molar concentration. Reduces the damage caused by the change of the electrolyte concentration and the production of ice crystals in the process of cell re-freezing storage. The extracellular protective agent is hydroxyethyl starch, taurine, trehalose and the like, the nutritional energy substances are L-glutamine dipeptide, glutamine, sodium pyruvate, glucose and the like, the antioxidant is ascorbic acid, reduced glutathione and the like, and the intracellular protective agent is DMSO, glycerol, propylene glycol and the like.
1. Preparing a cell freezing solution (the following percentage contents are calculated by mass):
experimental group 2: formulation and configuration procedure, see experimental group 2 of example 1.
Experimental group 9:25g trehalose, 25g glucose, 100g hydroxyethyl starch, 10g L-glutamine dipeptide, 3g sodium pyruvate, 3g ascorbic acid and 0.125g taurine.
Experimental group 10:25g trehalose, 25g glucose, 100g hydroxyethyl starch, 10g L-glutamine propyl dipeptide, 3g sodium pyruvate, 3g ascorbic acid and 0.125g taurine.
Experimental group 11:25g trehalose, 25g glucose, 100g hydroxyethyl starch, 10g L-glutamine dipeptide, 3g sodium pyruvate, 3g ascorbic acid and 0.125g taurine.
Experimental group 12:25g trehalose, 25g glucose, 100g hydroxyethyl starch, 10g L-glutamine dipeptide, 3g sodium pyruvate, 3g ascorbic acid and 0.125g taurine.
Control group 2:25g trehalose, 25g glucose, 100g hydroxyethyl starch, 10g L-glutamine dipeptide, 3g sodium pyruvate, 3g ascorbic acid and 0.125g taurine.
The solution of the experimental group 9 consisted of 5% DMSO and 95% PBS by volume.
The solution of the experimental group 10 consisted of 10% glycerol and 90% PBS by volume.
The dissolution solution of the experimental group 11 consisted of 5% DMSO, 5% glycerol and 90% PBS by volume ratio.
The dissolution of the experimental group 12 consisted of 5% glycerol, 5% propylene glycol and 90% PBS by volume.
The solution of control 2 was PBS.
Accurately weighing the components of the experimental groups 9-12 or the control group 2 respectively, stirring and dissolving the components by using corresponding dissolving liquid, and fixing the volume to 1L by using the dissolving liquid; adjusting the pH value to 7.2-7.6, and fixing the volume; filtering and sterilizing by using a 0.22 mu m filter to obtain the cell frozen stock solution.
Traditional control group: formulation and formulation procedure, see conventional control of example 1.
2. Experimental methods
The cells used in the cryopreservation experiment are NIH3T3 cells, the NIH3T3 cells are adherent cells, and the density of the cryopreserved NIH3T3 cells is 8 multiplied by 10 5 ～5×10 6 one/mL, preferably 4X 10 6 Each/mL, in this example, the NIH3T3 cells were cultured in DMEM medium, and the experimental group 2, the experimental groups 9 to 12, and the control group 2 were frozen in a non-programmed manner in the same manner as in example 1.
The conventional control group was frozen using the same procedure as in step (4) of example 1.
3. Results of the experiment
The viability and recovery rate of NIH3T3 cells after being thawed after being frozen for 1 month are shown in Table 2.
As shown in Table 2, it can be seen from control 2 that the intracellular protective agents such as propylene glycol, glycerol, and DMSO greatly affect the viability rate of the frozen cells. From the overall results of the examples, different intracellular protective agents have protective effect on cell cryopreservation, the average cell survival rate of the experimental group 11 is higher than that of the experimental group 12, and the average cell survival rate of the experimental group 2 is far higher than that of the experimental groups 9-12, so that the intracellular protective agent in the cell cryopreservation solution disclosed by the invention is preferably DMSO.
TABLE 2 Effect of intracellular protectant classes and ratios on cell program cryopreservation
| Test grouping | Average cell viability rate | Average cell recovery |
| Experimental group 2 | 94.12％ | 78.26％ |
| Experimental group 9 | 82.91 | 72.34％ |
| Experimental group 10 | 77.62％ | 67.59％ |
| Experimental group 11 | 86.25％ | 70.62％ |
| Experimental group 12 | 80.53％ | 71.68％ |
| Control group 2 | 26.21％ | 30.12％ |
| Conventional control group | 92.03％ | 72.38％ |
Example 3 cell cryopreservation stability test
The cell freezing is to put the cells into a low-temperature environment to reduce the basic metabolism of the cells and make the cells enter a temporary hibernation state, thereby achieving the purposes of long-term storage or transportation and the like.
1. Preparing a cell freezing medium:
experimental group 2: formulation and configuration procedure, see experimental group 2 of example 1.
Traditional control group: formulation and formulation steps, see conventional control of example 1.
2. Experimental methods
(1) The frozen experimental cells are MG-63 cells and Hela cells respectively, and the density of the frozen MG-63 cells and Hela cells is 8 multiplied by 10 5 ～5×10 6 one/mL, preferably 3X 10 6 MG-63 cells were adherent cells per mL, MEM was used for MG-63 cells in this example, and non-programmed cryopreservation was used for Experimental group 2 in the same manner as in example 1. The conventional control group was frozen in the same manner as in step (4) of example 1.
(2) Resuscitating the cells after 1 week, 1 month and 3 months of cryopreservation, respectively; the recovery experiment procedure is the same as that of example 1, and the detection and calculation methods of the average cell viability rate and the average cell recovery rate are the same as those of example 1.
3. Results of the experiment
As shown in tables 3 and 4, the cell cryopreserved solution of the experimental group 2 in example 1 of the present invention had good stability; after 3 months of cryopreservation of MG-63 cells and Hela cells, the stability of the cell cryopreservation solution of the experimental group 2 in the embodiment 1 of the invention is obviously superior to that of the traditional control group.
TABLE 3Hela cell cryopreservation stability test
TABLE 4MG-63 cell cryopreservation stability test
Example 4 Effect of cell cryopreservation solution on cryopreservation of Primary cells
1. Preparing a cell freezing medium:
example (b): formulation and configuration procedure, see experimental group 2 of example 1.
Traditional control group: formulation and formulation steps, see conventional control of example 1.
2. Experimental method
Isolating primary cells of mice and rats, including primary rat mesenchymal cells, primary rat cranial cells, primary rat myofibroblasts, and primary mouse myofibroblasts. The density of the frozen primary cells is 0.5-1 multiplied by 10 6 Per mL, run 2 was frozen in a non-programmed manner using the same procedures as in example 1 (DMEM media was used for primary cells).
The traditional control group was frozen using a program.
3. Results of the experiment
The survival rate and the anchorage rate of the recovered primary cells after being frozen and stored for 1 month are shown in the table 5. The effect of the cell cryopreservation solution of the experimental group 2 in the embodiment 1 of the invention in primary rat cranial cells and primary mouse myofibroblasts is obviously superior to that of the traditional control group.
TABLE 5 Primary cell cryopreservation test
Example 5 Effect of cell cryopreservation solution on conventional cell cryopreservation
1. Preparing cell freezing medium
Example (b): formulation and configuration procedure, see experimental group 2 of example 1.
Traditional control group: formulation and formulation procedure, see conventional control of example 1.
The same category of non-programmed cell cryopreserved from commercial companies A and B was prepared.
2. Experimental method
The cells used in the cryopreservation experiment are SP2/0, hek293, NIH3T3, hela, MG-63, 143b and HGC27 cells which are all adherent cells, wherein the density of the cryopreserved cells is 3-4 multiplied by 10 6 Per mL, frozen stocks of Experimental group 2 and commercial companies A and B were frozen in a non-programmed manner in the same manner as in example 1 (wherein SP2/0, hek293, hela and 143b used DMEM medium, MG-63 used MEM medium, and HGC27 used RPMI-1640 medium).
The traditional control group was frozen using a program.
And respectively planting the recovered SP2/0, hek293, NIH3T3, hela and MG-63 cells into a T25 cell culture flask, and photographing the next day to observe the recovered cell state.
3. Results of the experiment
The average cell viability rate and the average cell recovery rate after recovery after 1 month of cryopreservation are shown in Table 6, and the cell cryopreservation solution of the experimental group 2 in the embodiment 1 of the invention can exert good cryopreservation effect in SP2/0, hek293, NIH3T3, hela, MG-63, 143b and HGC27 cell cryopreservation, and the cell viability rate is more than 90%. While the activity rate of the traditional group in Hela, hek293, MG-63 and 143b cells is more than 90%, but the freezing survival rate of SP2/0, NIH3T3 and HGC27 is less than 90%. In addition, compared with the cell freezing solutions of the company A and the company B, the embodiment of the patent has more remarkable freezing effect on 2 sensitive cells such as SP2/0 and NIH3T3, and the recovery survival rate is far higher than that of the cell freezing solutions of the company A and the company B.
As shown in FIGS. 1A to E and FIGS. 2A to E, the cell culture solutions of the experimental group 2 in example 1 of the present invention were in a good state for cell state observation after thawing the frozen SP2/0, hek293, NIH3T3, hela and MG-63 cells. However, referring to the analysis results in table 6, the resuscitation effect of the examples of this patent is superior to that of the conventional control group. The observation effect of the frozen stock solution of the company A and the company B on the market after recovery is shown in figures 3A to E and figures 4A to E, although the recovery state of Hela, MG63 and Hek293 cells is good, the cell amount and the state after recovery of NIH3T3 and SP2/0 are obviously poor.
TABLE 6 conventional cell cryopreservation test
It should be finally noted that the above examples are only intended to illustrate the technical solutions of the present invention, and not to limit the scope of the present invention, and that other variations and modifications based on the above description and thought may be made by those skilled in the art, and that all embodiments need not be exhaustive. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.
Claims (10)
1. A composition comprising DMSO, PBS buffer, hydroxyethyl starch, glucose, trehalose, L-glutamine dipeptide, sodium pyruvate, ascorbic acid, and taurine.
2. The composition of claim 1, wherein the composition is a mixture of DMSO and PBS buffer containing 50 to 150g/L hydroxyethyl starch, 24.9 to 25.1g/L glucose, 0 to 40g/L trehalose, 9.9 to 10.1 g/L-glutamine dipeptide, 2.9 to 3.1g/L sodium pyruvate, 2.9 to 3.1g/L ascorbic acid, 0 to 0.125g/L taurine, and the volume ratio of the DMSO to the PBS buffer is 1 to 2:18 to 19.
3. The composition as claimed in claim 1, wherein the composition is a mixture of DMSO and PBS buffer containing 100g/L hydroxyethyl starch, 25g/L glucose, 25g/L trehalose, 10 g/L-glutamine dipeptide, 3g/L sodium pyruvate, 3g/L ascorbic acid, 0.125g/L taurine, and the volume ratio of DMSO to PBS buffer is 1:9.
4. use of a composition according to any one of claims 1 to 3 for the preparation of a cell cryopreservation solution.
5. A cell cryopreservation solution comprising the composition according to any one of claims 1 to 3.
6. The use of the cell cryopreservation solution of claim 5 in the preparation of a kit for cryopreserving cells.
7. The use of claim 6, wherein the cell is an animal cell.
8. The use of claim 7, wherein the animal cell is a mammalian cell.
9. A kit for cryopreserving cells, comprising the cell cryopreserving solution according to claim 5 or 6.
10. A method for freezing cells, which comprises resuspending the cells in the cell freezing solution of claim 5 or 6, and directly freezing the cells without performing gradient cooling.
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