CN115152743A - Preparation method of human umbilical cord mesenchymal stem cell serum-free frozen stock solution - Google Patents
Preparation method of human umbilical cord mesenchymal stem cell serum-free frozen stock solution Download PDFInfo
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- CN115152743A CN115152743A CN202210815654.4A CN202210815654A CN115152743A CN 115152743 A CN115152743 A CN 115152743A CN 202210815654 A CN202210815654 A CN 202210815654A CN 115152743 A CN115152743 A CN 115152743A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
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Abstract
The invention relates to the technical field of stem cell culture, and discloses a preparation method of a human umbilical cord mesenchymal stem cell serum-free frozen stock solution, which comprises the following steps: weighing 100 parts by volume of the mesenchymal stem cell serum-free culture medium, 10-30 parts by volume of a nutrient additive, 4-10 parts by volume of an impermeable cryoprotectant and 1-3 parts by volume of a permeable cryoprotectant, mixing, filtering, sterilizing and storing to obtain the human umbilical cord mesenchymal stem cell serum-free cryopreservation liquid, which does not contain animal serum and other heterologous proteins, reduces the possibility of contamination of the human umbilical cord mesenchymal stem cells, does not use dimethyl sulfoxide, avoids the potential clinical toxicity risk, maintains the biological characteristics and the survival rate of the human umbilical cord mesenchymal stem cells, effectively ensures the differentiation capacity of the cells, is simple to operate, has good cryopreservation effect, can be directly used as an auxiliary material, and has the characteristic of convenient use.
Description
Technical Field
The invention relates to the technical field of stem cell culture, in particular to a preparation method of a human umbilical cord mesenchymal stem cell serum-free frozen stock solution.
Background
The human umbilical cord mesenchymal stem cell is a multifunctional stem cell separated from an umbilical cord, is derived from Wharton's jelly in a newborn umbilical cord, has the characteristics of rich content, pure cells and low immunogenicity, is an original cell, has strong differentiation capacity, can be differentiated into a plurality of tissue cells, is easy to obtain materials, collect and store in a freezing way, and has wide clinical application prospect and research value.
Human umbilical cord mesenchymal stem cells are widely applied clinically, but cannot be applied in a large scale due to the limit value of freezing conditions, the cell freezing is an important means for long-term preservation of cells, a cell freezing solution protects the cells in the freezing process through a freezing protective agent, and damage of freezing ice crystals to the cells is reduced.
Disclosure of Invention
In order to solve the technical problems, the invention provides a preparation method of the human umbilical cord mesenchymal stem cell serum-free frozen stock solution, which avoids the possible risk of the human umbilical cord mesenchymal stem cell frozen stock solution containing serum in the clinical use process and solves the interference and pathogen pollution of the conventional serum frozen stock solution on cell differentiation.
In order to achieve the purpose, the technical scheme of the invention is as follows: a preparation method of a human umbilical cord mesenchymal stem cell serum-free frozen stock solution comprises the following steps:
step one, preparing raw materials: weighing 100 parts of mesenchymal stem cell serum-free culture medium, 10-30 parts of nutrient additive, 4-10 parts of non-permeable cryoprotectant and 1-3 parts of permeable cryoprotectant in the biological safety cabinet according to parts by volume;
step two, mixing: sequentially adding the raw materials weighed in the step one into a sterile reagent bottle, and stirring and mixing uniformly in a biological safety cabinet to obtain a mixed solution;
step three, filtering and sterilizing: filtering and sterilizing the mixed solution obtained in the step two through a 0.2-micron filter membrane, transferring the mixed solution into a new sterile reagent bottle, and detecting microorganisms and mycoplasma in the mixed solution in the sterile reagent bottle to obtain qualified negative mixed solution for later use;
step four, storage: and (5) sealing the sterile reagent bottle filled with the qualified detection mixed liquid in the third step, and refrigerating for storage.
As a further scheme of the invention: in the first step, the non-permeable cryoprotectant comprises one or more of hydroxyethyl starch, trehalose, sucrose and sodium carboxymethyl cellulose.
As a further scheme of the invention: in the first step, the osmotic cryoprotectant comprises one or two of glycerol and ethylene glycol.
As a further scheme of the invention: the nutritional additives in the first step comprise amino acid and vitamin, wherein the volume ratio of the amino acid to the vitamin is 10.
As a further scheme of the invention: the amino acid comprises one or more of valine, leucine, alanine, glutamic acid, serine and aspartic acid.
As a further scheme of the invention: the vitamins include one or more of vitamin C, vitamin B6, and vitamin B12.
As a further scheme of the invention: the temperature of the refrigeration storage is 2-8 ℃.
As a further scheme of the invention: the human umbilical cord mesenchymal stem cell serum-free frozen stock solution is applied to frozen storage of human umbilical cord mesenchymal stem cells.
As a further scheme of the invention: the human umbilical cord mesenchymal stem cell serum-free frozen stock solution is applied to frozen stock of human umbilical cord mesenchymal stem cells, and comprises the following steps:
preparing the human umbilical cord mesenchymal stem cells for frozen storage, washing by using PBS buffer solution, adding pancreatin for digestion until more than 85% of the human umbilical cord mesenchymal stem cells fall off, adding the prepared human umbilical cord mesenchymal stem cell serum-free frozen storage solution to stop digestion, carrying out centrifugal treatment, removing supernate, storing at-80 ℃ overnight, transferring into a liquid nitrogen environment for long-term frozen storage, and keeping at-196 ℃.
Compared with the prior art, the invention has the beneficial effects that:
the serum-free culture medium for the human umbilical cord mesenchymal stem cells is used, animal serum and other heterologous proteins are not contained, the possibility of pollution of the human umbilical cord mesenchymal stem cells is reduced, dimethyl sulfoxide is not used, the potential clinical toxicity risk is avoided, specific nutrient additives are added into the serum-free culture medium, the efficiency of amplification of the human umbilical cord mesenchymal stem cells is improved, the biological characteristics of the human umbilical cord mesenchymal stem cells are maintained, the survival rate of the human umbilical cord mesenchymal stem cells is increased, and the differentiation capacity of the cells is effectively ensured.
The human umbilical cord mesenchymal stem cell serum-free cryopreservation liquid prepared by the invention can better maintain the biological function of the human umbilical cord stem cells, can be directly cryopreserved without programmed cryopreservation, is simple to operate, has good cryopreservation effect, still maintains quite high activity and excellent differentiation capacity after cryopreservation and recovery of the human umbilical cord mesenchymal stem cells, can be directly used as an auxiliary material in clinical use, and has the characteristic of convenient use.
Of course, it is not necessary for any one product in which the invention is practiced to achieve all of the above-described technical effects simultaneously.
Drawings
FIG. 1 is a graph comparing growth curves of human umbilical cord mesenchymal stem cells cultured after resuscitation using different cryopreservation solutions.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments, and all other embodiments obtained by a person of ordinary skill in the art without any creative work based on the embodiments of the present invention belong to the protection scope of the present invention.
Example 1
A preparation method of a human umbilical cord mesenchymal stem cell serum-free frozen stock solution comprises the following steps:
weighing 100 parts of a mesenchymal stem cell serum-free culture medium, 10 parts of a nutrient additive valine, 2 parts of vitamin C, 4 parts of a non-permeable cryoprotectant hydroxyethyl starch and 1 part of a permeable cryoprotectant glycerol in a biosafety cabinet according to parts by volume; sequentially adding the weighed raw materials into a sterile reagent bottle, and uniformly stirring and mixing in a biological safety cabinet to obtain a mixed solution; filtering the obtained mixed solution through a 0.2-micron filter membrane for sterilization, transferring the mixed solution into a new sterile reagent bottle, detecting microorganisms and mycoplasma in the mixed solution in the sterile reagent bottle, sealing the sterile reagent bottle after the mixed solution is qualified, and refrigerating the sterile reagent bottle at the temperature of 2 ℃.
Example 2
A preparation method of a human umbilical cord mesenchymal stem cell serum-free frozen stock solution comprises the following steps:
weighing 100 parts of a mesenchymal stem cell serum-free culture medium, 10 parts of nutrient additives valine, 4 parts of glutamic acid, 2 parts of vitamin C, 3 parts of vitamin B6, 4 parts of non-permeable cryoprotectant hydroxyethyl starch, 2 parts of sucrose, 1 part of permeable cryoprotectant glycerol and 1 part of ethylene glycol in a biological safety cabinet according to parts by volume; sequentially adding the weighed raw materials into a sterile reagent bottle, and uniformly stirring and mixing in a biological safety cabinet to obtain a mixed solution; filtering the obtained mixed solution through a 0.2-micron filter membrane for sterilization, transferring the mixed solution into a new sterile reagent bottle, detecting microorganisms and mycoplasma in the mixed solution in the sterile reagent bottle, sealing the sterile reagent bottle after the mixed solution is qualified, and refrigerating the sterile reagent bottle at 4 ℃.
Example 3
A preparation method of a human umbilical cord mesenchymal stem cell serum-free frozen stock solution comprises the following steps:
weighing 100 parts of a mesenchymal stem cell serum-free culture medium, 10 parts of nutrient additives valine, 7 parts of glutamic acid, 2 parts of vitamin C, 5 parts of vitamin B6, 4 parts of non-permeable cryoprotectant hydroxyethyl starch, 4 parts of sucrose, 1 part of permeable cryoprotectant glycerol and 2 parts of ethylene glycol in a biological safety cabinet according to parts by volume; sequentially adding the weighed raw materials into a sterile reagent bottle, and stirring and mixing uniformly in a biological safety cabinet to obtain a mixed solution; filtering the obtained mixed solution through a 0.2-micron filter membrane for sterilization, transferring the mixed solution into a new sterile reagent bottle, detecting microorganisms and mycoplasma in the mixed solution in the sterile reagent bottle, sealing the sterile reagent bottle after the mixed solution is qualified, and refrigerating the sterile reagent bottle at the temperature of 6 ℃.
Example 4
A preparation method of a human umbilical cord mesenchymal stem cell serum-free frozen stock solution comprises the following steps:
weighing 100 parts of a mesenchymal stem cell serum-free culture medium, 20 parts of a nutrient additive valine, 10 parts of vitamin C, 10 parts of non-permeable cryoprotectant hydroxyethyl starch and 3 parts of permeable cryoprotectant glycerol in a biological safety cabinet according to parts by volume; sequentially adding the weighed raw materials into a sterile reagent bottle, and stirring and mixing uniformly in a biological safety cabinet to obtain a mixed solution; filtering the obtained mixed solution through a 0.2-micron filter membrane for sterilization, transferring the mixed solution into a new sterile reagent bottle, detecting microorganisms and mycoplasma in the mixed solution in the sterile reagent bottle, sealing the sterile reagent bottle after the mixed solution is qualified, and refrigerating the sterile reagent bottle at the temperature of 8 ℃.
Example 5
The human umbilical cord mesenchymal stem cells are subjected to cryopreservation treatment by using the human umbilical cord mesenchymal stem cell serum-free cryopreservation solution prepared in the embodiment 1, and the method comprises the following steps of:
taking human umbilical cord mesenchymal stem cell serum-free frozen stock solution precooled in a medical refrigerator for 60min, cleaning the human umbilical cord mesenchymal stem cells by using PBS buffer solution, adding pancreatin for digestion until more than 85% of human umbilical cord mesenchymal stem cells fall off, adding the prepared human umbilical cord mesenchymal stem cell serum-free frozen stock solution to terminate digestion, carrying out centrifugal treatment, removing supernatant, storing at-80 ℃ for overnight, transferring into a liquid nitrogen environment for long-term frozen stock at the temperature of-196 ℃, and obtaining human umbilical cord mesenchymal stem cell preservation solution.
Example 6
The human umbilical cord mesenchymal stem cells are subjected to cryopreservation treatment by using the human umbilical cord mesenchymal stem cell serum-free cryopreservation liquid prepared in the example 2, and the specific operation steps are consistent with those in the example 5.
Example 7
The human umbilical cord mesenchymal stem cells are subjected to cryopreservation treatment by using the human umbilical cord mesenchymal stem cell serum-free cryopreservation solution prepared in the example 3, and the specific operation steps are kept consistent with those in the example 5.
Example 8
The human umbilical cord mesenchymal stem cells are subjected to cryopreservation treatment by using the human umbilical cord mesenchymal stem cell serum-free cryopreservation solution prepared in the example 4, and the specific operation steps are consistent with those in the example 5.
Comparative example 1
A preparation method of a human umbilical cord mesenchymal stem cell serum-free frozen stock solution comprises the following steps:
weighing 100 parts of a mesenchymal stem cell serum-free culture medium, 10 parts of a nutrient additive valine, 7 parts of glutamic acid, 4 parts of a non-permeable cryoprotectant hydroxyethyl starch, 4 parts of sucrose, 1 part of a permeable cryoprotectant glycerol and 2 parts of ethylene glycol in a biosafety cabinet according to parts by volume; sequentially adding the weighed raw materials into a sterile reagent bottle, and uniformly stirring and mixing in a biological safety cabinet to obtain a mixed solution; filtering the obtained mixed solution through a 0.2-micron filter membrane for sterilization, transferring the mixed solution into a new sterile reagent bottle, detecting microorganisms and mycoplasma in the mixed solution in the sterile reagent bottle, sealing the sterile reagent bottle after the mixed solution is qualified, and refrigerating the sterile reagent bottle at the temperature of 6 ℃.
Comparative example 2
A preparation method of a human umbilical cord mesenchymal stem cell serum-free frozen stock solution comprises the following steps:
weighing 100 parts of a mesenchymal stem cell serum-free culture medium, 10 parts of nutrient additives, namely valine, 7 parts of glutamic acid, 1 part of osmotic cryoprotectant, and 2 parts of ethylene glycol in parts by volume in a biological safety cabinet; sequentially adding the weighed raw materials into a sterile reagent bottle, and stirring and mixing uniformly in a biological safety cabinet to obtain a mixed solution; filtering the obtained mixed solution through a 0.2-micron filter membrane for sterilization, transferring the mixed solution into a new sterile reagent bottle, detecting microorganisms and mycoplasma in the mixed solution in the sterile reagent bottle, sealing the sterile reagent bottle after the mixed solution is qualified, and refrigerating the sterile reagent bottle at the temperature of 6 ℃.
Comparative example 3
The human umbilical cord mesenchymal stem cells are subjected to cryopreservation treatment by using the human umbilical cord mesenchymal stem cell serum-free cryopreservation solution prepared in the comparative example 1, and the specific operation steps are kept consistent with those in the examples 5-8.
Comparative example 4
The human umbilical cord mesenchymal stem cells are subjected to cryopreservation treatment by using the human umbilical cord mesenchymal stem cell serum-free cryopreservation liquid prepared in the comparative example 2, and the specific operation steps are kept consistent with those in the examples 5-8.
Comparative example 5
The specific operation steps of the cryopreservation treatment of the umbilical cord mesenchymal stem cells by using the common stem cell cryopreservation liquid containing fetal bovine serum are kept consistent with those of the examples 5-8.
The mesenchymal stem cell serum-free culture medium used in the examples and comparative examples of the present invention was purchased from Wuhan Punuo Seiensis Life technologies, inc., and the catalog number is CM-SC01.
The cell survival rates of the human umbilical cord mesenchymal stem cells in the embodiments 5-8 and the comparative example 5 after cryopreservation recovery are recorded as A + grade when the survival rate is more than or equal to 98%, recorded as A grade when the survival rate is more than or equal to 90% but less than 98%, recorded as B grade when the survival rate is more than or equal to 85% but less than 90%, and recorded as C grade when the survival rate is less than 85%, and the detection results are shown in Table 1.
TABLE 1 cell survival rate after recovery of human umbilical cord mesenchymal stem cells
The cell survival rate result after the recovery of the human umbilical cord mesenchymal stem cells shows that the survival rate of the human umbilical cord mesenchymal stem cells after the recovery of the human umbilical cord mesenchymal stem cell serum-free frozen stock solution prepared by the technical scheme of the invention is obviously higher than that of the scheme in the comparative example, and the human umbilical cord mesenchymal stem cell serum-free frozen stock solution has excellent effect.
The effect of the sample in example 7 is obviously higher than that of examples 5, 6 and 8, the human umbilical cord mesenchymal stem cell preservation solution in example 7 and comparative example 5 is taken, recovery, subculture and expansion are carried out, the growth condition of the recovered cell culture is recorded, and the recorded result is shown in table 2.
TABLE 2 growth record table of human umbilical cord mesenchymal stem cell after recovery
The results show that the cell culture growth condition of the human umbilical cord mesenchymal stem cells frozen and stored by the serum-free frozen stock solution prepared by the invention is obviously higher than the cell culture growth condition of the human umbilical cord mesenchymal stem cells frozen and stored by the conventional frozen stock solution after being thawed.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (8)
1. A preparation method of a human umbilical cord mesenchymal stem cell serum-free frozen stock solution is characterized by comprising the following steps: the preparation method comprises the following steps:
step one, preparing raw materials: weighing 100 parts of mesenchymal stem cell serum-free culture medium, 10-30 parts of nutrient additive, 4-10 parts of non-permeable cryoprotectant and 1-3 parts of permeable cryoprotectant according to parts by volume;
step two, mixing: sequentially adding the raw materials weighed in the step one into a sterile reagent bottle, and uniformly stirring and mixing to obtain a mixed solution;
step three, filtering and sterilizing: filtering and sterilizing the mixed solution obtained in the step two through a 0.2-micron filter membrane, transferring the mixed solution into a new sterile reagent bottle, and detecting microorganisms and mycoplasma in the mixed solution in the sterile reagent bottle to obtain qualified negative mixed solution for later use;
step four, storage: and sealing the sterile reagent bottle filled with the qualified detection mixed solution in the third step, and refrigerating for storage.
2. The method for preparing the serum-free cryopreservation solution for the human umbilical cord mesenchymal stem cells according to claim 1, wherein the non-permeable cryoprotectant in the first step comprises one or more of hydroxyethyl starch, trehalose, sucrose and sodium carboxymethyl cellulose.
3. The method for preparing the serum-free cryopreservation solution for the human umbilical cord mesenchymal stem cells according to claim 1, wherein the osmotic cryoprotectant in the first step comprises one or two of glycerol and ethylene glycol.
4. The method for preparing a serum-free cryopreservation solution for human umbilical cord mesenchymal stem cells according to claim 1, wherein the nutritional additives in the first step comprise amino acids and vitamins, and the volume ratio of the amino acids to the vitamins is 10.
5. The method for preparing the serum-free cryopreservation liquid of the human umbilical cord mesenchymal stem cells as claimed in claim 4, wherein the amino acid comprises one or more of valine, leucine, alanine, glutamic acid, serine and aspartic acid.
6. The method for preparing the human umbilical cord mesenchymal stem cell serum-free frozen stock solution according to claim 4, wherein the vitamins include one or more of vitamin C, vitamin B6 and vitamin B12.
7. The method for preparing the serum-free cryopreservation liquid for the human umbilical cord mesenchymal stem cells according to claim 1, wherein the temperature for cryopreservation is 2-8 ℃.
8. The application of the human umbilical cord mesenchymal stem cell serum-free cryopreservation liquid in the cryopreservation of human umbilical cord mesenchymal stem cells according to any one of claims 1 to 7.
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| CN115885978A (en) * | 2022-12-30 | 2023-04-04 | 成都锦欣博悦生物科技有限公司 | Deciduous tooth mesenchymal stem cell cryopreservation liquid and cryopreservation method thereof |
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| CN115885978A (en) * | 2022-12-30 | 2023-04-04 | 成都锦欣博悦生物科技有限公司 | Deciduous tooth mesenchymal stem cell cryopreservation liquid and cryopreservation method thereof |
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