WO2024026996A1

WO2024026996A1 - Cell preservation solution and cell preservation method

Info

Publication number: WO2024026996A1
Application number: PCT/CN2022/120197
Authority: WO
Inventors: 唐伟
Original assignee: 华院计算技术(上海)股份有限公司
Priority date: 2022-08-02
Filing date: 2022-09-21
Publication date: 2024-02-08
Also published as: CN115349515A

Abstract

One aspect of the embodiments relates to a cell preservation solution, comprising: 20-50 mM of sucrose; 20-50 mM of mannitol; 10-50 mM of lactobionic acid; 5-20 mM of glucose; 1-3 mM of adenosine; 1-5 mM of reduced glutathione; 1-2 mM of dextran-40; 10-30 mM of potassium dihydrogen phosphate; 10-30 mM of potassium carbonate; 10-30 mM of potassium chloride; 5-30 mM of sodium chloride; 10-60 mM of sodium hydroxide; 10-60 mM of potassium hydroxide; 0-20 mM of vitamin E; 0-25 mM of 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid; and 0-10% of dimethyl sulfoxide. Another aspect of the embodiments relates to a cell preservation method, which uses the cell preservation solution of the present application to store cells.

Description

Cell preservation solution, cell preservation method

This application claims priority to the Chinese patent application filed with the China Patent Office on August 2, 2022, with the application number 202210922177.1 and the invention name "Cell Preservation Solution, Cell Preservation Method", the entire content of which is incorporated into this application by reference. .

Technical field

The present invention relates to the field of modern regenerative medicine, and in particular to a cell preservation solution and a cell preservation method.

Background technique

Modern regenerative medicine, such as gene therapy, tumor cell therapy, new antibody therapy, stem cell regenerative therapy, etc., is an emerging medical field. Its characteristic is to transform isolated living cells into therapeutic cells and then reinfuse/transplant them into into the patient's body to achieve therapeutic purposes. Generally speaking, in vitro living cells must be cryopreserved and cryopreserved to ensure that the viability and functionality of the cells are maintained during storage and transportation. In other words, cell cryopreservation and cryopreservation technology can be said to be an indispensable part of modern regenerative medicine.

The development of cell cryopreservation and cryopreservation technology needs to be based on full research and understanding of cell cryobiology. In order to effectively cryopreserve and freeze cells, it is necessary to avoid the damage to cells caused by cryopreservation and freezing according to the cryobiological characteristics of cells to the greatest extent, and to maintain cell viability and functionality. Specifically, under normal physiological temperature, the intracellular and extracellular fluids are in a state of isotonicity and isotonicity. Cell metabolism is active and needs to consume oxygen and nutrients while removing a large amount of free radicals and peroxides produced to maintain normal physiological state. However, in low-temperature refrigeration and freezing, cells may experience varying degrees of damage, affecting normal physiological activities and even death. The damage caused to cells by cryogenic refrigeration and freezing may be multifaceted, such as osmotic damage, mechanical damage, chemical damage, etc. For example, during the process of refrigeration and freezing, ice crystals may first form in the extracellular space environment, the interstitial liquid is concentrated, and the osmotic pressure rises, causing the water in the cells to leak out through the cell membrane and the electrolyte concentration in the cells. Increases lead to pH changes, protein denaturation, lysosome destruction, loss of membrane protein function, membrane leakage and rupture, etc., ultimately leading to cell death. At the same time, further cooling may produce ice crystals in cells, which may pierce or squeeze cell membranes, organelles, etc., resulting in mechanical damage to cell membranes and death. On the other hand, large amounts of free radicals and peroxides in cells can also induce cell apoptosis in low-temperature refrigeration and freezing environments.

Existing cell preservation solutions and cell preservation methods can prevent cell damage caused by cryogenic refrigeration and freezing to a certain extent, but they still have shortcomings. For example, when existing cell preservation solutions and cell preservation methods are used in clinical applications, there may be risks of adverse reactions to the human body, and may lead to intracellular deoxyribonucleic acid (DNA) distortion and protein denaturation, and The human body may have an adverse reaction to the preserved cells. In addition, some components of existing cell preservation solutions not only have limited sources and are expensive, but also have potential dangers of contamination by viruses and other microorganisms, and may cause side effects such as hypersensitivity to the human body.

Contents of the invention

The object of the present invention is to provide improved cell preservation solutions and cell preservation methods.

On the one hand, embodiments of the present invention relate to a cell preservation solution, which includes: 20mM to 50mM sucrose; 20mM to 50mM mannitol; 10mM to 50mM lactobionic acid; 5mM to 20mM glucose; 1mM to 3mM adenosine; 1mM to 5mM reduced form Glutathione; 1mM～2mM dextran-40; 10mM～30mM potassium dihydrogen phosphate; 10mM～30mM potassium carbonate; 10mM～30mM potassium chloride; 5mM～30mM sodium chloride; 10mM～60mM sodium hydroxide; 10mM～60mM Potassium hydroxide; 0mM～20mM vitamin E; 0mM～25mM 4-hydroxyethylpiperazineethanesulfonic acid; and 0%～10% dimethyl sulfoxide.

In some embodiments, the cell preservation solution includes 0.5mM to 20mM vitamin E and 0% dimethyl sulfoxide.

In some embodiments, the cell preservation solution includes 2mM to 5mM vitamin E.

In some embodiments, the cell preservation solution includes 0mM to 20mM 4-hydroxyethylpiperazineethanesulfonic acid.

In some embodiments, the cell preservation solution includes 0mM vitamin E and >0% dimethyl sulfoxide.

In some embodiments, the cell preservation solution includes 5% to 10% dimethyl sulfoxide.

In some embodiments, the pH value of the cell preservation solution is in the range of 7.0 to 8.0.

In some embodiments, the osmotic pressure of the cell preservation solution is 300 to 400 milliosmole (mosm).

In some embodiments, the cell preservation solution includes water.

Another aspect of embodiments of the present invention relates to a cell preservation method, which uses the cell preservation solution described in the present application to preserve cells.

In some embodiments, the cell preservation method preserves the cells at 2°C to 8°C or -80°C to -196°C.

In some embodiments, the cells include one or more of stem cells, immune cells, and tumor cells.

In some embodiments, the cells include human colon cancer cells.

When technical conditions permit, the technical solutions of the embodiments in this application can be combined in any way.

The present application will be further described below in conjunction with the accompanying drawings.

Description of the drawings

Figure 1 is a graph of experimental data of cell metabolic activity obtained in Experimental Example 5.

Figure 2 is a graph of cell proliferation activity experimental data obtained in Experimental Example 5.

Figure 3 is a graph of experimental data of cell metabolic activity obtained in Experimental Example 10.

Figure 4 is a graph of cell proliferation activity experimental data obtained in Experimental Example 10.

Detailed ways

The present invention will be further described below in conjunction with specific embodiments. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the protection scope of the present invention. In practical applications, improvements and adjustments made by those skilled in the art based on the present invention still fall within the protection scope of the present invention.

The cell preservation solution according to the embodiment of the present invention has clear ingredients, stable performance, safety and reliability, and can be directly used in the human body. The content of dimethylsulfoxide (DMSO) in the cell preservation solution of the embodiment of the present invention is low or 0. When used in clinical applications, there is less risk of adverse reactions to the human body and less risk of causing intracellular DNA (deoxyribonucleic acid, DNA) distortion and protein denaturation, the body is less likely to have adverse reactions to its preserved cells. In addition, the cell preservation solution according to the embodiment of the present invention has a wide range of ingredients, is cheap, and does not contain exogenous animal serum, exogenous proteins, etc., making it less likely to be contaminated by viruses and other microorganisms, and less likely to cause hypersensitivity to the human body. Wait for side effects.

In the embodiments of the present invention, unless otherwise specified, numerical values may include measurement errors, precision errors, measurement errors and other errors, for example, errors within the range of plus or minus 5%. For example, 10% may include a value in the range of 10%×(1±5%), that is, a value in the interval from 9.5% to 10.5%.

In the embodiments of the present invention, unless otherwise specified, the numerical range may include any sub-range therein, for example, 0%-10% may include 5%-10%, 0%-5%, 3%-8%, and so on.

The sucrose can serve as a non-permeable extracellular cryoprotectant to protect cell membranes from cold shock. The sucrose can also be a source of energy for cell metabolism during cell storage.

The mannitol can be used as an extracellular non-permeable substance to increase extracellular osmotic pressure, and can also be used as an effective hydroxide free radical scavenger.

The lactobionic acid can act as a non-permeable anion and can be used to counteract cell swelling during cold temperatures. The lactobionic acid can be biocompatible, can be a strong chelator of calcium and iron, and can help reduce cell damage due to calcium influx and free radical formation.

The glucose can be the main energy source for cell metabolism during cell storage. In the mitochondria, carbon dioxide and water are generated through the anaerobic glycolysis pathway or aerobic and tricarboxylic acid cycles, and adenosine triphosphate (ATP) is released to provide cells with Life activities provide energy.

The adenosine can be a substrate for the regeneration of adenosine triphosphate (ATP) in cell mitochondria, and can participate in providing energy for cell life activities.

The reduced glutathione can be an important antioxidant and hydroxyl radical scavenger in cells, protecting sulfhydryl groups in intracellular proteins and enzymes and other molecules, and protecting cell membranes from damage caused by free radicals. The reduced glutathione can also be a cofactor of glutathione peroxidase, which can promote the metabolism of lipid peroxides and hydrogen peroxide.

The dextran-40 can be a non-permeable extracellular cryoprotectant, which can offset the colloid osmotic pressure generated by negatively charged proteins and other metabolites in cells at low temperatures, thereby preventing cells from cracking due to water absorption and expansion.

The vitamin E can be an antioxidant that can scavenge intracellular oxygen free radicals at low temperatures, thereby preventing free radicals from damaging cell membranes. The vitamin E also inhibits the release of nitric oxide. The vitamin E also protects cells from freezing-induced apoptosis.

The 4-hydroxyethylpiperazine ethanesulfonic acid (N-2-Hydroxyethylpiperazine-N-2-Ethane Sulfonic Acid, HEPES) can be a large sulfonic acid biological pH liquid, which can have excellent buffering capacity at low temperatures, Prevents acidosis and does not penetrate into cells, helping to prevent osmotic swelling.

The dimethyl sulfoxide (DMSO) can be a permeable cell protective agent that can quickly penetrate the cell membrane and enter the cells, lower the freezing point, delay the cryopreservation process, increase the intracellular ion concentration, and reduce the formation of intracellular ice crystals, thereby Reduce cell damage. The corresponding content and concentration percentage of the dimethyl sulfoxide can be a volume percentage, that is, 0% to 10% can mean that the number of ml of the dimethyl sulfoxide in 100 ml of the cell preservation solution is 0 to 10.

The potassium dihydrogen phosphate, the potassium carbonate, the potassium chloride, the sodium chloride, the sodium hydroxide, and the potassium hydroxide can respectively be electrolytes to maintain intracellular pH levels and cell The sodium potassium ion pump works.

The cell preservation solution of the embodiment of the present application can be used for short-term freezing of cells at low temperatures such as 2°C to 8°C to maintain cell viability and functionality, or for long-term freezing of cells at deep low temperatures such as -80°C to -196°C to maintain cell viability and functionality. Maintain cell viability and functionality.

In this way, it can be helpful for the cell preservation solution to maintain cell viability and functionality during cryogenic storage of cells at, for example, 2° C. to 8° C. for, for example, 5 days, 7 days, or more. Moreover, the dimethylsulfoxide (DMSO) content in the cell preservation solution is 0. When used clinically, there is less risk of adverse reactions to the human body and less risk of causing intracellular deoxyribonucleic acid (DNA). ) distortion and protein denaturation, the body is less likely to have adverse reactions to its preserved cells.

In this way, it can be beneficial for the cell preservation solution to scavenge intracellular oxygen free radicals when preserving cells at a low temperature of, for example, 2°C to 8°C, thereby preventing damage to the cell membrane by free radicals, inhibiting the release of nitric oxide, and protecting the cells. Cells are protected from freezing-induced apoptosis.

In this way, it can help the cell preservation solution to have excellent buffering capacity when storing cells at a low temperature of 2°C to 8°C, prevent acidosis, and will not penetrate into the cells, helping to prevent osmotic swelling. .

In this way, it can be advantageous that the cell preservation solution can quickly penetrate the cell membrane during cryopreservation of cells at a cryogenic temperature of -80°C to -196°C for months, years, decades, or more. Entering cells, lowering the freezing point, delaying the cryopreservation process, while increasing intracellular ion concentration and reducing the formation of intracellular ice crystals, thereby reducing cell damage and maintaining cell survival rate and functionality.

In this way, it can be helpful for the cell preservation solution to have excellent buffering capacity when cryopreserving cells at a cryogenic temperature of, for example, -80°C to -196°C, prevent acidosis, and will not penetrate into the cells, which is helpful. To prevent infiltration and swelling.

In this way, the cell preservation solution can quickly penetrate the cell membrane and enter the cells when cryopreserving cells at, for example, -80°C to -196°C, lowering the freezing point, delaying the cryopreservation process, and at the same time increasing the intracellular ion concentration, reducing The formation of intracellular ice crystals, thereby reducing cell damage.

In this way, it can help the cell preservation solution to provide a physiological environment suitable for the cells.

In this way, the cell preservation solution can provide a physiological environment suitable for the cells. The osmotic pressure may be the sum of moles of each component in the cell preservation solution.

In some embodiments, the cell preservation solution includes water.

In this way, it can help the cell preservation solution to provide a physiological environment suitable for the cells. The other components of the cell preservation solution other than water may be mixed in water to form a mixed solution.

The cell preservation solution used in the cell preservation method according to the embodiment of the present invention has clear ingredients, stable performance, safety and reliability, and can be directly used in the human body. The content of dimethylsulfoxide (DMSO) in the cell preservation solution used in the cell preservation method according to the embodiment of the present invention is low or 0. When used in clinical applications, it is unlikely to cause adverse reactions to the human body. It is less likely to cause intracellular deoxyribonucleic acid (DNA) distortion and protein denaturation, and the human body is less likely to have an adverse reaction to its preserved cells. In addition, the cell preservation solution used in the cell preservation method according to the embodiments of the present invention has a wide range of ingredients, is low in price, does not contain exogenous animal serum, and is less susceptible to the potential risk of contamination by viruses and other microorganisms, and is not easily contaminated. Cause side effects such as hypersensitivity to the human body.

In this way, it can be helpful to ensure that the cell viability and functionality are maintained during storage and transportation in a short time or a long time. For example, the cells can be stored refrigerated at 2°C to 8°C for 5 days, 7 days or longer using the cell preservation method. Alternatively, the cells can be cryopreserved using the cell preservation method at -80°C to -196°C for months, years, decades or longer.

In some embodiments, the cell preservation method may include centrifuging and collecting the cells to be preserved, and then resuspending the cells in the cell preservation solution so that the cell concentration reaches, for example, 2×10 ⁶ /ml to 1×10 ⁷ /ml. , placed in a refrigerator at 2°C to 8°C for storage for up to 5 days, 7 days or longer.

In some embodiments, the cell preservation method may include adhering the cells to be preserved in a culture dish to a required number, aspirating off the cell culture medium, adding the cell preservation medium, and placing the cells at 2°C to 8°C. Store in the refrigerator for up to 5 days, 7 days or longer.

In some embodiments, the cell preservation method may include centrifuging and collecting the cells to be preserved, and then resuspending the cells in the cell preservation solution so that the cell concentration reaches 2×10 ⁶ /ml to 1×10 ⁷ /ml, Move it into a freezing tube, place it at a temperature of -80°C and cool it at a cooling rate of about 1°C/min. After overnight, move it to liquid nitrogen (-196°C) for long-term storage. The storage time can last for decades.

In this way, it can help ensure that one or more of the stem cells, immune cells, and tumor cells remain active and functional during storage and transportation.

In some embodiments, the cells include human colon cancer cells.

In this way, it can help ensure that the human colon cancer cells maintain viability and functionality during storage and transportation.

The cell preservation method and the rewarming method of cells stored in refrigerated or cryopreserved cells in the embodiments of the present application may include: centrifuging and collecting the refrigerated or cryopreserved cells, aspirating off the cell preservation solution, and using Resuspend the cells in cell culture medium and continue to culture the cells.

The cell preservation method and the function detection method of cells stored in refrigerated or cryopreserved cells in the embodiments of the present application may include: after rewarming and culturing the preserved cells for 1 day, detecting them using, for example, ALAMAR BLUE ^TM cell viability detection reagent. Cell metabolic activity. Within 1 day after cells are rewarmed, there may be delayed cell apoptosis. The cells tend to be stable after 1 day, so detection after 1 day can more truly reflect cell activity.

When the cells stored in the cell preservation method, the cell preservation solution, and the cryopreservation solution involved in the embodiments of this application need to be used clinically, they can be resuspended and directly reinfused into the body.

The experimental examples in this application are mainly used to help understand the embodiments of the present invention and are not intended to limit the scope of the claims. Referring to the experimental examples described below, it can be seen that the cell preservation solution in the embodiment of the present application has a simple formula, lower cost, and performance comparable to or even better than commercially available products.

Experimental examples 1-4

Weigh out sucrose, mannitol, lactobionic acid, glucose, adenosine, reduced glutathione, dextran-40, and phosphoric acid according to the components and concentrations of the cell preservation solution corresponding to Experimental Examples 1-4 shown in Table 1 below. Potassium dihydrogen, potassium carbonate, potassium chloride, sodium chloride, potassium hydroxide, vitamin E, 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid, HEPES, Prepare four mixed solutions with purified water, titrate the pH to 7.6 with sodium hydroxide, and filter with a 0.2um sterile filter membrane. The filtrate obtained by filtration is the four cell preservation solutions of Experimental Examples 1-4.

Table 1

Experimental example 5

with those purchased on the market

The quality of the cell preservation solutions in Experimental Examples 1-4 can be tested by comparing the preservation solution with the standard.

Specifically, the human colon cancer cell line (HCT116WT) was cultured in a culture dish. After digestion with trypsin solution, the cell suspension was collected, centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, and the cell preservation solutions of Experimental Examples 1-4 were used. sample and

Resuspend the cells in the preservation solution sample (control group), aliquot them into sterile cryopreservation tubes at a cell concentration of 2×10 ⁶ /tube, place them in a 4°C refrigerator, and store them in the refrigerator for 5 days.

Then, take out some of the cryovials, collect the cells by centrifugation, aspirate the preservation solution, resuspend the cells in cell culture medium, and use a Countess 3 automatic cell counter to morphologically detect the percentage of viable cells. The results are shown in Table 2 below. It can be seen that the survival rate of cells preserved using the cell preservation solution samples of Experimental Examples 1-4 is as high as over 94%, which is comparable to or even higher than the survival rate of cells preserved using the control group. However, the cell preservation solution of the embodiment of the present invention is simpler and lower in cost than the formula of the control group.

Table 2

样本sample	细胞存活率％(形态学)Cell viability % (morphology)
实验示例1Experimental example 1	94.794.7
实验示例2Experimental example 2	95.395.3
实验示例3Experimental example 3	96.296.2
实验示例4Experimental example 4	96.696.6
对比组comparison group	94.494.4

On the other hand, use the cell preservation solution sample of Experimental Example 1-4 and the

Cells in the preservation solution sample (control group) that had been refrigerated at 4°C for 5 days were moved to a 96-well culture dish for recovery and culture. One day later, ALAMAR BLUE ^TM cell viability detection reagent was used to detect cell metabolic activity, and Sartorius IncuCyte real-time live cells were used simultaneously. The imaging analyzer records cell proliferation activity. Subsequently, remove the ALAMAR BLUE ^TM cell viability detection reagent solution, re-add fresh culture medium, resuscitate and culture, repeat the ALAMAR BLUE ^TM cell viability detection reagent to detect cell metabolic activity experiment after 3 days, and simultaneously use Sartorius IncuCyte real-time live cell imaging analysis The instrument records cell proliferation activity. Then, remove the ALAMAR BLUE ^TM cell viability detection reagent solution, add fresh culture medium again, resuscitate and culture. After 5 days, repeat the ALAMAR BLUE ^TM cell viability detection reagent to detect cell metabolic activity, and use Sartorius IncuCyte real-time live cells simultaneously. The imaging analyzer records cell proliferation activity.

The cell metabolic activity experimental data on

days

1, 3, and 5 of recovery obtained by the above ALAMAR BLUE ^TM cell viability detection reagent test are shown in Figure 1. The experimental data on cell proliferation activity recorded by Sartorius IncuCyte real-time live cell imaging analyzer are shown in Figure 1. in Figure 2. Referring to Figure 1, the metabolic function of the cells preserved with the cell preservation solution samples of Experimental Examples 1-4 was effectively restored, and the number of absorption units increased as the number of recovery days increased. As shown in Figure 2, cells preserved with the cell preservation fluid samples of Experimental Examples 1-4 had normal growth, differentiation, and proliferation functions, indicating that the refrigerated cells were effectively recovered, and the degree of cell fusion was higher as the recovery days increased. It can be seen that the performance of the cell preservation solutions of the embodiments of the present invention is comparable to or better than that of the comparison group.

Experimental example 6-9

Weigh out sucrose, mannitol, lactobionic acid, glucose, adenosine, reduced glutathione, dextran-40, and phosphate according to the components and concentrations of the cell preservation solution corresponding to Experimental Examples 6-9 shown in Table 3 below. Potassium dihydrogen, potassium carbonate, potassium chloride, sodium chloride, potassium hydroxide, 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid, HEPES), dimethyl sulfoxide, prepare four mixed solutions with purified water, titrate the pH to 7.6 with sodium hydroxide, and filter with a 0.2um sterile filter membrane. The filtrate obtained by filtration is the four cell preservation solutions in Experimental Examples 6-9.

table 3

Experimental example 10

Use those purchased on the market

CS5 cell cryopreservation solution and

The CS10 cell cryopreservation solution is used for benchmarking to measure the quality of the cell preservation solutions in Experimental Examples 6-9.

Specifically, the experimental cell line HCT116WT was cultured in a culture dish. After digestion with trypsin solution, the cell suspension was collected, centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, and the cell preservation solution samples of Experimental Examples 6-9 and

CS5 cell cryopreserved solution sample (comparison group 1) and

CS10 cell cryopreservation solution sample (comparison group 2) resuspends the cells, aliquots into sterile cryopreservation tubes at a cell concentration of 2×10 ⁶ /tube, and places them at a temperature of -80°C at a rate of approximately 1°C/min. The temperature was reduced at a cooling rate, and then transferred to liquid nitrogen (-196°C) for freezing and storage overnight.

After 2 months of storage, take out the cryopreservation tube and quickly rewarm it at 37°C. Stop rewarming when the ice crystals are about to completely melt. After centrifugation to collect, remove the cell preservation solution and cryopreservation solution, and reuse them with cell culture medium. The cells were suspended and the percentage of viable cells was morphologically determined using a Countess 3 automatic cell counter. The results are shown in Table 4 below. It can be seen that the survival rate of cells preserved using the cell preservation solution samples of Experimental Examples 6-9 is as high as more than 95%, which is comparable to or even higher than the survival rate of cells preserved using

Comparative Groups

1 and 2. However, the cell preservation solution of the embodiment of the present invention is simpler and lower in cost than the formulas of

Comparative Groups

1 and 2.

Table 4

样本sample	细胞存活率％(形态学)Cell viability % (morphology)
实验示例6Experimental example 6	95.795.7
实验示例7Experimental example 7	97.297.2
实验示例8Experimental example 8	96.996.9
实验示例9Experimental example 9	95.795.7
对比组1 Comparison group 1	94.994.9
对比组2 Comparison group 2	95.695.6

On the other hand, move the above-mentioned cell preservation solution samples of Experimental Examples 6-9 and the cells of

Comparative Groups

1 and 2 that have been cryopreserved for 2 months to a 96-well culture dish at an amount of 4000 cells/well. After one day, use ALAMAR BLUE ^TM cell viability detection reagent detects cell metabolic activity, and Sartorius IncuCyte real-time live cell imaging analyzer is used to simultaneously record cell proliferation activity. Subsequently, remove the ALAMAR BLUE ^TM cell viability detection reagent solution, re-add fresh culture medium, resuscitate and culture, repeat the ALAMAR BLUE ^TM cell viability detection reagent to detect cell metabolic activity experiment after 3 days, and simultaneously use Sartorius IncuCyte real-time live cell imaging analysis The instrument records cell proliferation activity. Then, remove the ALAMAR BLUE ^TM cell viability detection reagent solution, add fresh culture medium again, resuscitate and culture. After 5 days, repeat the ALAMAR BLUE ^TM cell viability detection reagent to detect cell metabolic activity, and use Sartorius IncuCyte real-time live cells simultaneously. The imaging analyzer records cell proliferation activity.

The cell metabolic activity experimental data obtained from the above-mentioned ALAMAR BLUE ^TM cell viability detection reagent test experiment on the 1st, 3rd and 5th days of recovery are shown in Figure 3. The 1st and 3rd days of recovery recorded by the Sartorius IncuCyte real-time live cell imaging analyzer , 5-day cell proliferation activity experimental data are shown in Figure 4. Please refer to Figure 3. The metabolic function of the cells preserved with the cell preservation solution samples of Experimental Examples 6-9 is effectively restored, and the number of absorption units increases as the number of recovery days increases. As shown in Figure 4, the cells preserved with the cell preservation solution samples of Experimental Examples 6-9 have normal growth, differentiation, and proliferation functions, indicating that frozen cells are effectively revived, and the degree of cell fusion becomes higher as the number of days of recovery increases. It can be seen that the performance of the cell preservation solution of the embodiment of the present invention is comparable to or even better than that of the

comparison groups

1 and 2.

The various specific embodiments described above and shown in the drawings are only used to illustrate the present invention and are not exhaustive of the present invention. Within the scope of the basic technical idea of the present invention, any changes made to the present invention by those of ordinary skill in the relevant technical fields are within the protection scope of the present invention.

Claims

A cell preservation solution, characterized by including:

20mM～50mM sucrose;

20mM～50mM mannitol;

10mM～50mM lactobionic acid;

5mM～20mM glucose;

1mM～3mM adenosine;

1mM～5mM reduced glutathione;

1mM～2mM dextran-40;

10mM～30mM potassium dihydrogen phosphate;

10mM～30mM potassium carbonate;

10mM～30mM potassium chloride;

5mM～30mM sodium chloride;

10mM～60mM sodium hydroxide;

10mM～60mM potassium hydroxide;

0mM～20mM Vitamin E;

0mM～25mM 4-hydroxyethylpiperazineethanesulfonic acid; and

0% ~ 10% dimethyl sulfoxide.
The cell preservation solution according to claim 1, characterized in that it contains 0.5mM to 20mM vitamin E and 0% dimethyl sulfoxide.
The cell preservation solution according to claim 2, characterized in that it contains 2mM to 5mM vitamin E.
The cell preservation solution according to any one of claims 1 to 3, characterized in that it includes 0mM to 20mM 4-hydroxyethylpiperazineethanesulfonic acid.
The cell preservation solution according to claim 1, characterized in that it includes 0mM vitamin E and >0% dimethyl sulfoxide.
The cell preservation solution according to claim 5, characterized in that it contains 0mM to 20mM 4-hydroxyethylpiperazineethanesulfonic acid.
The cell preservation solution according to claim 5 or 6, characterized in that it contains 5% to 10% dimethyl sulfoxide.
The cell preservation solution according to any one of claims 1 to 7, wherein the pH value is in the range of 7.0 to 8.0.
The cell preservation solution according to any one of claims 1 to 8, characterized in that the osmotic pressure is 300 to 400 milliosmole.
The cell preservation solution according to any one of claims 1 to 9, characterized in that it contains water.
A cell preservation method, characterized in that cells are preserved using the cell preservation solution according to any one of claims 1-10.
The cell preservation method according to claim 11, characterized in that the cells are preserved at 2°C to 8°C or -80°C to -196°C.
The cell preservation method according to claim 11 or 12, wherein the cells include one or more of stem cells, immune cells, and tumor cells.
The cell preservation method according to any one of claims 11-13, wherein the cells comprise human colon cancer cells.