Cell freezing medium

Abstract

The invention discloses a cell cryopreservation solution, and belongs to the technical field of cell cryopreservation, the cell cryopreservation solution comprises the following components by volume: 5%-15% of a protective agent, 1%-5% of human serum albumin, 1%-5% of AB serum and 75%-95% of a culture medium, and the sum of the proportions of the components is 100%; according to the cell cryopreservation liquid, the permeable cryoprotectant (glycerol and ethylene glycol) and the impermeable cryoprotectant (dextran) are mixed, so that formation of ice crystals inside and outside cells can be reduced conveniently in the cell cryopreservation process, that is, dextran can weaken the crystallization process of water and increase the viscosity of a solution, and then formation of ice crystals outside the cells is reduced; the glycerol and the ethylene glycol are matched, so that the gel can permeate into cells, formation of ice crystals in the cells is reduced, damage to the cells is effectively reduced, and the survival rate of the unfrozen cells is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of cell cryopreservation, and specifically to a cell cryopreservation solution. Background Art

[0002] Cell cryopreservation is a technique that places cells in a low-temperature environment to reduce cell metabolism for long-term storage. It is one of the main methods for cell preservation and plays a role in cell strain conservation. Currently, glycerol or dimethyl sulfoxide (DMSO) is mostly used as a cryoprotectant. These two substances can improve the water permeability of cell membranes. Coupled with slow freezing, water inside the cells can exude outside the cells, reducing the formation of ice crystals inside the cells, thereby reducing cell damage caused by ice crystal formation. When reviving cells, a rapid thawing method should be adopted, which can ensure that extracellular crystals melt within a very short time, avoiding cell damage caused by intracellular recrystallization due to slow thawing that allows water to penetrate into the cells; However, the above-mentioned cryoprotectants are all permeating cryoprotectants. Although they can reduce the formation of ice crystals inside the cells, during the freezing process, ice crystals will also form outside the cells, which will cause damage to the cells. At the same time, freeze storage will cause changes in the osmotic pressure inside and outside the cells, which is also likely to cause damage to the cells, resulting in a low survival rate of the thawed cells. Therefore, we have proposed a cell cryopreservation solution. Summary of the Invention

[0003] Aiming at the deficiencies proposed in the above background art, the present invention provides a cell cryopreservation solution, which solves the problems proposed in the above background art.

[0004] To achieve the above object, the present invention provides the following technical solution: A cell cryopreservation solution, the cryopreservation solution includes the following components: a cryoprotectant, human serum albumin, AB serum, and a culture medium. Among them, the volume fractions of each component are cryoprotectant (5%-15%), human serum albumin (1%-5%), AB serum (1%-5%), and culture medium (75%-95%), and the total ratio of each component is 100%.

[0005] Preferably, in terms of volume fraction percentage, the ratios of each component in the cryopreservation solution are as follows: cryoprotectant 10%, human serum albumin 3%, AB serum 2%, and culture medium 85%, and the total ratio of each component is 100%; Human serum albumin can maintain the constancy of plasma colloid osmotic pressure, ensure the exchange between intracellular fluid, extracellular fluid, and tissue fluid, and play a stabilizing role in colloidal protection for globulins. AB serum can increase the growth rate of most human-derived cells and is applicable to the culture of various human-derived cells with a lower usage concentration than mixed serum.

[0006] Preferably, the cryoprotectant is composed of a permeating cryoprotectant and a non-permeating cryoprotectant.

[0007] Preferably, the permeating cryoprotectant is one or both of glycerol and ethylene glycol, and the non-permeating cryoprotectant is dextran; Glycerol can penetrate into cells, reduce the formation of ice crystals inside cells, and thus protect cells from damage. The addition of glycerol lowers the freezing point of the solution, making cells less susceptible to water sublimation during freezing; Ethylene glycol can penetrate into cells, create a certain molar concentration inside and outside cells, reduce the concentration of electrolytes in the unfrozen solution inside and outside cells, and thus protect cells from damage by high-concentration electrolytes. At the same time, the water inside cells will not overly exude, avoiding excessive dehydration and shrinkage of cells; Dextran can combine with water molecules in the solution to undergo a hydration effect, thereby weakening the crystallization process of water, increasing the viscosity of the solution, and further reducing the formation of ice crystals. By reducing the formation of ice crystals, dextran can play a protective role in cells and help improve the survival rate of cells after cryopreservation.

[0008] Preferably, the volume fraction percentage of the permeating cryoprotectant is 70%, the volume fraction percentage of the non-permeating cryoprotectant is 30%, and the total ratio of the two is 100%.

[0009] Preferably, the culture medium is a basal medium, and the basal medium is any one of high-glucose DMEM, low-glucose DMEM, or RPMI-1640.

[0010] Preferably, the preparation method of the cell cryopreservation solution includes the following steps: S1. First, add human serum albumin and AB serum to the culture medium and mix them to obtain the cryopreservation solution matrix; S2. Then, add the cryoprotectant to the cryopreservation solution matrix prepared in step S1 to obtain the cell cryopreservation solution.

[0011] Preferably, the cryoprotectant needs to be added to the cryopreservation solution matrix slowly in multiple times and mixed.

[0012] Preferably, the usage method of the cell cryopreservation solution includes the following steps: (1). Use a centrifuge to process the cells to be cryopreserved, remove the supernatant after centrifugation, and slowly add the prepared cell cryopreservation solution; (2). Mix the liquid in step (1), perform sub-packaging, and then cryopreserve the sub-packaged liquid; (3). When it is necessary to detect the cryopreserved cells, first take out the cryopreservation solution, thaw it, and then measure the survival quantity of the thawed liquid.

[0013] Preferably, the rotation speed of the centrifuge is 1500 rmp, and the centrifugation time is 5 min.

[0014] Compared with the prior art, the present invention provides a cell cryopreservation solution, which has the following beneficial effects: In this cell cryopreservation solution, by using a mixture of a permeating cryoprotectant (glycerol and ethylene glycol) and a non-permeating cryoprotectant (dextran), during the process of cell cryopreservation, it is convenient to reduce the formation of ice crystals inside and outside the cells. That is, dextran can weaken the crystallization process of water, increase the viscosity of the solution, and thus reduce the formation of extracellular ice crystals. The combination of glycerol and ethylene glycol can penetrate into the cells to reduce the formation of intracellular ice crystals, effectively reducing cell damage, and thus improving the survival rate of cells after thawing; In this cell cryopreservation solution, by using human serum albumin and AB serum, during the process of cell cryopreservation, it can ensure the communication between the intracellular fluid, extracellular fluid and tissue fluid, maintain the stability of the osmotic pressure inside and outside the cells, play a protective role on the cells, and further improve the survival rate of cells after thawing. Specific embodiments

[0015] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0016] Embodiment 1:

[0017] A cell cryopreservation solution, and the cryopreservation solution includes the following components: a cryoprotectant, human serum albumin, AB serum, and a culture medium (the culture medium is a basal medium, and the basal medium is any one of high-glucose DMEN, low-glucose DMEN, or RPMI-1640). Among them, in terms of volume fraction percentage, the proportions of the components in the cryopreservation solution are as follows: 5% cryoprotectant, 5% human serum albumin, 5% AB serum, and 85% culture medium, and the total proportion of the components is 100%; The cryoprotectant is composed of a permeating cryoprotectant with a volume fraction percentage of 70% and a non-permeating cryoprotectant with a volume fraction percentage of 30%. Among them, the permeating cryoprotectant is a mixture of glycerol and ethylene glycol, and the volume fraction percentages of glycerol and ethylene glycol are each 50%, and the non-permeating cryoprotectant is dextran; The preparation and use method of the above cell cryopreservation solution includes the following steps: S1. First, add human serum albumin and AB serum to the culture medium for mixing to obtain a cryopreservation solution matrix; S2. Then, add the cryoprotectant to the cryopreservation liquid matrix obtained in step S1 in multiple portions slowly and mix them to obtain the cell cryopreservation liquid. S3. Use a centrifuge with a rotational speed of 1500 rmp to process the cells to be cryopreserved for 5 minutes, then remove the supernatant after centrifugation and slowly add the prepared cell cryopreservation liquid. S4. Mix the liquid in step S3, perform aliquoting, and then cryopreserve the aliquoted liquid. S5. When it is necessary to detect the cryopreserved cells, first take out the cryopreservation liquid, thaw it, and then measure the survival quantity of the thawed liquid.

[0018] Example 2:

[0019] The difference between this example and Example 1 is that the permeating cryoprotectant is glycerol.

[0020] Example 3:

[0021] The difference between this example and Example 1 is that the permeating cryoprotectant is ethylene glycol.

[0022] Example 4:

[0023] A cell cryopreservation liquid, and the cryopreservation liquid comprises the following components: cryoprotectant, human serum albumin, AB serum, and a culture medium (the culture medium is a basal medium, and the basal medium is any one of high-glucose DMEM, low-glucose DMEM, or RPMI-1640). Among them, in terms of volume fraction percentage, the proportion of each component in the cryopreservation liquid is as follows: cryoprotectant 10%, human serum albumin 3%, AB serum 2%, and culture medium 85%. The total proportion of each component is 100%. The cryoprotectant is composed of 70% of a permeating cryoprotectant and 30% of a non-permeating cryoprotectant in terms of volume fraction percentage. Among them, the permeating cryoprotectant is a mixture of glycerol and ethylene glycol, and the volume fraction percentage of glycerol and ethylene glycol is each 50%. The non-permeating cryoprotectant is dextran. The preparation and usage method of the above cell cryopreservation liquid comprises the following steps: S1. First, add human serum albumin and AB serum to the culture medium and mix them to prepare the cryopreservation liquid matrix. S2. Then, add the cryoprotectant to the cryopreservation liquid matrix obtained in step S1 in multiple portions slowly and mix them to obtain the cell cryopreservation liquid. S3. Use a centrifuge with a rotational speed of 1500 rmp to process the cells to be cryopreserved for 5 minutes, then remove the supernatant after centrifugation and slowly add the prepared cell cryopreservation liquid. S4. Mix the liquid in step S3, then perform sub-packaging, and then freeze the sub-packaged liquid. S5. When it is necessary to detect the cryopreserved cells, first take out the cryopreservation solution, thaw it, and then measure the survival quantity of the thawed liquid.

[0024] Example Five:

[0025] The difference between this example and Example Four is that the permeating cryoprotectant is glycerol.

[0026] Example Six:

[0027] The difference between this example and Example Four is that the permeating cryoprotectant is ethylene glycol.

[0028] Example Seven:

[0029] A cell cryopreservation solution, and the cryopreservation solution comprises the following components: a protectant, human serum albumin, AB serum, and a culture medium (the culture medium is a basal medium, and the basal medium is any one of high-glucose DMEN, low-glucose DMEN, or RPMI-1640). Among them, in terms of volume fraction percentage, the proportion of each component in the cryopreservation solution is as follows: the protectant is 13%, human serum albumin is 1%, AB serum is 1%, and the culture medium is 85%. The total proportion of each component is 100%; The protectant is composed of a permeating cryoprotectant accounting for 70% by volume fraction percentage and a non-permeating cryoprotectant accounting for 30% by volume fraction percentage. Among them, the permeating cryoprotectant is a mixture of glycerol and ethylene glycol, and the volume fraction percentage of glycerol and ethylene glycol is each 50%, and the non-permeating cryoprotectant is dextran; The preparation and usage method of the above cell cryopreservation solution comprises the following steps: S1. First add human serum albumin and AB serum to the culture medium for mixing to obtain the cryopreservation solution matrix; S2. Then add the protectant to the cryopreservation solution matrix obtained in step S1 in several times slowly and mix it to obtain the cell cryopreservation solution; S3. Use a centrifuge with a rotation speed of 1500 rmp to process the cells to be cryopreserved for 5 minutes, then remove the supernatant after centrifugation, and slowly add the prepared cell cryopreservation solution; S4. Mix the liquid in step S3, then perform sub-packaging, and then freeze the sub-packaged liquid; S5. When it is necessary to detect the cryopreserved cells, first take out the cryopreservation solution, thaw it, and then measure the survival quantity of the thawed liquid.

[0030] Example Eight:

[0031] The difference between this embodiment and Embodiment Seven is that the permeating cryoprotectant is glycerol.

[0032] Embodiment Nine:

[0033] The difference between this embodiment and Embodiment Seven is that the permeating cryoprotectant is ethylene glycol.

[0034] By comparing the cell cryopreservation solutions prepared by the methods of Embodiments One to Nine above, it can be seen that the cell cryopreservation solution prepared by the method of Embodiment Four has the best number of surviving cells after thawing, and the comparison results of Embodiments One to Nine are shown in the following table: Example 1 Example 2 Example 3 Example 4 Example 5 Example 6 Example 7 Example 8 Example 9 Number of surviving cells Around 80% Around 70% Around 70% Around 88% Around 76% Around 76% Around 82% Around 74% Around 74% In the above solution, it should be noted that: in terms of volume fraction percentage, the proportions of the components in the cryopreservation solution are as follows: cryoprotectant 10%, human serum albumin 3%, AB serum 2%, and culture medium 85%. The total proportion of the components is 100%, and the cell cryopreservation solution prepared in this way has the best number of surviving cells after thawing detection.

[0035] Although the embodiments of the present invention have been shown and described, for those of ordinary skill in the art, it can be understood that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principle of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A cell cryopreservation solution, characterized in that, The cryopreservation solution comprises the following components: a cryoprotectant, human serum albumin, AB serum, and a culture medium. Among them, the volume fractions of the components are cryoprotectant (5%-15%), human serum albumin (1%-5%), AB serum (1%-5%), and culture medium (75%-95%), and the total ratio of the components is 100%.

2. The cell cryopreservation solution according to claim 1, characterized in that, By volume fraction percentage, the ratios of the components in the cryopreservation solution are as follows: cryoprotectant 10%, human serum albumin 3%, AB serum 2%, and culture medium 85%, and the total ratio of the components is 100%.

3. The cell cryopreservation solution according to claim 1, wherein The cryoprotectant is composed of a permeating cryoprotectant and a non-permeating cryoprotectant.

4. A cell cryopreservation solution according to claim 3, wherein, The permeating cryoprotectant is one or both of glycerol and ethylene glycol, and the non-permeating cryoprotectant is dextran.

5. A cell cryopreservation solution according to claim 4, wherein, The volume fraction percentage of the permeating cryoprotectant is 70%, the volume fraction percentage of the non-permeating cryoprotectant is 30%, and the total ratio of the two is 100%.

6. The cryopreservation solution according to claim 1, characterized in that, The culture medium is a basal medium, and the basal medium is any one of high-glucose DMEN, low-glucose DMEN, or RPMI-1640.

7. A cell cryopreservation solution according to claim 1, wherein The preparation method of the cell cryopreservation solution comprises the following steps: S1. First, add human serum albumin and AB serum to the culture medium for mixing to obtain a cryopreservation solution matrix; S2. Then, add the cryoprotectant to the cryopreservation solution matrix prepared in step S1 to obtain a cell cryopreservation solution.

8. A cell cryopreservation solution according to claim 7, wherein, The cryoprotectant needs to be added to the cryopreservation solution matrix slowly in multiple times and mixed.

9. A cell cryopreservation solution according to claim 1, wherein, The usage method of the cell cryopreservation solution comprises the following steps: (1). Use a centrifuge to process the cells to be cryopreserved, remove the supernatant after centrifugation, and slowly add the prepared cell cryopreservation solution; (2). Mix the liquid in step (1), perform sub-packaging, and then cryopreserve the sub-packaged liquid; (3). When it is necessary to detect the cryopreserved cells, first take out the cryopreservation solution, thaw it, and then measure the survival quantity of the thawed liquid.

10. A cell cryopreservation solution according to claim 9, characterized in that, The rotation speed of the centrifuge is 1500 rmp, and the centrifugation time is 5 min.