CN117084236A - A cryopreservation solution that improves the survival rate of stem cells and its preparation process - Google Patents

Abstract

The invention relates to the technical field of cell cryopreservation solutions, and in particular to a cryopreservation solution that improves the survival rate of stem cells and its preparation process. The invention provides such a cryopreservation solution that improves the survival rate of stem cells and a preparation process thereof, which includes the following steps: preparing an extracellular cryoprotectant, preparing an intracellular cryoprotectant, preparing a stabilizer, preparing PBS buffer and Bacteria mixed filling. The present invention prepares extracellular cryoprotectant and intracellular cryoprotectant, and uses non-permeable components and natural permeable components to compound, hydroxyethyl starch can weaken osmotic damage and improve the stability of cells in a hypertonic environment. Survival rate, proline has good biocompatibility, can regulate the balance of intracellular and intracellular osmotic pressure and inhibit oxidative damage. The prepared cryopreservation solution does not contain DMSO and serum, which reduces cytotoxicity and thus improves the survival rate of stem cells.

Description

A cryopreservation solution that improves the survival rate of stem cells and its preparation process

Technical field

The invention relates to the technical field of cell cryopreservation solutions, and in particular to a cryopreservation solution that improves the survival rate of stem cells and its preparation process.

Background technique

Cell cryopreservation is a technology that places cells in a low-temperature environment to reduce cell metabolism for long-term storage. It is usually necessary to use cryopreservation solution to freeze cells. Common cryopreservation solutions will add dimethyl sulfoxide DMSO. However, DMSO has a certain degree of cytotoxicity, which may cause irreversible damage to cell structure and function, affect the survival rate of stem cells, and lead to adverse reactions after cell transplantation in clinical patients.

Currently, stem cell cryopreservation solutions use non-permeable components such as hydroxyethyl starch and trehalose, which have good antifreeze properties and the ability to inhibit osmotic damage, and can stabilize the cell membrane structure under low temperature conditions. However, non-permeable components It is difficult for particles to enter cells and the effect of preserving cells is limited. Therefore, there is an urgent need to develop a cryopreservation solution that improves the survival rate of stem cells and its preparation process. By using non-permeable components and natural permeable components, stem cells can be preserved. Maintain similar morphology and vitality to fresh cells, thereby meeting the cryopreservation requirements of cells for clinical treatment.

Contents of the invention

In view of the above-mentioned shortcomings of the prior art, the object of the present invention is to provide a cryopreservation solution that improves the survival rate of stem cells and a preparation process thereof.

A cryopreservation solution preparation process that improves the survival rate of stem cells specifically includes the following steps:

S1: Preparation of extracellular cryoprotectant

Add hydroxyethyl starch to the serum-free culture medium, filter and sterilize the hydroxyethyl starch solution through a sterile filter membrane, and obtain an extracellular cryoprotectant;

S2: Preparation of intracellular cryoprotectant

Add proline to the serum-free medium to obtain a proline solution with a concentration of 20% to 22%. Filter and sterilize the proline solution through a 0.20-0.22μm sterile filter to obtain intracellular cryopreservation protection. agent;

S3: Preparation of Stabilizer

Add methylcellulose into the sterilization container, heat it through a heating device, and perform sterilization and disinfection through ultraviolet lamps. The methylcellulose enters the stirring container through a sterile pipeline, and the methylcellulose in the mixing container is cooled through an ice water bath. After adding the serum-free culture medium, stir the methylcellulose evenly with a stirrer, put the stirring container into a freezing device for storage, melt the methylcellulose solution through a water bath device to obtain a stabilizer, and use it to sterile packaging equipment. The stabilizer is put into separate containers, and the packaged stabilizer is stored through a freezing device;

S4: Prepare PBS buffer

Weigh NaCl, KH ₂ P0 ₄ and Na ₂ HP0 ₄ ·12H ₂ 0 into the quantitative container through an electronic scale, then add distilled water to the quantitative container, dilute to volume to obtain PBS buffer, add the prepared PBS buffer to the autoclave Perform high-pressure sterilization in a sterilization container, and put the sterilized PBS buffer into an insulated container for storage;

S5: Aseptic mixing and filling

Melt the aliquoted stabilizer at 2℃-4℃, then mix the extracellular cryoprotectant, intracellular cryoprotectant, stabilizer and PBS buffer in a volume ratio of (5-6): (2-3): (1-2): (2-3) Perform aseptic mixing and filling to obtain a frozen storage solution. The final concentration of hydroxyethyl starch is 5%-7%, and the final concentration of proline is 4%. ~4.4%, the final concentration of methylcellulose is 1%-1.2%.

Further, step S1 prepares an extracellular cryoprotectant, specifically including the following steps:

S1.1: Add hydroxyethyl starch to the serum-free medium to obtain a hydroxyethyl starch solution with a concentration of 10% to 14%;

S1.2: Filter and sterilize the hydroxyethyl starch solution through a 0.20-0.22 μm sterile filter membrane to obtain an extracellular cryoprotectant.

Further, step S3 prepares a stabilizer, specifically including the following steps:

S3.1: Add methylcellulose into the sterilization container, heat the methylcellulose through the heating device, and sterilize the methylcellulose through ultraviolet lamp;

S3.2: The sterilized methylcellulose enters the stirring container through a sterile pipeline. Use an ice water bath to cool the methylcellulose in the stirring container to 37°C-38°C. After adding serum-free medium, the methylcellulose The concentration of cellulose is 5%-6%. Stir the methylcellulose evenly with a stirrer. Place the stirring container containing methylcellulose into a freezing device and place it at -22°C to -20°C. 12h-14h;

S3.3: Place the stirring container in a water bath device to melt the methylcellulose and culture medium at 2°C-4°C to obtain the stabilizer;

S3.4: Put the stabilizer into the packaging container through the sterile packaging equipment, and store the packaged stabilizer at -22°C ~ -20°C through the freezing device.

Further, step S4 prepares PBS buffer solution, specifically including the following steps:

S4.1: Weigh NaCl, KH ₂ P0 ₄ and Na ₂ HP0 ₄ ·12H ₂ 0 into the quantitative container using an electronic scale, then add distilled water to the quantitative container, and dilute to volume to obtain PBS buffer. In the PBS buffer, NaCl, The mass ratio of KH ₂ P0 ₄ and Na ₂ HP0 ₄ ·12H ₂ 0 is (8.9-9): (0.14-0.15): (2-2.1);

S4.2: Add the prepared PBS buffer solution into the high-pressure sterilization container, and perform high-pressure sterilization of the PBS solution. The sterilization temperature is 121°C-122°C. Use a timer. After high-pressure sterilization for 15-20 minutes, wait for 4 seconds. Store the sterilized PBS buffer in an insulated container at ℃-6℃.

Further, when performing step S3.3 and maintaining the temperature of the stirring container at 2°C-4°C through a water bath device, step S4.2 can be performed simultaneously, and the PBS buffer in step S4.2 and step S3. The stirring vessels in 3 are placed in two connected water baths, and the PBS buffer in step S4.2 is first exposed to cold water. After the cold water leaves the water bath where the PBS buffer is located, it passes through the insulation pipe. Enter the stirring container in step S3.3, and finally return to the refrigeration device.

Further, the sterile filter membrane is specifically a cellulose acetate filter membrane.

A cryopreservation solution that improves the survival rate of stem cells is prepared by applying a cryopreservation solution preparation process that improves the survival rate of stem cells.

The beneficial effects are: 1. By preparing extracellular cryoprotectant and intracellular cryoprotectant, and using non-permeable components and natural permeable components, hydroxyethyl starch can weaken osmotic damage and improve cell stability. Survival rate in a hypertonic environment. Proline has good biocompatibility and can regulate the balance of intracellular and intracellular osmotic pressure and inhibit oxidative damage. The prepared cryopreservation solution does not contain DMSO and serum, which reduces cytotoxicity and thereby improves stem cells. Survival rate.

2. The present invention adds methylcellulose. Methylcellulose has the effect of adjusting the viscosity of cells and is resistant to acid, alkali, microorganisms and heat. It is used to reduce damage caused by changes in osmotic pressure during cell cryopreservation and accessory processes. The aqueous solution of cellulose is very stable, which is beneficial to the long-term storage of cryopreservation solutions.

3. The present invention places the PBS buffer solution and the stirring container in two connected water baths respectively, so that the PBS buffer solution and the stirring container can be bathed in the water bath device at the same time, thereby utilizing the heat generated during the high-pressure sterilization process. The heat is transferred to the stirring container filled with methylcellulose solution, which is energy-saving and environmentally friendly.

Description of the drawings

Figure 1 is a process flow chart of a cryopreservation solution preparation process used in an embodiment of the present invention to improve the survival rate of stem cells.

Detailed ways

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only some of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of the present invention.

Example 1: A cryopreservation solution preparation process that improves the survival rate of stem cells, as shown in Figure 1, specifically includes the following steps:

S1: Preparation of extracellular cryoprotectant:

Add hydroxyethyl starch to the serum-free culture medium to obtain a hydroxyethyl starch solution with a concentration of 14%. Hydroxyethyl starch is a biodegradable amylopectin derivative and has good antifreeze properties and film stabilization effects. and the ability to inhibit osmotic damage;

Filter and sterilize the hydroxyethyl starch solution through a 0.22 μm sterile filter membrane to obtain the extracellular cryoprotectant. The sterile filter membrane is specifically a cellulose acetate filter membrane.

S2: Preparation of intracellular cryoprotectant

Add proline to the serum-free culture medium to obtain a proline solution with a concentration of 22%. Filter and sterilize the proline solution through a 0.22 μm sterile filter membrane to obtain an intracellular cryoprotectant. Sterile filter The membrane is specifically a cellulose acetate filter membrane, which has good biocompatibility with proline and has strong ability to inhibit osmotic damage;

S3: Preparation of Stabilizer:

Add methylcellulose into a sterilization container, heat the methylcellulose through a heating device, and sterilize the methylcellulose through ultraviolet lamps. Ultraviolet rays can destroy the molecular structure of microbial cells and cause cell death, achieving Bactericidal effect;

The sterilized methylcellulose enters the stirring container through a sterile pipeline. The methylcellulose in the stirring container is cooled to 37°C through an ice-water bath. After adding serum-free culture medium, the concentration of methylcellulose is 6%. , stir the methylcellulose evenly with a stirrer, put the stirring container containing methylcellulose into the freezing device, and place it at -22°C for 12h;

Place the stirring container in a water bath device to melt the methylcellulose and culture medium at 2°C to obtain the stabilizer;

Put the stabilizer into the packaging container through the sterile packaging equipment, and store the packaged stabilizer at -22°C through the freezing device.

S4: Prepare PBS buffer solution:

Weigh NaCl, KH ₂ P0 ₄ and Na ₂ HP0 ₄ ·12H ₂ 0 into the quantitative container using an electronic scale, then add distilled water to the quantitative container, and dilute to volume to obtain PBS buffer. NaCl, KH ₂ P0 ₄ in the PBS buffer The mass ratio of Na ₂ HP0 ₄ ·12H ₂ 0 is 8.9:0.14:2;

Add the prepared PBS buffer solution into the high-pressure sterilization container, autoclave the PBS solution at a sterilization temperature of 121°C, and use high-pressure saturated steam to denature the proteins and nucleic acids in the microorganisms, thereby killing the microorganisms and timing the After 20 minutes of high-pressure sterilization, put the sterilized PBS buffer into an insulated container and store it at 4°C.

S5: Aseptic mixing and filling

Melt the aliquoted stabilizer at 2°C, then mix the extracellular cryoprotectant, intracellular cryoprotectant, stabilizer and PBS buffer in a volume ratio of 5:2:1:2. The bacteria are mixed and filled to obtain a frozen storage solution. The final concentration of hydroxyethyl starch is 5%, the final concentration of proline is 4%, and the final concentration of methylcellulose is 1%.

When performing step S3 to maintain the temperature of the stirring vessel at 2°C using a water bath device, step S4 can be performed simultaneously, and the PBS buffer in step S4 and the stirring vessel in step S3 are placed in two connected water baths. , and let the PBS buffer in step S4 first contact the cold water, so that the heat in the high-pressure sterilized PBS buffer is transferred to the water bath. After the cold water leaves the water bath where the PBS buffer is located, pass The thermal insulation pipe enters the stirring container in step S3, so that the heat in the water bath is transferred to the stirring container, thereby utilizing the heat in the high-pressure sterilization process, and finally the water returns to the refrigeration device.

A cryopreservation solution that improves the survival rate of stem cells is prepared by applying a cryopreservation solution preparation process that improves the survival rate of stem cells.

Example 2: A cryopreservation solution preparation process that improves the survival rate of stem cells, as shown in Figure 1, specifically includes the following steps:

S1: Preparation of extracellular cryoprotectant:

Add hydroxyethyl starch to the serum-free culture medium to obtain a hydroxyethyl starch solution with a concentration of 10%. Hydroxyethyl starch is a biodegradable amylopectin derivative and has good antifreeze properties and film stabilization effects. and the ability to inhibit osmotic damage;

Filter and sterilize the hydroxyethyl starch solution through a 0.22 μm sterile filter membrane to obtain the extracellular cryoprotectant. The sterile filter membrane is specifically a cellulose acetate filter membrane.

S2: Preparation of intracellular cryoprotectant

Add proline to the serum-free culture medium to obtain a proline solution with a concentration of 20%. Filter and sterilize the proline solution through a 0.22 μm sterile filter membrane to obtain an intracellular cryoprotectant. Sterile filter The membrane is specifically a cellulose acetate filter membrane, which has good biocompatibility with proline and has strong ability to inhibit osmotic damage;

S3: Preparation of Stabilizer:

Add methylcellulose into a sterilization container, heat the methylcellulose through a heating device, and sterilize the methylcellulose through ultraviolet lamps. Ultraviolet rays can destroy the molecular structure of microbial cells and cause cell death, achieving Bactericidal effect;

The sterilized methylcellulose enters the stirring container through a sterile pipeline. The methylcellulose in the stirring container is cooled to 37°C through an ice-water bath. After adding serum-free medium, the concentration of methylcellulose is 5%. , stir the methylcellulose evenly with a stirrer, put the stirring container containing methylcellulose into the freezing device, and place it at -22°C for 12h;

Place the stirring container in a water bath device to melt the methylcellulose and culture medium at 2°C to obtain the stabilizer;

Put the stabilizer into the packaging container through the sterile packaging equipment, and store the packaged stabilizer at -22°C through the freezing device.

S4: Prepare PBS buffer solution:

Weigh NaCl, KH ₂ P0 ₄ and Na ₂ HP0 ₄ ·12H ₂ 0 into the quantitative container using an electronic scale, then add distilled water to the quantitative container, and dilute to volume to obtain PBS buffer. NaCl, KH ₂ P0 ₄ in the PBS buffer The mass ratio of Na ₂ HP0 ₄ ·12H ₂ 0 is 9:0.15:2.1;

Add the prepared PBS buffer into the high-pressure sterilization container, and autoclave the PBS solution at a sterilization temperature of 121°C-122°C. Use high-pressure saturated steam to denature the proteins and nucleic acids in the microorganisms, thereby killing the microorganisms. , and at the same time, the timer counts. After high-pressure sterilization for 20 minutes, put the sterilized PBS buffer into an insulated container and store it at 4°C.

S5: Aseptic mixing and filling

Melt the aliquoted stabilizer at 2°C, then mix the extracellular cryoprotectant, intracellular cryoprotectant, stabilizer and PBS buffer in a volume ratio of 6:3:2:3. The bacteria are mixed and filled to obtain a frozen storage solution. The final concentration of hydroxyethyl starch is 7%, the final concentration of proline is 4.4%, and the final concentration of methylcellulose is 1.2%.

When performing step S3 to maintain the temperature of the stirring vessel at 2°C using a water bath device, step S4 can be performed simultaneously, and the PBS buffer in step S4 and the stirring vessel in step S3 are placed in two connected water baths. , and let the PBS buffer in step S4 first contact the cold water, so that the heat in the high-pressure sterilized PBS buffer is transferred to the water bath. After the cold water leaves the water bath where the PBS buffer is located, pass The thermal insulation pipe enters the stirring container in step S3, so that the heat in the water bath is transferred to the stirring container, thereby utilizing the heat in the high-pressure sterilization process, and finally the water returns to the refrigeration device.

A cryopreservation solution that improves the survival rate of stem cells is prepared by applying a cryopreservation solution preparation process that improves the survival rate of stem cells.

Example 3: A cryopreservation solution preparation process that improves the survival rate of stem cells, as shown in Figure 1, specifically includes the following steps:

S1: Preparation of extracellular cryoprotectant:

Add hydroxyethyl starch to the serum-free culture medium to obtain a hydroxyethyl starch solution with a concentration of 14%. Hydroxyethyl starch is a biodegradable amylopectin derivative and has good antifreeze properties and film stabilization effects. and the ability to inhibit osmotic damage;

Filter and sterilize the hydroxyethyl starch solution through a 0.20 μm sterile filter membrane to obtain the extracellular cryoprotectant. The sterile filter membrane is specifically a cellulose acetate filter membrane.

S2: Preparation of intracellular cryoprotectant

Add proline to the serum-free culture medium to obtain a proline solution with a concentration of 22%. Filter and sterilize the proline solution through a 0.20 μm sterile filter membrane to obtain an intracellular cryoprotectant. Sterile filter The membrane is specifically a cellulose acetate filter membrane, which has good biocompatibility with proline and has strong ability to inhibit osmotic damage;

S3: Preparation of Stabilizer:

Add methylcellulose into a sterilization container, heat the methylcellulose through a heating device, and sterilize the methylcellulose through ultraviolet lamps. Ultraviolet rays can destroy the molecular structure of microbial cells and cause cell death, achieving Bactericidal effect;

The sterilized methylcellulose enters the stirring container through a sterile pipeline. The methylcellulose in the stirring container is cooled to 38°C through an ice-water bath. After adding serum-free culture medium, the concentration of methylcellulose is 6%. , stir the methylcellulose evenly with a stirrer, put the stirring container containing methylcellulose into the freezing device, and place it at -20°C for 14h;

Place the stirring container in a water bath device to melt the methylcellulose and culture medium at 4°C to obtain the stabilizer;

The stabilizer is loaded into the packaging container through the sterile packaging equipment, and the packaged stabilizer is stored at -20°C through the freezing device.

S4: Prepare PBS buffer solution:

Weigh NaCl, KH ₂ P0 ₄ and Na ₂ HP0 ₄ ·12H ₂ 0 into the quantitative container using an electronic scale, then add distilled water to the quantitative container, and dilute to volume to obtain PBS buffer. NaCl, KH ₂ P0 ₄ in the PBS buffer The mass ratio of Na ₂ HP0 ₄ ·12H ₂ 0 is 8.9:0.14:2;

Add the prepared PBS buffer solution into the high-pressure sterilization container, autoclave the PBS solution at a sterilization temperature of 122°C, and use high-pressure saturated steam to denature the proteins and nucleic acids in the microorganisms, thereby killing the microorganisms and timing the After 15 minutes of high-pressure sterilization, put the sterilized PBS buffer into an insulated container and store it at 6°C.

S5: Aseptic mixing and filling

Melt the aliquoted stabilizer at 4°C, then mix the extracellular cryoprotectant, intracellular cryoprotectant, stabilizer and PBS buffer in a volume ratio of 5:2:1:2. The bacteria are mixed and filled to obtain a frozen storage solution. The final concentration of hydroxyethyl starch is 5%, the final concentration of proline is 4%, and the final concentration of methylcellulose is 1%.

When performing step S3 to maintain the temperature of the stirring vessel at 4°C using a water bath device, step S4 can be performed simultaneously, and the PBS buffer in step S4 and the stirring vessel in step S3 are placed in two connected water baths. , and let the PBS buffer in step S4 first contact the cold water, so that the heat in the high-pressure sterilized PBS buffer is transferred to the water bath. After the cold water leaves the water bath where the PBS buffer is located, pass The thermal insulation pipe enters the stirring container in step S3, so that the heat in the water bath is transferred to the stirring container, thereby utilizing the heat in the high-pressure sterilization process, and finally the water returns to the refrigeration device.

A cryopreservation solution that improves the survival rate of stem cells is prepared by applying a cryopreservation solution preparation process that improves the survival rate of stem cells.

The above embodiments only illustrate the principles and effects of the present invention, but are not intended to limit the present invention. Anyone familiar with this technology can modify or change the above embodiments without departing from the spirit and scope of the invention. Therefore, all equivalent modifications or changes made by those with ordinary knowledge in the technical field without departing from the spirit and technical ideas disclosed in the present invention shall still be covered by the claims of the present invention.

Claims (7)

1. The preparation process of the frozen stock solution for improving the survival rate of the stem cells is characterized by comprising the following steps of:

s1: preparation of extracellular cryopreservation protectant

Adding hydroxyethyl starch into a serum-free culture medium, and filtering and sterilizing the hydroxyethyl starch solution through a sterile filter membrane to obtain an extracellular cryopreservation protective agent;

s2: preparation of intracellular cryopreservation protectant

Adding proline into a serum-free culture medium to obtain a proline solution with the concentration of 20% -22%, and filtering and sterilizing the proline solution through a sterile filter membrane with the concentration of 0.20-0.22 mu m to obtain the intracellular cryopreservation protective agent;

s3: preparation of the stabilizer

Adding methyl cellulose into a disinfection container, heating by a heating device, sterilizing by an ultraviolet lamp, enabling the methyl cellulose to enter a stirring container through a sterile pipeline, cooling the methyl cellulose in the stirring container through an ice-water bath, uniformly stirring the methyl cellulose through a stirrer after adding a serum-free culture medium, placing the stirring container into a freezing device for storage, melting a methyl cellulose solution through a water bath device to obtain a stabilizer, filling the stabilizer into a sub-packaging container through sterile sub-packaging equipment, and storing the sub-packaged stabilizer through the freezing device;

s4: preparation of PBS buffer

Weighing NaCl, KH2P04 and Na2HP 04.12H2 into a quantitative container through an electronic scale, adding distilled water into the quantitative container, fixing the volume to obtain PBS buffer solution, adding the prepared PBS buffer solution into an autoclave for autoclaving, and filling the sterilized PBS buffer solution into a heat preservation container for preservation;

s5: sterile mixing and filling

Thawing the packaged stabilizer at 2-4deg.C, and mixing extracellular cryoprotectant, intracellular cryoprotectant, stabilizer and PBS buffer solution at volume ratio of (5-6): (2-3): (1-2): (2-3) carrying out aseptic mixing and filling to obtain frozen stock solution, wherein the final concentration of hydroxyethyl starch is 5% -7%, the final concentration of proline is 4% -4.4%, and the final concentration of methyl cellulose is 1% -1.2%.

2. The process for preparing the cryopreservation liquid for improving the survival rate of stem cells according to claim 1, wherein the step S1 is characterized by preparing an extracellular cryopreservation protective agent, and specifically comprises the following steps:

s1.1: adding hydroxyethyl starch into a serum-free culture medium to obtain a hydroxyethyl starch solution with the concentration of 10% -14%;

s1.2: filtering and sterilizing the hydroxyethyl starch solution by a sterile filter membrane with the diameter of 0.20-0.22 mu m to obtain the extracellular cryopreservation protective agent.

3. The process for preparing the frozen stock solution for improving the survival rate of the stem cells according to claim 1, wherein the step S3 is used for preparing the stabilizer, and specifically comprises the following steps:

s3.1: adding methyl cellulose into a disinfection container, heating the methyl cellulose by a heating device, and sterilizing the methyl cellulose by an ultraviolet lamp;

s3.2: the sterilized methyl cellulose enters a stirring container through a sterile pipeline, the methyl cellulose in the stirring container is cooled to 37-38 ℃ through an ice-water bath, after a serum-free culture medium is added, the concentration of the methyl cellulose is 5-6%, the methyl cellulose is uniformly stirred through a stirrer, the stirring container filled with the methyl cellulose is placed in a refrigerating device, and the stirring container is placed for 12-14 h at the temperature of minus 22-minus 20 ℃;

s3.3: placing the stirring container in a water bath device to enable the methylcellulose and the culture medium to be melted at the temperature of 2-4 ℃ to obtain a stabilizer;

s3.4: the stabilizer is filled into a split charging container through sterile split charging equipment, and the split charged stabilizer is stored at the temperature of minus 22 ℃ to minus 20 ℃ through a refrigerating device.

4. The process for preparing a frozen stock solution for improving the survival rate of stem cells according to claim 3, wherein the step S4 is to prepare PBS buffer solution, and specifically comprises the following steps:

s4.1: weighing NaCl and KH by electronic scale ₂ P0 ₄ And Na (Na) ₂ HP0 ₄ ·12H ₂ 0 to a quantitative container, adding distilled water to the quantitative container, and fixing the volume to obtain PBS buffer solution, wherein NaCl and KH are contained in the PBS buffer solution ₂ P0 ₄ And Na (Na) ₂ HP0 ₄ ·12H ₂ 0 in a mass ratio of (8.9-9): (0.14-0.15): (2-2.1);

s4.2: adding the prepared PBS buffer solution into an autoclave, autoclaving the PBS solution at 121-122 ℃, timing by a timer, autoclaving for 15-20min, and filling the sterilized PBS buffer solution into a heat preservation container at 4-6 ℃ for preservation.

5. The process for preparing a frozen stock solution for improving the survival rate of stem cells according to claim 4, wherein when the temperature of the stirring container is kept at 2-4 ℃ in the step S3.3 through a water bath device, the step S4.2 can be synchronously performed, the PBS buffer solution in the step S4.2 and the stirring container in the step S3.3 are respectively placed in two communicated water baths, the PBS buffer solution in the step S4.2 is contacted with cold water firstly, and after the cold water leaves from the water bath in which the PBS buffer solution is located, the cold water enters the stirring container in the step S3.3 through a heat preservation pipeline and finally returns to the refrigerating device.

6. The process for preparing a frozen stock solution for improving the survival rate of stem cells according to claim 2, wherein the sterile filter membrane is a cellulose acetate filter membrane.

7. A frozen stock solution for improving the survival rate of stem cells, which is prepared by the frozen stock solution preparation process for improving the survival rate of stem cells according to any one of claims 1 to 6.