Cell cryopreservation liquid, cell cryopreservation method and composition

Abstract

The invention provides a cell cryopreservation solution, a cell cryopreservation method and a composition. The cell cryopreservation liquid comprises 60-80 v / v% of a basic culture medium; 3-15 v / v% of fetal calf serum; 0.1 to 0.3 w / v% of chitosan quaternary ammonium salt; 6-17 v / v% of a cryoprotectant; 2-5 w / v% of polyethylene glycol; 1 to 10.5 mM of an antioxidant; 0.1 to 0.4 w / v% of cholesterol; the buffer agent is used for maintaining the pH value of the cell freezing medium to be 7.2 to 7.6. The cell cryopreservation liquid is efficient and low in toxicity, and the survival rate of cells and functional integrity after resuscitation can be remarkably improved by adopting the cell cryopreservation liquid to cryopreserve the cells.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell preservation, and in particular relates to a cell freezing solution, a cell freezing method and a composition. Background Art

[0002] Cell cryopreservation technology, as an indispensable part of biological and medical research, is crucial for long-term preservation of cell lines, maintenance of biodiversity, and promotion of disease treatment and regenerative medicine. Cryoprotectants (CPAs) are one of the key elements in cell cryopreservation technology. Cryoprotectants reduce cell damage caused by freezing by reducing the formation of ice crystals in cells. Dimethyl sulfoxide (DMSO) and glycerol are two commonly used cryoprotectants, especially DMSO, which is widely used in cell cryopreservation schemes due to its high antifreeze ability. The standard cryopreservation formula currently used usually contains 80% basal culture medium, 10% fetal bovine serum and 10% DMSO. DMSO performs well in inhibiting ice crystal formation, but high concentrations of DMSO itself have certain toxicity to cells, which is particularly prominent during cell cryopreservation and recovery. High concentrations of DMSO can cause problems such as changes in cell membrane permeability and inhibition of enzyme activity, thereby affecting cell viability and subsequent functional performance. Especially for some sensitive cell types, the toxic effect of DMSO is more obvious, limiting the preservation and application potential of these cells. In addition, the peculiar smell and potential skin irritation of DMSO also bring inconvenience to laboratory operators, and DMSO must be quickly diluted or removed after thawing to reduce continued damage to cells.

[0003] In addition, cell freezing methods based on traditional cryopreservation solutions usually require frequent replenishment of liquid nitrogen to maintain extremely low temperatures, which is not only cumbersome to operate, but also has high requirements for hardware equipment.

[0004] In view of the above problems, developing efficient and low-toxic cell freezing solution formulas and improving freezing efficiency and cell vitality after recovery are of great significance to the development of cell freezing technology. Summary of the invention

[0005] The present invention provides the following technical solutions to solve all or part of the problems of the prior art:

[0006] In a first aspect, the present invention provides a cell freezing solution, the cell freezing solution comprising:

[0007] Basal medium, 60-80 v / v%;

[0008] Fetal bovine serum, 3-15 v / v%;

[0009] Chitosan quaternary ammonium salt, 0.1-0.3w / v%;

[0010] Cryoprotectants, 6-17 v / v%;

[0011] Polyethylene glycol, 2-5 w / v%;

[0012] Antioxidants, 1-10.5 mM;

[0013] Cholesterol, 0.1-0.4w / v%;

[0014] A buffer is used to maintain the pH value of the cell freezing solution at 7.2-7.6.

[0015] The cell freezing solution provided by the present invention can provide good protection for cells in the freezing state and during the recovery process, effectively reduce the damage and death of frozen cells, and improve the survival rate of cells after multiple freezing and thawing.

[0016] In addition, the polyethylene glycol in the cell freezing solution provided by the present invention can reduce cell membrane damage. 2-5w / v% polyethylene glycol can reduce cell dehydration without causing toxicity problems. The cholesterol therein also has the effect of enhancing cell membrane stability. 0.1-0.4w / v% cholesterol can improve the cold resistance of the cell membrane.

[0017] In some embodiments, the cryoprotectant comprises glycerol and / or dimethyl sulfoxide (DMSO).

[0018] In some preferred embodiments, the cryoprotectant includes glycerol and dimethyl sulfoxide, the content of the glycerol is 3-8 v / v% of the cell freezing solution, and the content of the dimethyl sulfoxide is 3-9 v / v% of the cell freezing solution.

[0019] In the cryopreservation solution system provided by the present invention, the combined use of glycerol and DMSO in the above-mentioned content will have a better effect than the use of either alone. DMSO as the main cryoprotectant can effectively prevent the formation of ice crystals and protect the cell membrane and internal structure. Glycerol as an auxiliary cryoprotectant can reduce the concentration of DMSO used to reduce toxicity.

[0020] In some embodiments, the antioxidant comprises vitamin E and / or N-acetyl-L-cysteine.

[0021] In some preferred embodiments, the antioxidant includes vitamin E and N-acetyl-L-cysteine, the content of the vitamin E is 0.1-0.5mM, and the content of the N-acetyl-L-cysteine ​​is 1-10mM. Vitamin E and N-acetylcysteine ​​are compounded as antioxidants to reduce cell damage caused by oxidative stress during freezing, and the compounded concentration of the two can provide protection while avoiding toxic reactions. And in the freezing solution system provided by the present invention, the compounded use of the two is more effective than using them alone.

[0022] In some embodiments, the cell freezing solution further comprises recombinant human laminin, and the content of the recombinant human laminin is 1-5 ug / ml.

[0023] In some embodiments, the recombinant human laminin is recombinant human laminin-521 (e.g., A29249). The present invention finds that adding recombinant human laminin-521 to the above-mentioned cell freezing system can quickly promote cell adhesion, resume growth, and activate cell survival signaling pathways after cell recovery. In particular, in the cryopreservation solution in which the antioxidant vitamin E exists, the effect of reducing oxidative stress damage to cells after recovery is more obvious. During freezing and thawing, cells are susceptible to stress signals (such as excessive generation of ROS), which in turn activate the apoptosis pathway. Vitamin E can effectively reduce the generation of ROS through its antioxidant properties, while recombinant human laminin-521 can further inhibit the activation of apoptosis signals by promoting extracellular matrix-cell signaling. When vitamin E is combined with recombinant human laminin-521, it can not only protect cells from the oxidative stress level, but also prevent cell damage during freezing by maintaining cell-matrix interactions. The two-pronged approach is better than a single mechanism of action. This can protect the cell membrane, reduce cell death, promote cell function recovery, improve the freezing effect, and especially improve the health of cells after repeated freezing and thawing.

[0024] In some embodiments, the basal culture medium is DMEM culture medium or RPMI-1640 culture medium, but is not limited thereto. Other culture media available in the art may also be used, or a suitable culture medium may be selected according to the type of cells to be frozen, as long as the basal culture medium can provide the basic nutrients required for cell survival and maintain the basic metabolic function of the cells during the freezing process.

[0025] In some embodiments, the buffer is HEPES, and the amount of HEPES is 10-20 mM. Usually, the pH condition required for cell cryopreservation is 7.2-7.6, so other buffers that can provide this pH environment can also be used, or when there are special requirements for the acid-base environment based on the different types of cells to be cryopreserved, they can also be specifically selected according to the cells to be cryopreserved.

[0026] On the other hand, the present invention also provides a cell freezing method, wherein the method uses the cell freezing solution described in any of the above technical solutions to freeze cells.

[0027] In some embodiments, the cell freezing method includes: suspending cells in the cell freezing solution described in any of the above technical solutions to obtain a cell suspension; program cooling the cell suspension until the temperature drops below -80°C, storing it in an environment below -80°C or transferring it to liquid nitrogen for long-term storage.

[0028] The cell concentration in the cell suspension is, for example, between 1-10×10^6 cells / mL, and can of course be appropriately adjusted according to actual conditions, such as cell characteristics.

[0029] On the other hand, the present invention also provides a composition, comprising the cell freezing solution described in any one of the technical solutions and cells frozen in the cell freezing solution.

[0030] The cell freezing solution provided by the present invention is suitable for the freezing of most types of cells including conventional cell lines and primary cell lines, such as human umbilical cord mesenchymal stem cells, HeLa cells, A549, H9C2, H1299, H2126, BEAS-2B, etc., and has universality and wide application.

[0031] Compared with the prior art, the present invention has at least the following beneficial effects:

[0032] (1) The cell freezing solution provided by the present invention can provide good protection for cells during the freezing state and the recovery process, effectively reduce the damage and death of frozen cells, and improve the survival rate of frozen cells;

[0033] (2) Furthermore, the present invention has found that cell cryopreservation solution containing recombinant human laminin-521 can promote cell adhesion and resumption of growth after cell recovery, further reduce cell death, and promote cell function recovery; especially in a freezing solution system where vitamin E and recombinant human laminin-521 coexist, the synergistic use of the two can improve the health of cells after recovery after repeated freezing and thawing. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is a schematic diagram of flow cytometric analysis of cells using the cell freezing solution of Example 1 after violent freezing and thawing with liquid nitrogen for 10 times, and then labeling living cells with Calcein green fluorescent staining;

[0035] Figure 2 This is a schematic diagram of flow cytometric analysis of cells using the cell freezing solution of Comparative Example 1 after violent freezing and thawing with liquid nitrogen for 10 times, and then labeling living cells with Calcein green fluorescent staining;

[0036] Figure 3This is a schematic diagram of flow cytometric analysis of cells using the cell freezing solution of Example 1 after violent freezing and thawing with liquid nitrogen for 10 times, and marking dead cells by PI red fluorescent staining;

[0037] Figure 4 This is a schematic diagram of flow cytometric analysis of cells using the cell freezing solution of Comparative Example 1 after violent freezing and thawing with liquid nitrogen for 10 times, and then marking dead cells with PI red fluorescent staining. DETAILED DESCRIPTION

[0038] The technical solutions in the specific embodiments of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments.

[0039] Unless otherwise specified, the raw materials and reagents used in the present invention are all commercially available.

[0040] Example 1

[0041] Under sterile conditions, 64v / v% DMEM basal medium was taken, 10v / v% fetal bovine serum was added to the DMEM basal medium, and the mixture was fully mixed until it was completely dissolved; then 0.2w / v% chitosan quaternary ammonium salt (purchased from McLean, item number 850216) was added thereto, and stirred until it was fully and completely dissolved; 6v / v% glycerol was slowly added to mix it evenly to avoid excessive local concentration; then 5v / v% DMSO was added dropwise under stirring, and the addition under stirring was to avoid solution instability caused by sudden temperature change; then 4w / v% % polyethylene glycol 6000, 0.5mM vitamin E, 5mM N-acetyl-L-cysteine, 0.31w / v% cholesterol, in the process of adding, the added substances are completely dissolved and evenly distributed before adding the next substance, and finally HEPES buffer 20mM is added to maintain the pH stability of the freezing solution; after all the above combinations are mixed evenly, sterile filtration is performed using a 0.1 / 0.22 micron filter membrane to remove possible particles and microorganisms, and then the filtered cell freezing solution is divided into sterile containers, sealed and labeled, and stored in a -20°C refrigerator for use. The composition of the cell freezing solution of Example 1 is shown in Table 1:

[0042] Table 1 Composition of cell freezing solution in Example 1

[0043]

[0044] The cells were frozen using the above-prepared cell freezing solution, specifically: the HeLa cells to be frozen were taken, the culture medium was removed by centrifugation, the cells were resuspended in the above-prepared cell freezing solution to obtain a cell suspension, and the cell concentration was about 5×10^6 cells / mL, and the cell suspension was divided into cryopreservation tubes, each tube was 2 mL; a programmed cooling instrument was used to freeze the cells to -80°C at a cooling rate of 1°C per minute, and then the cells were quickly transferred to a liquid nitrogen tank for storage.

[0045] Cell recovery: When cells need to be recovered, remove the cryovials from liquid nitrogen and thaw quickly in a 37°C water bath for about 1-2 minutes until the ice is completely melted; immediately after thawing, transfer the cell suspension to a centrifuge tube containing preheated basal culture medium and remove the supernatant by centrifugation to remove DMSO; then resuspend the cells in fresh culture medium and culture in a 37°C, 5% CO2 incubator to observe the cell recovery.

[0046] Comparative Example 1

[0047] The conventional DMSO cell freezing solution was used as comparative example 1 for comparison. The composition of the conventional DMSO cell freezing solution is shown in Table 2:

[0048] Table 2 Cell freezing solution composition of Comparative Example 1

[0049] DMEM basal culture Fetal bovine serum DMSO 80v / v% 10v / v% 10v / v%

[0050] The cells were cryopreserved using the conventional DMSO cell cryopreservation solution, and the cryopreservation method and cell recovery operation were the same as those in Example 1.

[0051] The present invention uses the cell freezing solution of Example 1 and Comparative Example 1 to freeze cells, and performs liquid nitrogen violent freezing and thawing treatment on the cells 10 times, and compares and analyzes the survival and death of the two groups of cells by flow cytometry.

[0052] Figure 1 This is a schematic diagram of flow cytometric analysis of cells using the cell freezing solution of Example 1 after violent freezing and thawing with liquid nitrogen for 10 times, and then labeling living cells with Calcein green fluorescent staining. Figure 2 This is a schematic diagram of flow cytometric analysis of cells prepared by the cell freezing solution of Comparative Example 1 after violent freezing and thawing with liquid nitrogen for 10 times, and then labeling living cells with Calcein green fluorescent staining.

[0053] pass Figure 2 It can be seen that Figure 2The Q12 quadrant (lower left quadrant) shows 16.2% of the cells, which do not show obvious green fluorescence signals, that is, these cells may be dead cells or cell fragments; the Q9 quadrant (upper left quadrant) shows 0.42% of the cells, which show low green fluorescence and high side scatter signals, representing unstained complex cells, which may be early apoptotic or damaged cells; the Q11 quadrant (lower right quadrant) shows 61.0% of the cells, which show high green fluorescence signals and are stained living cells, indicating that the cell freezing solution of Comparative Example 1 still has a certain proportion of living cells after freezing and thawing; the Q10 quadrant (upper right quadrant) shows 22.4% of the cells, which are cells with strong green fluorescence and complex structure, which may be early apoptotic or complex living cells.

[0054] pass Figure 1 It can be seen that Figure 1 The Q12 quadrant (lower left quadrant) shows 12.6% of cells, which do not show green fluorescence and may be dead cells or cell fragments. Compared with Comparative Example 1, the proportion of unstained cells is reduced; the Q9 quadrant (upper left quadrant) shows 0.29% of cells, which also show low green fluorescence and high side scatter signals, and the proportion of unstained cells is slightly lower; the Q11 quadrant (lower right quadrant) shows 75.5% of cells, which show strong green fluorescence signals, indicating that Example 1 significantly improves the survival rate of cells compared with Comparative Example 1, which is significantly higher than 61.0% of Comparative Example 1; the Q10 quadrant (upper right quadrant) shows 11.6% of cells, which are cells with high green fluorescence and complex structure, which is lower than Comparative Example 1, indicating that there are fewer apoptotic cells.

[0055] Comparative analysis of the live cell ratios (Q10+Q11 quadrants) of Example 1 and Comparative Example 1 shows that the live cell ratio of Comparative Example 1 is 22.4%+61.0%=83.4%, and after freeze-thaw treatment, a relatively high proportion of cells are still in an early apoptotic state. The live cell ratio of Example 1 is 11.6%+75.5%=87.1%. In comparison, the cell freezing solution of Example 1 can better maintain the activity of cells, and the proportion of early apoptotic and damaged cells is relatively low.

[0056] After 10 violent freeze-thaw treatments with liquid nitrogen, the flow cytometry results showed that the proportion of live cells in Example 1 was higher than that in Comparative Example 1. In particular, in the Q11 quadrant (stained live cells), the proportion of live cells in Example 1 reached 75.5%, which was significantly better than 61.0% in Comparative Example 1. At the same time, the proportion of apoptotic cells (Q10 quadrant) was also significantly reduced in Example 1, indicating that the cell freezing solution of Example 1 can effectively reduce cell damage and maintain a higher cell survival rate.

[0057] Figure 3This is a schematic diagram of flow cytometric analysis of cells using the cell freezing solution of Example 1 after violent freezing and thawing with liquid nitrogen for 10 times, and then marking dead cells with PI red fluorescent staining. Figure 4 This is a schematic diagram of flow cytometric analysis of cells using the cell freezing solution of Comparative Example 1 after violent freezing and thawing with liquid nitrogen for 10 times, and then marking dead cells with PI red fluorescent staining.

[0058] pass Figure 4 It can be seen that Figure 4 The Q8 quadrant (lower left quadrant) shows that 73.8% of the cells are not stained, indicating that they are living cells; the Q5 quadrant (upper left quadrant) shows that 4.81% of the cells are unstained but have complex internal structures, which may be early apoptotic cells or complex living cells; the Q6 quadrant (upper right quadrant) shows that 14.5% of the cells show high red fluorescence signals, indicating that they are dead cells, and these cells have a more complex structure, which may be late apoptotic cells; the Q7 quadrant (lower right quadrant) shows that 6.89% of the cells show high red fluorescence signals, but their internal structures are simple, usually dead cells or cell fragments.

[0059] pass Figure 3 It can be seen that Figure 3 The Q8 quadrant (lower left quadrant) shows 89.7% of the cells, which are not stained, indicating that they are viable cells, which is significantly higher than 73.8% of Comparative Example 1, indicating that the cell freezing solution of Example 1 can better protect cell survival; the Q5 quadrant (upper left quadrant) shows that 1.11% of the cells are not stained but have a complex internal structure, and the proportion is small, indicating that there are fewer apoptotic or complex cells, further illustrating the cell protection effect of the cell freezing solution of Example 1; the Q6 quadrant (upper right quadrant) shows 9.14% of the cells, which are dead cells with high red fluorescence signals, and the proportion is significantly lower than 14.5% of Comparative Example 1, indicating that the cell freezing solution of Example 1 reduces the formation of dead cells; the Q7 quadrant (lower right quadrant) shows 0.072% of the cells, which have almost no structure or are dead cells or fragments with simple structure, which is significantly lower than 6.89% of Comparative Example 1, proving that the cell freezing solution of Example 1 greatly reduces cell death and damage.

[0060] By comparing and analyzing the dead cell ratios (Q6+Q7 quadrants) of Example 1 and Comparative Example 2, the dead cell ratio of Comparative Example 1 was 14.5%+6.89%=21.39%, indicating that after 10 freeze-thaw treatments, Comparative Example 1 had a higher cell mortality rate; the dead cell ratio of Example 1 was 9.14%+0.072%=9.21%, which was significantly reduced compared with Comparative Example 1, indicating that its cell protection effect during multiple freeze-thaw processes was significantly better than that of Comparative Example 1.

[0061] It can be seen that according to the results of flow cytometry, the cell group using the cell freezing solution of Example 1 has a significantly reduced cell mortality rate and a significantly improved survival rate after 10 violent freeze-thaw treatments with liquid nitrogen. In particular, the proportion of surviving cells in the Q8 quadrant reached 89.7%, which is significantly higher than 73.8% in Comparative Example 1. In addition, the proportion of dead cells (Q6+Q7) was significantly reduced in the cell freezing solution of Example 1, which was only 9.21%, much lower than 21.39% in Comparative Example 1, indicating that the cell freezing solution of Example 1 greatly reduced cell death and damage, and provided a more excellent cell protection effect.

[0062] Example 2

[0063] Example 2 is basically the same as Example 1, except that the formula of the cell freezing solution in Example 2 is as shown in Table 3:

[0064] Table 3 Composition of cell freezing solution in Example 2

[0065]

[0066] The freezing effect of the cell freezing solution in Example 2 was verified by the same freezing method and detection method as in Example 1. Flow cytometric analysis showed that after the cell group using the cell freezing solution in Example 2 was subjected to 10 violent freezing and thawing treatments with liquid nitrogen, the proportion of live cells was 85.7% and the proportion of dead cells was 9.2%.

[0067] Example 3

[0068] Example 3 is basically the same as Example 1, except that the formula of the cell freezing solution in Example 3 is as shown in Table 4:

[0069] Table 4 Composition of cell freezing solution in Example 1

[0070]

[0071] The freezing effect of the cell freezing solution in Example 3 was verified by the same freezing method and detection method as in Example 1. Flow cytometric analysis showed that after the cell group using the cell freezing solution in Example 3 was subjected to 10 violent freezing and thawing treatments with liquid nitrogen, the proportion of live cells was 91.5% and the proportion of dead cells was 7.1%.

[0072] Example 4

[0073] The only difference between Example 4 and Example 1 is that the cell freezing solution of Example 4 contains 1 ug / mL of recombinant human laminin-521 (A29249), and the specific composition is shown in Table 5:

[0074] Table 5 Composition of cell freezing solution of Example 4

[0075]

[0076] The freezing effect of the cell freezing solution in Example 4 was verified by the same freezing method and detection method as in Example 1. Flow cytometric analysis showed that after the cell group using the cell freezing solution of Example 4 was subjected to 10 violent freezing and thawing treatments with liquid nitrogen, the proportion of live cells was 89.6% and the proportion of dead cells was 7.66%.

[0077] Example 5

[0078] The only difference between Example 5 and Example 1 is that the cell freezing solution of Example 5 contains 5ug / mL of recombinant human laminin-521 (A29249), and the specific composition is shown in Table 6:

[0079] Table 6 Composition of cell freezing solution in Example 5

[0080]

[0081] The freezing effect of the cell freezing solution in Example 5 was verified by the same freezing method and detection method as in Example 1. Flow cytometric analysis showed that after the cell group using the cell freezing solution in Example 5 was subjected to 10 violent freezing and thawing treatments with liquid nitrogen, the proportion of live cells was 95.7% and the proportion of dead cells was 2.86%.

[0082] Example 6

[0083] The only difference between Example 6 and Example 1 is that the cell freezing solution of Example 6 contains 3ug / mL of recombinant human laminin-521 (A29249), and the specific composition is shown in Table 7:

[0084] Table 7 Composition of cell freezing solution of Example 6

[0085]

[0086] The freezing effect of the cell freezing solution in Example 6 was verified using the same freezing method and detection method as in Example 1. Flow cytometric analysis showed that after the cell group using the cell freezing solution in Example 6 was subjected to 10 violent freezing and thawing treatments with liquid nitrogen, the proportion of live cells was 92.7% and the proportion of dead cells was 5.26%.

[0087] Comparative Example 2

[0088] The composition of the cell freezing solution of Comparative Example 2 is shown in Table 8:

[0089] Table 8 Cell freezing solution composition of comparative example 2

[0090]

[0091] The freezing effect of the cell freezing solution in Comparative Example 2 was verified by the same freezing method and detection method as in Example 1. Flow cytometric analysis showed that after the cell group using the cell freezing solution of Comparative Example 2 was subjected to 10 violent freeze-thaw treatments with liquid nitrogen, the proportion of live cells was 89.3% and the proportion of dead cells was 7.91%.

[0092] By comparing Example 4 with Comparative Example 2, it is found that the freezing effect of Example 4 is better. This may be because vitamin E can effectively reduce the generation of stress signals during repeated freezing and thawing of cells through its antioxidant properties, and recombinant human laminin-521 can inhibit the activation of cell apoptosis signals by promoting extracellular matrix-cell signal transduction. The combination of the two can not only protect cells from oxidative stress, but also prevent cell damage during freezing by maintaining cell-matrix interaction.

[0093] Comparative Example 3

[0094] The composition of the cell freezing solution of Comparative Example 3 is shown in Table 9:

[0095] Table 9 Cell freezing solution composition of comparative example 3

[0096]

[0097] The freezing effect of the cell freezing solution in Comparative Example 3 was verified by the same freezing method and detection method as in Example 1. Flow cytometric analysis showed that after the cell group using the cell freezing solution of Comparative Example 3 was subjected to 10 violent freeze-thaw treatments with liquid nitrogen, the proportion of live cells was 84.3% and the proportion of dead cells was 11.9%.

[0098] By comparing Example 4 with Comparative Example 3, it is found that the freezing effect of Example 4 is better. This may be because chitosan quaternary ammonium salt, as a cationic polysaccharide, can interact with the cell membrane, provide physical protection, and reduce the mechanical damage of freezing to the cell membrane; and recombinant human laminin-521 further enhances the stability of the cell membrane and reduces the change of cell membrane permeability by binding to the cell membrane; the combination of the two not only reduces the damage of ice crystals to the cell membrane during the freezing process, but also can further repair the membrane damage after recovery, so that the cells have a stronger recovery ability when experiencing low temperature stress, and have a certain synergistic effect.

[0099] Comparative Example 4

[0100] 100 g of a 0.05 g / ml chitosan quaternary ammonium salt aqueous solution and 0.5 g of sodium carboxymethyl cellulose were rapidly stirred and mixed at 5000 rpm for 24 hours to obtain a mixed solution; the mixed solution was placed in a dialysis bag with a molecular weight cutoff of 400 Da, and the dialysis bag was placed in deionized water for dialysis treatment. The deionized water was replaced every 2 hours during the dialysis process, and the total dialysis time was controlled to be 60 hours; after the dialysis was completed, the liquid in the dialysis bag was mixed evenly with 200 g of a 1 wt% polyvinyl alcohol solution, and finally freeze-dried at -20°C for 24 hours to obtain a polymer-modified polysaccharide.

[0101] The difference between Comparative Example 4 and Example 1 is that the chitosan quaternary ammonium salt in Example 1 is replaced by the polymer-modified polysaccharide prepared above, and the rest is the same as Example 1.

[0102] The present invention has found through further systematic research that when the polymer modified polysaccharide prepared by chitosan quaternary ammonium salt, sodium carboxymethyl cellulose and polyvinyl alcohol is added to the cell freezing solution, the freezing solution will become viscous or even have flocculent precipitation during repeated freezing and thawing, thereby affecting the uniformity of the freezing solution, causing the freezing solution to be unstable and its protective function for cells to decrease. This may be because when the cationic chitosan quaternary ammonium salt combines with the anionic sodium carboxymethyl cellulose, a charge neutralization reaction occurs, causing the two to form an insoluble complex and produce instability.

[0103] The various aspects, embodiments, features and examples of the present invention should be considered as illustrative in all aspects and are not intended to limit the present invention, the scope of the present invention is defined only by the claims. Other embodiments, modifications and uses will be apparent to those skilled in the art without departing from the spirit and scope of the claimed invention.

[0104] In addition, the inventors of this case also referred to the aforementioned embodiments and conducted experiments with other raw materials, process operations, and process conditions described in this specification, and obtained relatively ideal results.

[0105] Although the present invention has been described with reference to illustrative embodiments, it will be appreciated by those skilled in the art that various other changes, omissions and / or additions may be made without departing from the spirit and scope of the present invention and that the elements of the embodiments may be replaced by substantial equivalents. In addition, many modifications may be made without departing from the scope of the present invention to adapt specific circumstances or materials to the teachings of the present invention. Therefore, it is not intended herein to limit the present invention to the disclosed specific embodiments for performing the present invention, but it is intended that the present invention will include all embodiments within the scope of the appended claims. In addition, unless specifically stated, any use of the terms first, second, etc. does not indicate any order or importance, but rather uses the terms first, second, etc. to distinguish one element from another.

[0106] The above description is only a specific embodiment of the present invention, so that those skilled in the art can understand or implement the present invention. Various modifications to these embodiments will be obvious to those skilled in the art, and the present invention will not be limited to the embodiments shown herein, but should conform to the widest scope consistent with the principles and novel features applied herein.

Claims

1. A cell freezing solution, characterized in that: The cell freezing solution comprises: Basal medium, 60-80 v / v%; Fetal bovine serum, 3-15 v / v%; Chitosan quaternary ammonium salt, 0.1-0.3w / v%; Cryoprotectants, 6-17 v / v%; Polyethylene glycol, 2-5 w / v%; Antioxidants, 1-10.5 mM; Cholesterol, 0.1-0.4w / v%; A buffer is used to maintain the pH value of the cell freezing solution at 7.2-7.

6.

2. The cell freezing solution according to claim 1, characterized in that: The cryoprotectant includes glycerol and / or dimethyl sulfoxide.

3. The cell freezing solution according to claim 2, characterized in that: The cryoprotectant comprises glycerol and dimethyl sulfoxide, wherein the content of the glycerol is 3-8 v / v% of the cell freezing solution, and the content of the dimethyl sulfoxide is 3-9 v / v% of the cell freezing solution.

4. The cell freezing solution according to claim 1, characterized in that: The antioxidant includes vitamin E and / or N-acetyl-L-cysteine.

5. The cell freezing solution according to claim 4, characterized in that: The antioxidant comprises vitamin E and N-acetyl-L-cysteine, the content of the vitamin E is 0.1-0.5 mM, and the content of the N-acetyl-L-cysteine ​​is 1-10 mM.

6. The cell freezing solution according to claim 1, characterized in that: It also includes recombinant human laminin, wherein the content of the recombinant human laminin is 1-5 ug / ml.

7. The cell freezing solution according to claim 6, characterized in that: The recombinant human laminin is recombinant human laminin-521.

8. The cell freezing solution according to claim 1, characterized in that: The basal culture medium is DMEM culture medium or RPMI-1640 culture medium; and / or, the buffer is HEPES, and the content of HEPES is 10-20 mM.

9. A cell freezing method, characterized in that: The cells are cryopreserved using the cell cryopreservation solution according to any one of claims 1 to 8.

10. A composition, characterized in that The invention comprises the cell freezing solution as described in any one of claims 1 to 8 and cells frozen in the cell freezing solution.

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