CN112741081B - Programmed cooling method for human umbilical cord mesenchymal stem cells with excellent freezing and cooling effects - Google Patents
Programmed cooling method for human umbilical cord mesenchymal stem cells with excellent freezing and cooling effects Download PDFInfo
- Publication number
- CN112741081B CN112741081B CN202110126859.7A CN202110126859A CN112741081B CN 112741081 B CN112741081 B CN 112741081B CN 202110126859 A CN202110126859 A CN 202110126859A CN 112741081 B CN112741081 B CN 112741081B
- Authority
- CN
- China
- Prior art keywords
- cooling
- freezing
- cells
- stem cells
- umbilical cord
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- 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
-
- 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
- A01N1/0278—Physical preservation processes
- A01N1/0284—Temperature processes, i.e. using a designated change in temperature over time
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Dentistry (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Environmental Sciences (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Hair brushThe invention relates to the technical field of stem cells, in particular to a programmed cooling method for human umbilical cord mesenchymal stem cells with excellent cryopreservation and cooling effects. A programmed cooling method for human umbilical cord mesenchymal stem cells with excellent freezing and cooling effects comprises the following steps: providing a frozen stock solution, and taking pretreated human umbilical cord mesenchymal stem cells in a ratio of 1 × 107Adding pre-cooled freezing medium into per ml of cell density, after re-suspending, sucking 1-5 ml of cell suspension into a programmed cooling instrument for programmed cooling and freezing. After the novel freezing solution is adopted and the program cooling step is matched, the survival rate of the cells before and after the cells are frozen is not obviously reduced and is more than 97 percent, and in addition, the number of the cells still keeps higher concentration.
Description
Technical Field
The invention relates to the technical field of stem cells, in particular to a programmed cooling method for human umbilical cord mesenchymal stem cells with excellent cryopreservation and cooling effects.
Background
The human umbilical cord mesenchymal stem cell is a pluripotent stem cell separated from an umbilical cord, has obvious clinical application advantages compared with mesenchymal stem cells from other sources, such as easy material taking, easy collection, freezing storage, relative purity and the like, has higher differentiation potential, can be differentiated in multiple directions, such as osteoblasts, chondrocytes, heparinoid cells, insulin-like cells, nerve cells, myocardial cells and the like, and has no ethical problem. At present, human umbilical cord mesenchymal stem cells have been applied in the clinical field and have achieved encouraging therapeutic effects.
The seed bank of the human umbilical cord mesenchymal stem cells is widely applied clinically, but the seed bank has the problem that the seed bank cannot be applied in a large scale due to the limitation of freezing conditions, no exogenous pollution is generated in the freezing process of the stem cells, which is the key for the safety and reliability of the umbilical cord mesenchymal stem cells in clinical treatment application, on one hand, the fetal bovine serum in the conventional freezing solution contains various undefined cell growth factors and can interfere the differentiation of the cells, on the other hand, the stem cells cultured by animal serum are clinically applied and can cause the cross infection of pathogens, so the human umbilical cord mesenchymal stem cell serum-free freezing solution with clear components needs to be developed.
The long-term activity of the human umbilical cord mesenchymal stem cell seed bank cells is closely related to the freezing environment, the freezing method has important influence on the human umbilical cord mesenchymal stem cells, the morphology, proliferation, differentiation, apoptosis and aging of the stem cells can be directly influenced when the freezing method is changed, the biological characteristics and genetic stability of the cells can be changed, and the optimization of the freezing condition of the stem cells plays a positive role in the standardized application of the stem cell technology.
Disclosure of Invention
Unless otherwise indicated, implied from the context, or customary in the art, all parts and percentages herein are by weight and the testing and characterization methods used are synchronized with the filing date of the present application. To the extent that a definition of a particular term disclosed in the prior art is inconsistent with any definitions provided herein, the definition of the term provided herein controls.
The words "preferred", "preferably", "more preferred", and the like, in the present invention, refer to embodiments of the invention that may provide certain benefits, under certain circumstances. However, other embodiments may be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, nor is it intended to exclude other embodiments from the scope of the invention. The sources of components not mentioned in the present invention are all commercially available.
The inventor provides a programmed cooling method for human umbilical cord mesenchymal stem cells with excellent cryopreservation and cooling effects after earnest research for solving the problems, and after the programmed cooling step is matched in the presence of a special cryopreservation solution, the survival rate of the cells before and after the cells are cryopreserved is not obviously reduced, the survival rate is more than 97%, and in addition, the cell number is still kept at a high concentration.
The programmed cooling method of human umbilical cord mesenchymal stem cells comprises the steps of providing a freezing storage solution, and taking pretreated human umbilical cord mesenchymal stem cells at a ratio of 1 multiplied by 107Adding pre-cooled freezing medium into per ml of cell density, after re-suspending, sucking 1-5 ml of cell suspension into a programmed cooling instrument for programmed cooling and freezing.
Cell cryopreservation is an important means for maximally preserving cell viability. At present, dimethyl sulfoxide is commonly used as a protective agent, and is combined with water molecules to lower the freezing point and reduce the formation of ice crystals in cells, thereby reducing the damage of the ice crystals.
However, the commonly used dimethyl sulfoxide has certain toxicity to cells, and researches show that the commonly used dimethyl sulfoxide can be combined with proteins to cause protein denaturation so as to cause harm; therefore, the invention obtains a new frozen stock solution after research on the basis, and the content of dimethyl sulfoxide can be greatly reduced.
Specifically, the preferable freezing storage solution at least comprises human serum albumin, dimethyl sulfoxide, compound electrolyte solution, sodium chloride solution and glucose solution.
Among them, the preferred human albumin can replace serum to further protect cells and reduce the toxicity of dimethyl sulfoxide to the cells, and in addition, can provide certain nutrition, so that the cells still maintain better activity after fast melting. In addition, the risk of viral infection due to the addition of serum is avoided, and most preferably, the human serum albumin is 20% human serum albumin.
The frozen stock solution preferably comprises, by volume percentage, at least 20-50% of human serum albumin, 5% of dimethyl sulfoxide, 40-60% of compound electrolyte solution, 1-10% of sodium chloride solution and 1-10% of glucose solution.
The basic core of the cryopreservation method is to try to use different components with lower content of dimethyl sulfoxide to partially replace the dosage ratio of dimethyl sulfoxide by the impermeable protective agent.
The research results show that the frozen stock solution obtained by compounding the preferred components in the proper range has a good frozen stock effect, and the frozen stock solution is preferably subjected to a proper temperature reduction procedure to further ensure that the survival rate reaches more than 97 percent.
The human serum albumin in the frozen stock solution can be purchased or obtained by low-temperature separation and extraction of healthy human plasma, and the invention is not limited in any way; the dimethyl sulfoxide is preferably dimethyl sulfoxide of molecular biochemical grade (BR), such as dimethyl sulfoxide of WAK-Chemie.
The compound electrolyte liquid is water, a supplement source of electrolyte and an alkalizer; preferably, the compound electrolyte is compound electrolyte injection, wherein sodium chloride, potassium chloride and magnesium chloride are chloride salts which are used together for maintaining the system balance of the ionic osmotic pressure and the colloid osmotic pressure, and the sodium gluconate and the sodium acetate are coordinated to form a proper physiological environment to be beneficial to the survival of cells.
The sodium chloride solution is preferably sodium chloride injection, is 0.9% sodium chloride aqueous solution, and can be used as normal saline to maintain cell osmotic pressure. The glucose solution is preferably glucose injection solution for providing nutrition.
Further, the frozen stock solution of the invention preferably also comprises 1-10% of dextran 40 glucose solution, and preferably is dextran 40 glucose injection. The weight average molecular weight of the dextran 40 is 32000-42000, so that osmotic pressure and PH can be maintained, and an environment suitable for cell survival is provided.
Further, the frozen stock solution of the invention preferably also comprises hydroxypropyl-beta-cyclodextrin, preferably 40% hydroxypropyl-beta-cyclodextrin aqueous solution; and the volume percentage of the 40 percent hydroxypropyl-beta-cyclodextrin water solution in the frozen stock solution is 0.5 to 5 percent.
In the method of the present invention, the human umbilical cord mesenchymal stem cells are first pretreated and then mixed with the frozen stock solution, wherein the pretreatment step includes digestion and termination of the digestion step.
The digestion step is to add enzyme into the human umbilical cord mesenchymal stem cells of no more than 5 generations for digestion for 1-3 min, preferably the enzyme comprises 0.25% trypsin with the concentration of 0.2ml/cm2In addition, in order to shorten the digestion time, a compound enzyme can be adopted, for example, the compound enzyme can be digested by trypsin and EDTA together, and the ratio of the two can be any ratio. And the step of terminating digestion is to add a complete culture medium of 5-10 times of digestive juice to terminate enzymolysis.
Researches on the frozen stock solution find that the frozen stock solution has different compositions and different phase change points, and the activity of protecting cells to the maximum extent in the cooling process can be ensured by regulating the temperature to be gradient and slowly cooling in the cooling process.
Generally speaking, the temperature reduction process is a gradual gradient temperature reduction process, and the gradient temperature reduction process is essentially in freezing storage, because the dosage of the protective agent dimethyl sulfoxide is small, a small amount of ice crystals still cause cell damage in cells.
In further research, the invention changes the gradient of temperature reduction, a single gradient type temperature reduction method is not adopted in the temperature reduction process, but a proper temperature rise process is adopted when the temperature is reduced to a certain temperature point, so that the damage degree of the cells in the frozen storage is greatly reduced, and finally, the temperature reduction program which can be divided into 6 steps is optimized according to the experimental result of the cell survival rate.
Specifically, the preferable 6-step cooling procedure is as follows:
the different formula of the frozen liquid, the phase change point of the frozen liquid is different when the temperature is reduced by the program, so that the phase change point of the preferable frozen liquid is in the stage from the lowest temperature to the temperature rise, the more latent heat absorption of the frozen liquid is, the best freezing effect is achieved, and the final stock rate is more than 97%.
Has the advantages that: after the novel freezing solution is adopted and the program cooling step is matched, the survival rate of the cells before and after the cells are frozen is not obviously reduced and is more than 97 percent, and in addition, the number of the cells still keeps higher concentration.
Drawings
FIG. 1 is a temperature programmed curve of example 1.
Detailed Description
The present invention is described in detail below with reference to examples, which are provided for the purpose of further illustration only and are not to be construed as limiting the scope of the present invention, and the insubstantial modifications and adaptations thereof by those skilled in the art based on the teachings of the present invention will still fall within the scope of the present invention.
Example 1
1.1 preparation of freezing stock solution:
the formula is as follows: 5ml of DMSO, 56ml of compound electrolyte injection, 25ml of 20% human serum albumin, 8.5ml of dextran 40 glucose injection, 3ml of 0.9% sodium chloride injection and 2.5ml of 10% glucose injection. The preparation method comprises the following steps: preparing a centrifugal tube, adding 5ml of DMSO solution, adding 56ml of compound electrolyte injection, 8.5ml of dextran 40 glucose injection, 3ml of 0.9% sodium chloride injection and 2.5ml of 10% glucose injection, and shaking while adding; after mixing, 25ml of 20% human serum albumin was added slowly dropwise while shaking the centrifuge tube. After preparation, the components are fully and uniformly mixed and placed in a refrigerator with the temperature of 2-8 ℃ for precooling for at least 30 min.
1.2 cryopreservation of human umbilical cord mesenchymal stem cells:
adding 0.2ml/cm into the human umbilical cord mesenchymal stem cells of no more than 5 generations2Digesting with 0.25% trypsin for 1-3 min, adding complete culture medium 6 times of digestive juice to stop enzymolysis, and counting at 1 × 107Adding precooled freezing solution into/ml cell density, after resuspending, sucking 3ml cell suspension for freezing, freezing 3 tubes marked as A1, A2 and A3 respectively, preparing 1 tube marked as A0, adding 3ml freezing solution for inserting a temperature probe of a program cooling instrument for temperature measurement. After the freezing is finished, transporting the 4 freezing pipes to a program cooling instrument at a low temperature of 2-8 ℃ for program cooling, wherein the transportation time is generally controlled within 15 min; and after the programmed cooling is finished, transferring the sample into a liquid nitrogen tank for storage.
The cooling procedure is as follows: (FIG. 1 is the temperature programmed curve of example 1)
1.3 cell recovery:
cells were revived on day 3 after cryopreservation. 3 50ml centrifuge tubes were taken and labeled: a1, A2 and A3. During recovery: sucking 20ml of complete culture medium into a 50ml centrifuge tube, placing the frozen cells in a 37 ℃ water bath, adding the cells into the corresponding centrifuge tube after the cells are dissolved, carrying out heavy suspension, balancing, centrifuging for 5min at 400g, removing the supernatant, adding the precipitate into 20ml of complete culture medium, and mixing uniformly.
1.4, detection:
1.4.1 sampling before cryopreservation:
cell number, survival rate: before the final centrifugation and after adding the freezing medium for re-suspension, 100 mul of cell suspension is respectively taken to be applied to a Countstar cell fluorescence analyzer, and the cell number and the survival rate are obtained by machine calculation.
1.4.2 sampling after cryopreservation:
(ii) number of cells and survival rate: after the cells were thawed and lysed, 100. mu.l of cell suspension was taken for detection.
1.5 analysis
1.5.1 cell count results:
after frozen, the A group has no significant difference in cell concentration and survival rate (P is more than 0.05). See table 1 for details.
TABLE 1 results of cell count of each group
| Group of | Cell concentration (individual/ml) | Survival rate (%) |
| Before freezing | 1.08×107 | 99.3 |
| Group A after cryopreservation | 1.06×107 | 97.3 |
Example 2
1.1 preparation of freezing stock solution:
the formula is as follows: 5ml of DMSO, 56ml of compound electrolyte injection, 25ml of 20% human serum albumin, 6.5ml of dextran 40 glucose injection, 3ml of 0.9% sodium chloride injection, 2.5ml of 10% glucose injection and 2.5ml of 40% hydroxypropyl-beta-cyclodextrin aqueous solution.
The preparation method comprises the following steps: preparing a centrifugal tube, adding 5ml of DMSO solution, then adding 56ml of compound electrolyte injection, 6.5ml of dextran 40 glucose injection, 3ml of 0.9% sodium chloride injection, 2.5ml of 10% glucose injection and 2.5ml of 40% hydroxypropyl-beta-cyclodextrin aqueous solution, and shaking while adding; after mixing, 25ml of 20% human serum albumin was added slowly dropwise while shaking the centrifuge tube. After preparation, the components are fully and uniformly mixed and placed in a refrigerator with the temperature of 2-8 ℃ for precooling for at least 30 min.
1.2 cryopreservation of human umbilical cord mesenchymal stem cells:
adding 0.25% trypsin of 0.2ml/cm2 into human umbilical cord mesenchymal stem cells of no more than 5 generations for digestion for 1-3 min, adding a complete culture medium of 6 times of digestion solution for terminating enzymolysis, then adding precooled frozen stock solution with the cell density of 1 multiplied by 107/ml according to the counting result, after heavy suspension, sucking 3ml of cell suspension for freezing, freezing 3 tubes marked as B1, B2 and B3 respectively, preparing 1 tube marked as B0 additionally, adding 3ml of frozen stock solution, and inserting a temperature probe of a program cooling instrument for temperature measurement. After the freezing is finished, transporting the 4 freezing pipes to a program cooling instrument at a low temperature of 2-8 ℃ for program cooling, wherein the transportation time is generally controlled within 15 min; and after the programmed cooling is finished, transferring the sample into a liquid nitrogen tank for storage.
The cooling procedure is as follows:
1.3 cell recovery:
cells were revived on day 3 after cryopreservation. 3 50ml centrifuge tubes were taken and labeled: b1, B2 and B3. During recovery: sucking 20ml of complete culture medium into a 50ml centrifuge tube, placing the frozen cells in a 37 ℃ water bath, adding the cells into the corresponding centrifuge tube after the cells are dissolved, carrying out heavy suspension, balancing, centrifuging for 5min at 400g, removing the supernatant, adding the precipitate into 20ml of complete culture medium, and mixing uniformly.
1.4, detection:
1.4.1 sampling before cryopreservation:
cell number, survival rate: before the final centrifugation and after adding the freezing medium for re-suspension, 100 mul of cell suspension is respectively taken to be applied to a Countstar cell fluorescence analyzer, and the cell number and the survival rate are obtained by machine calculation.
1.4.2 sampling after cryopreservation:
(ii) number of cells and survival rate: after the cells were thawed and lysed, 100. mu.l of cell suspension was taken for detection.
1.5 analysis
1.5.1 cell count results:
after frozen, the A group has no significant difference in cell concentration and survival rate (P is more than 0.05). See table 2 for details.
TABLE 2 results of cell count of each group
| Group of | Cell concentration (individual/ml) | Survival rate (%) |
| Before freezing | 1.10×107 | 98.9 |
| Group B after cryopreservation | 1.07×107 | 97.5 |
Example 3
1.1 preparation of freezing stock solution:
the formula is as follows: 10ml of DMSO, 56ml of compound electrolyte injection, 25ml of 20% human serum albumin, 6.5ml of dextran 40 glucose injection and 3ml of 0.9% sodium chloride injection.
The preparation method comprises the following steps: preparing a centrifugal tube, adding 5ml of DMSO solution, adding 56ml of compound electrolyte injection, 6.5ml of dextran 40 glucose injection and 3ml of 0.9% sodium chloride injection, and shaking while adding; after mixing, 25ml of 20% human serum albumin was added slowly dropwise while shaking the centrifuge tube. After preparation, the components are fully and uniformly mixed and placed in a refrigerator with the temperature of 2-8 ℃ for precooling for at least 30 min.
1.2 cryopreservation of human umbilical cord mesenchymal stem cells:
adding 0.25% trypsin of 0.2ml/cm2 into human umbilical cord mesenchymal stem cells of no more than 5 generations for digestion for 1-3 min, adding a complete culture medium of 6 times of digestion solution for terminating enzymolysis, then adding precooled frozen stock solution at a cell density of 1 multiplied by 107/ml according to a counting result, after heavy suspension, sucking 3ml of cell suspension for freezing, and freezing 3 tubes which are respectively marked as C1, C2 and C3, preparing 1 tube which is marked as C0, adding 3ml of frozen stock solution, and inserting a temperature probe of a program cooling instrument for temperature measurement. After the freezing is finished, transporting the 4 freezing pipes to a program cooling instrument at a low temperature of 2-8 ℃ for program cooling, wherein the transportation time is generally controlled within 15 min; and after the programmed cooling is finished, transferring the sample into a liquid nitrogen tank for storage.
The cooling procedure is as follows:
1.3 cell recovery:
cells were revived on day 3 after cryopreservation. 3 50ml centrifuge tubes were taken and labeled: c1, C2, C3. During recovery: sucking 20ml of complete culture medium into a 50ml centrifuge tube, placing the frozen cells in a 37 ℃ water bath, adding the cells into the corresponding centrifuge tube after the cells are dissolved, carrying out heavy suspension, balancing, centrifuging for 5min at 400g, removing the supernatant, adding the precipitate into 20ml of complete culture medium, and mixing uniformly.
1.4, detection:
1.4.1 sampling before cryopreservation:
cell number, survival rate: before the final centrifugation and after adding the freezing medium for re-suspension, 100 mul of cell suspension is respectively taken to be applied to a Countstar cell fluorescence analyzer, and the cell number and the survival rate are obtained by machine calculation.
1.4.2 sampling after cryopreservation:
(ii) number of cells and survival rate: after the cells were thawed and lysed, 100. mu.l of cell suspension was taken for detection.
1.5 analysis
1.5.1 cell count results:
after frozen, the A group has no significant difference in cell concentration and survival rate (P is more than 0.05). See table 3 for details.
TABLE 3 results of cell count of each group
| Group of | Cell concentration (individual/ml) | Survival rate (%) |
| Before freezing | 1.06×107 | 98.3 |
| After cryopreservation, group C | 0.98×107 | 91.0 |
Example 4
1.1 preparation of freezing stock solution:
the formula is as follows: 3ml of DMSO, 56ml of compound electrolyte injection, 25ml of 20% human serum albumin, 6.5ml of dextran 40 glucose injection, 3ml of 0.9% sodium chloride injection, 3.5ml of 10% glucose injection and 3.5ml of 40% hydroxypropyl-beta-cyclodextrin aqueous solution.
The preparation method comprises the following steps: preparing a centrifugal tube, adding 3ml of DMSO solution, adding 56ml of compound electrolyte injection, 6.5ml of dextran 40 glucose injection, 3ml of 0.9% sodium chloride injection, 3.5ml of 10% glucose injection and 3.5ml of 40% hydroxypropyl-beta-cyclodextrin aqueous solution, and shaking while adding; after mixing, 25ml of 20% human serum albumin was added slowly dropwise while shaking the centrifuge tube. After preparation, the components are fully and uniformly mixed and placed in a refrigerator with the temperature of 2-8 ℃ for precooling for at least 30 min.
1.2 cryopreservation of human umbilical cord mesenchymal stem cells:
adding 0.25% trypsin of 0.2ml/cm2 into human umbilical cord mesenchymal stem cells of no more than 5 generations for digestion for 1-3 min, adding a complete culture medium of 6 times of digestion solution for terminating enzymolysis, then adding precooled frozen stock solution with the cell density of 1 multiplied by 107/ml according to counting results, after heavy suspension, sucking 3ml of cell suspension for freezing, freezing 3 tubes respectively marked as D1, D2 and D3, preparing 1 tube additionally marked as D0, adding 3ml of frozen stock solution, and inserting a temperature probe of a program cooling instrument for temperature measurement. After the freezing is finished, transporting the 4 freezing pipes to a program cooling instrument at a low temperature of 2-8 ℃ for program cooling, wherein the transportation time is generally controlled within 15 min; and after the programmed cooling is finished, transferring the sample into a liquid nitrogen tank for storage.
The cooling procedure is as follows:
1.3 cell recovery:
cells were revived on day 3 after cryopreservation. 3 50ml centrifuge tubes were taken and labeled: d1, D2, D3. During recovery: sucking 20ml of complete culture medium into a 50ml centrifuge tube, placing the frozen cells in a 37 ℃ water bath, adding the cells into the corresponding centrifuge tube after the cells are dissolved, carrying out heavy suspension, balancing, centrifuging for 5min at 400g, removing the supernatant, adding the precipitate into 20ml of complete culture medium, and mixing uniformly.
1.4, detection:
1.4.1 sampling before cryopreservation:
cell number, survival rate: before the final centrifugation and after adding the freezing medium for re-suspension, 100 mul of cell suspension is respectively taken to be applied to a Countstar cell fluorescence analyzer, and the cell number and the survival rate are obtained by machine calculation.
1.4.2 sampling after cryopreservation:
(ii) number of cells and survival rate: after the cells were thawed and lysed, 100. mu.l of cell suspension was taken for detection.
1.5 analysis
1.5.1 cell count results:
after frozen, the A group has no significant difference in cell concentration and survival rate (P is more than 0.05). See table 4 for details.
TABLE 4 results of cell count of each group
| Group of | Cell concentration (individual/ml) | Survival rate (%) |
| Before freezing | 1.09×107 | 99.5 |
| D group after cryopreservation | 1.00×107 | 91.2 |
Example 5
1.1 preparation of freezing stock solution:
the formula is as follows: 5ml of DMSO, 56ml of compound electrolyte injection, 25ml of 20% human serum albumin, 6.5ml of dextran 40 glucose injection, 3ml of 0.9% sodium chloride injection, 2.5ml of 10% glucose injection and 2.5ml of 40% hydroxypropyl-beta-cyclodextrin aqueous solution.
The preparation method comprises the following steps: preparing a centrifugal tube, adding 5ml of DMSO solution, then adding 56ml of compound electrolyte injection, 6.5ml of dextran 40 glucose injection, 3ml of 0.9% sodium chloride injection, 2.5ml of 10% glucose injection and 2.5ml of 40% hydroxypropyl-beta-cyclodextrin aqueous solution, and shaking while adding; after mixing, 25ml of 20% human serum albumin was added slowly dropwise while shaking the centrifuge tube. After preparation, the components are fully and uniformly mixed and placed in a refrigerator with the temperature of 2-8 ℃ for precooling for at least 30 min.
1.2 cryopreservation of human umbilical cord mesenchymal stem cells:
adding 0.25% trypsin of 0.2ml/cm2 into human umbilical cord mesenchymal stem cells of no more than 5 generations for digestion for 1-3 min, adding a complete culture medium of 6 times of digestion solution for terminating enzymolysis, then adding precooled cryopreservation solution at a cell density of 1 multiplied by 107/ml according to a counting result, after heavy suspension, sucking 3ml of cell suspension for cryopreservation, and performing cryopreservation on 3 tubes respectively marked as E1, E2 and E3, preparing 1 tube additionally marked as E0, adding 3ml of cryopreservation solution, and using the cryopreservation solution for inserting a temperature probe of a program cooling instrument for temperature measurement. After the freezing is finished, transporting the 4 freezing pipes to a program cooling instrument at a low temperature of 2-8 ℃ for program cooling, wherein the transportation time is generally controlled within 15 min; and after the programmed cooling is finished, transferring the sample into a liquid nitrogen tank for storage.
The cooling procedure is as follows:
1.3 cell recovery:
cells were revived on day 3 after cryopreservation. 3 50ml centrifuge tubes were taken and labeled: e1, E2, E3. During recovery: sucking 20ml of complete culture medium into a 50ml centrifuge tube, placing the frozen cells in a 37 ℃ water bath, adding the cells into the corresponding centrifuge tube after the cells are dissolved, carrying out heavy suspension, balancing, centrifuging for 5min at 400g, removing the supernatant, adding the precipitate into 20ml of complete culture medium, and mixing uniformly.
1.4, detection:
1.4.1 sampling before cryopreservation:
cell number, survival rate: before the final centrifugation and after adding the freezing medium for re-suspension, 100 mul of cell suspension is respectively taken to be applied to a Countstar cell fluorescence analyzer, and the cell number and the survival rate are obtained by machine calculation.
1.4.2 sampling after cryopreservation:
(ii) number of cells and survival rate: after the cells were thawed and lysed, 100. mu.l of cell suspension was taken for detection.
1.5 analysis
1.5.1 cell count results:
after frozen, the A group has no significant difference in cell concentration and survival rate (P is more than 0.05). See table 5 for details.
TABLE 5 results of cell count of each group
| Group of | Cell concentration (individual/ml) | Survival rate (%) |
| Before freezing | 1.10×107 | 98.8 |
| After cryopreservation, group E | 0.98×107 | 88.9 |
Example 6
1.1 preparation of freezing stock solution:
the formula is as follows: 5ml of DMSO, 56ml of compound electrolyte injection, 25ml of 20% human serum albumin, 6.5ml of dextran 40 glucose injection, 3ml of 0.9% sodium chloride injection, 2.5ml of 10% glucose injection and 2.5ml of 40% hydroxypropyl-beta-cyclodextrin aqueous solution.
The preparation method comprises the following steps: preparing a centrifugal tube, adding 5ml of DMSO solution, then adding 56ml of compound electrolyte injection, 6.5ml of dextran 40 glucose injection, 3ml of 0.9% sodium chloride injection, 2.5ml of 10% glucose injection and 2.5ml of 40% hydroxypropyl-beta-cyclodextrin aqueous solution, and shaking while adding; after mixing, 25ml of 20% human serum albumin was added slowly dropwise while shaking the centrifuge tube. After preparation, the components are fully and uniformly mixed and placed in a refrigerator with the temperature of 2-8 ℃ for precooling for at least 30 min.
1.2 cryopreservation of human umbilical cord mesenchymal stem cells:
adding 0.25% trypsin of 0.2ml/cm2 into human umbilical cord mesenchymal stem cells of no more than 5 generations for digestion for 1-3 min, adding a complete culture medium of 6 times of digestion solution for terminating enzymolysis, then adding precooled frozen stock solution at a cell density of 1 multiplied by 107/ml according to a counting result, after heavy suspension, sucking 3ml of cell suspension for freezing, and freezing 3 tubes respectively marked as F1, F2 and F3, and additionally preparing 1 tube marked as F0, adding 3ml of frozen stock solution, and using the frozen stock solution for inserting a temperature probe of a program cooling instrument for temperature measurement. After the freezing is finished, transporting the 4 freezing pipes to a program cooling instrument at a low temperature of 2-8 ℃ for program cooling, wherein the transportation time is generally controlled within 15 min; and after the programmed cooling is finished, transferring the sample into a liquid nitrogen tank for storage.
The cooling procedure is as follows:
1.3 cell recovery:
cells were revived on day 3 after cryopreservation. 3 50ml centrifuge tubes were taken and labeled: f1, F2 and F3. During recovery: sucking 20ml of complete culture medium into a 50ml centrifuge tube, placing the frozen cells in a 37 ℃ water bath, adding the cells into the corresponding centrifuge tube after the cells are dissolved, carrying out heavy suspension, balancing, centrifuging for 5min at 400g, removing the supernatant, adding the precipitate into 20ml of complete culture medium, and mixing uniformly.
1.4, detection:
1.4.1 sampling before cryopreservation:
cell number, survival rate: before the final centrifugation and after adding the freezing medium for re-suspension, 100 mul of cell suspension is respectively taken to be applied to a Countstar cell fluorescence analyzer, and the cell number and the survival rate are obtained by machine calculation.
1.4.2 sampling after cryopreservation:
(ii) number of cells and survival rate: after the cells were thawed and lysed, 100. mu.l of cell suspension was taken for detection.
1.5 analysis
1.5.1 cell count results:
after frozen, the A group has no significant difference in cell concentration and survival rate (P is more than 0.05). See table 6 for details.
TABLE 6 results of cell count of each group
| Group of | Cell concentration (individual/ml) | Survival rate (%) |
| Before freezing | 1.01×107 | 98.5 |
| F group after cryopreservation | 0.95×107 | 92.7 |
Claims (3)
1. Freezing temperature reduction effectThe programmed cooling method of the human umbilical cord mesenchymal stem cells with excellent fruits is characterized by comprising the following steps: providing a frozen stock solution, and taking pretreated human umbilical cord mesenchymal stem cells in a ratio of 1 × 107Adding pre-cooled freezing medium into per ml of cell density, after re-suspending, sucking 1-5 ml of cell suspension into a programmed cooling instrument for programmed cooling freezing;
the frozen stock solution consists of 5% of human serum albumin, 5% of dimethyl sulfoxide, 40% -60% of compound electrolyte solution, 1% -10% of sodium chloride solution with the mass concentration of 0.9%, 1% -10% of glucose solution with the mass concentration of 10%, 1% -10% of dextran 40 glucose solution and 0.5% -5% of hydroxypropyl-beta-cyclodextrin in percentage by volume;
the procedure cooling specifically comprises the following steps: waiting at 20 ℃; cooling to-16 deg.C at 1 deg.C/min; cooling to-60 deg.C at 25 deg.C/min; heating to-18 ℃ at a speed of 10 ℃/min; cooling to-60 deg.C at 1 deg.C/min; cooling to-90 ℃ at a speed of 10 ℃/min.
2. The method of claim 1, wherein the pretreatment process comprises digestion and termination of the digestion step.
3. The method of claim 2, wherein the enzyme used in the digesting step comprises trypsin.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110126859.7A CN112741081B (en) | 2021-01-29 | 2021-01-29 | Programmed cooling method for human umbilical cord mesenchymal stem cells with excellent freezing and cooling effects |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110126859.7A CN112741081B (en) | 2021-01-29 | 2021-01-29 | Programmed cooling method for human umbilical cord mesenchymal stem cells with excellent freezing and cooling effects |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112741081A CN112741081A (en) | 2021-05-04 |
| CN112741081B true CN112741081B (en) | 2021-12-21 |
Family
ID=75653336
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110126859.7A Active CN112741081B (en) | 2021-01-29 | 2021-01-29 | Programmed cooling method for human umbilical cord mesenchymal stem cells with excellent freezing and cooling effects |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112741081B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113475501A (en) * | 2021-08-13 | 2021-10-08 | 深圳市北科生物科技有限公司 | Umbilical cord mesenchymal stem cell programmed cooling method |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102908364A (en) * | 2012-11-14 | 2013-02-06 | 青岛奥克生物开发有限公司 | Mesenchymal stem cell injection, as well as preparation method and application in preparing medicament for treating children dilated cardiomyopathy |
| CN104472473A (en) * | 2014-11-20 | 2015-04-01 | 黑龙江天晴干细胞股份有限公司 | Cryopreservation method of umbilical cord tissue blocks |
| CN106982821A (en) * | 2017-05-22 | 2017-07-28 | 安徽瑞杰赛尔生物科技有限公司 | Umbilical cord mesenchymal stem cells clinic freezes protection liquid composition and application thereof |
| CN107751186A (en) * | 2017-10-25 | 2018-03-06 | 北京科兴生物制品有限公司 | A kind of method of Rapid-Freezing Method and recovery cell |
| CN108471736A (en) * | 2016-01-14 | 2018-08-31 | 德普伊新特斯产品公司 | Composition and method for freezen protective HUTC |
| CN109820810A (en) * | 2019-03-18 | 2019-05-31 | 上海熙承生物科技有限公司 | A kind of beauty and skin care stem cell medicine and preparation method thereof |
| CN110810399A (en) * | 2019-11-21 | 2020-02-21 | 康妍葆(北京)干细胞科技有限公司 | Cell transportation protective solution, preparation method and application |
| EP3622959A1 (en) * | 2018-09-17 | 2020-03-18 | Roquette America, Inc. | Cyclodextrin for protecting cells upon cryopreservation |
-
2021
- 2021-01-29 CN CN202110126859.7A patent/CN112741081B/en active Active
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102908364A (en) * | 2012-11-14 | 2013-02-06 | 青岛奥克生物开发有限公司 | Mesenchymal stem cell injection, as well as preparation method and application in preparing medicament for treating children dilated cardiomyopathy |
| CN104472473A (en) * | 2014-11-20 | 2015-04-01 | 黑龙江天晴干细胞股份有限公司 | Cryopreservation method of umbilical cord tissue blocks |
| CN108471736A (en) * | 2016-01-14 | 2018-08-31 | 德普伊新特斯产品公司 | Composition and method for freezen protective HUTC |
| CN106982821A (en) * | 2017-05-22 | 2017-07-28 | 安徽瑞杰赛尔生物科技有限公司 | Umbilical cord mesenchymal stem cells clinic freezes protection liquid composition and application thereof |
| CN107751186A (en) * | 2017-10-25 | 2018-03-06 | 北京科兴生物制品有限公司 | A kind of method of Rapid-Freezing Method and recovery cell |
| EP3622959A1 (en) * | 2018-09-17 | 2020-03-18 | Roquette America, Inc. | Cyclodextrin for protecting cells upon cryopreservation |
| CN109820810A (en) * | 2019-03-18 | 2019-05-31 | 上海熙承生物科技有限公司 | A kind of beauty and skin care stem cell medicine and preparation method thereof |
| CN110810399A (en) * | 2019-11-21 | 2020-02-21 | 康妍葆(北京)干细胞科技有限公司 | Cell transportation protective solution, preparation method and application |
Also Published As
| Publication number | Publication date |
|---|---|
| CN112741081A (en) | 2021-05-04 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN112889813B (en) | Cryopreservation method of human umbilical cord mesenchymal stem cells with high cell survival rate | |
| CN108235981B (en) | Cell cryopreservation liquid capable of being used clinically | |
| EP3933032A1 (en) | Three-dimensional culture method for large-scale preparation of stem cells | |
| CN114557337B (en) | Protein-free non-program freezing solution of umbilical cord mesenchymal stem cells and preparation method thereof | |
| CN110684722A (en) | Preparation method of mesenchymal stem cells derived from placenta chorion plate tissue | |
| CN112741081B (en) | Programmed cooling method for human umbilical cord mesenchymal stem cells with excellent freezing and cooling effects | |
| CN116725003B (en) | Stem cell cryopreservation liquid and stem cell cryopreservation method | |
| CN112913833A (en) | Programmed cooling method for human umbilical cord mesenchymal stem cell working cell bank | |
| CN112741082A (en) | Method for detecting influence of cooling process on cryopreservation effect of human umbilical cord mesenchymal stem cells | |
| CN110074093B (en) | Programmed cooling and freezing method for human umbilical cord blood hematopoietic stem cells | |
| CN108029679B (en) | Freezing medium for freezing mononuclear cells | |
| CN106635952B (en) | A kind of cell recovery reagent | |
| CN106417260A (en) | Umbilical cord tissue cryoprotectant | |
| CN112655700B (en) | Application of frozen stock solution in gallbladder stem cells and recovery method of gallbladder stem cells | |
| CN114736855B (en) | High-purity extraction method of stem cell exosomes | |
| CN114891744B (en) | Freezing umbilical cord blood NK cell in-vitro amplification method | |
| CN110839612A (en) | Cord blood hematopoietic stem cell preserving fluid and preparation method thereof | |
| CN115943949A (en) | Cell cryopreservation solution and preparation method and application thereof | |
| CN108124853A (en) | A kind of store method of mescenchymal stem cell | |
| CN110192552B (en) | Application of glycylglycine conditioned medium in mesenchymal stem cell cryopreservation and preparation of commercial cryopreservation protective agent | |
| CN106479976A (en) | A kind of method of Cord blood megakaryoblast In vitro culture | |
| CN111838138A (en) | Stem cell cryopreservation liquid and preparation and use methods thereof | |
| CN111838132A (en) | Stem cell cryopreservation method and cell cryopreservation liquid used by same | |
| CN114762497B (en) | Cell cryopreservation liquid and cell cryopreservation method | |
| CN118355899A (en) | Frozen stock solution and preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20211203 Address after: 201100 4th floor, building 23, No. 1188, LIANHANG Road, Minhang District, Shanghai Applicant after: Huaxiayuan (Shanghai) Biotechnology Co.,Ltd. Address before: Unit A2, 2 / F, building 23, No. 1188, LIANHANG Road, Minhang District, Shanghai 201100 Applicant before: Huaxiayuan Cell Engineering Group Co., Ltd |
|
| TA01 | Transfer of patent application right | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |