CN111727962B - Stem cell cryopreservation liquid - Google Patents
Stem cell cryopreservation liquid Download PDFInfo
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- CN111727962B CN111727962B CN202010797418.5A CN202010797418A CN111727962B CN 111727962 B CN111727962 B CN 111727962B CN 202010797418 A CN202010797418 A CN 202010797418A CN 111727962 B CN111727962 B CN 111727962B
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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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- 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
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0664—Dental pulp stem cells, Dental follicle stem cells
Abstract
The invention belongs to the field of biological medicines, and relates to a stem cell cryopreservation solution. The stem cell cryopreservation liquid comprises a basic cryopreservation liquid and is added with phenethyl isothiocyanate, the concentration of the phenethyl isothiocyanate in the stem cell cryopreservation liquid is 0.1-10 mu M, and the basic cryopreservation liquid comprises 5v/v% -10 v/v% of DMSO and 90 v/v% -95v/v% of FBS; the stem cells are dental pulp stem cells. After the dental pulp stem cells cryopreserved by the cryopreservation liquid are recovered, the proliferation capacity of the dental pulp stem cells is remarkably improved, and the separation and culture efficiency of the dental pulp stem cells can be greatly improved. The dental pulp stem cells can be better suitable for clinical application. Has good practical significance.
Description
Technical Field
The invention belongs to the field of biological medicines, and relates to a stem cell cryopreservation solution.
Background
Reportedly, dental pulp stem cells can play a good role in repairing periodontitis in the treatment of periodontitis by the following action mechanisms: the local inflammation is inhibited and the local angiogenesis is promoted by implanting into local parts and differentiating into defective cells, secreting cell factors and chemotactic stem cells to the local parts.
The clinical application of the dental pulp stem cells relates to the freezing storage and recovery technology of the dental pulp stem cells, the freezing storage of the cells is a process of adding nutrient components and an anti-freezing protective agent DMSO into the cells which are grown and cultured at 37 ℃, and freezing the cells in liquid nitrogen for a long time at ultralow temperature in a gradual cooling mode.
The cryopreservation process significantly alters the thermodynamic, chemical and physical environment of the cells with the attendant risk of biological damage. The temperature change mainly depends on the boundary condition of heat conduction caused by a temperature reduction or rise method and is also influenced by the latent heat effect in the cell re-melting process. In order to minimize cell damage during freeze-thawing, the chemical and temperature manipulation processes must be further optimized. However, one or two cryoprotectants are added before the temperature is reduced, and are removed after dissolution.
At present, the cryopreservation of cells is performed by using a common commercial culture medium or serum, in addition to the cryopreservation of stem cells by using dimethyl sulfoxide (DMSO).
The dental pulp stem cells are easy to pollute in the preparation process, so that the number of the cells is small, and the growth is slow. Furthermore, the dental pulp mesenchymal stem cells are not easy to adhere to the wall, which causes cell loss. The existing freezing solutions still inevitably cause certain damage to dental pulp stem cells in the process of freezing the cells, so that the activity of the stem cells cannot be ensured, the cell proliferation rate is low, and the clinical utilization of the stem cells is influenced.
Disclosure of Invention
In some embodiments, the invention provides a cell cryopreservation solution capable of promoting cell proliferation after cryopreservation.
In some embodiments, the invention provides a cell cryopreservation solution which can obviously promote proliferation of dental pulp stem cells after cryopreservation.
In some embodiments, the invention provides a stem cell cryopreservation solution comprising phenethylisothiocyanate.
In some embodiments, the cell culture medium comprises 0.1-10 μ M phenethylisothiocyanate.
In some embodiments, the stem cell cryopreservation solution comprises a base cryopreservation solution added with phenethylisothiocyanate, wherein the concentration of the phenethylisothiocyanate in the stem cell cryopreservation solution is 0.1-10 μ M, and the base cryopreservation solution comprises 5v/v% to 10 v/v% of DMSO and 90 v/v% -95v/v% of FBS.
In some embodiments, the cryopreservation solution comprises the following components: 0.1-10 mu M of phenethylisothiocyanate, 5-15 v/v% of DMSO, and 90-95 v/v% of FBS; the stem cells are dental pulp stem cells.
The inventors found that when dental pulp cells are frozen in the frozen stock solution and then thawed, the proliferation capacity of the cells is significantly improved as compared with that of a normal frozen stock solution.
In some embodiments, the cryopreserved contains 0.01 to 30 μ M phenethylisothiocyanate.
In some embodiments, the cryopreserved contains 0.05 to 20 μ M phenethylisothiocyanate.
In some embodiments, the cryopreservation solution contains 0.1-20 μ M phenethylisothiocyanate.
In some embodiments, the cryopreserved contains 0.5 to 20 μ M phenethylisothiocyanate.
In some embodiments, the cryopreserved contains 0.5 to 10 μ M phenethylisothiocyanate.
In some embodiments, the cryopreserved contains 1-15 μ M phenethylisothiocyanate.
In some embodiments, the cryopreserved contains 1-10 μ M phenethylisothiocyanate.
In some embodiments, the cryopreserved contains 5-15 μ M phenethylisothiocyanate.
In some embodiments, the cryopreserved contains 5-10 μ M phenethylisothiocyanate.
In some embodiments, the cryopreserved contains phenylethyl isothiocyanate 0.1. mu.M, 0.2. mu.M, 0.3. mu.M, 0.4. mu.M, 0.5. mu.M, 1. mu.M, 2. mu.M, 3. mu.M, 4. mu.M, 5. mu.M, 6. mu.M, 7. mu.M, 8. mu.M, 9. mu.M, 10. mu.M.
In some embodiments, the cryopreservation solution further comprises 0.01-30 μ M resveratrol.
In some embodiments, the cryopreservation solution comprises 0.1-30 μ M resveratrol.
In some embodiments, the cryopreservation solution comprises 0.1-20 μ M resveratrol.
In some embodiments, the cryopreservation solution comprises 0.1-15 μ M resveratrol.
In some embodiments, the cryopreservation solution comprises 0.1-10 μ M resveratrol.
In some embodiments, the cryopreservation solution comprises 0.5-10 μ M resveratrol.
In some embodiments, the cryopreservation solution comprises 1-10 μ M resveratrol.
In some embodiments, the cryopreservation solution comprises 5-10 μ M resveratrol.
In some embodiments, the cryopreservation solution comprises resveratrol 0.1 μ M, 0.2 μ M, 0.3 μ M, 0.4 μ M, 0.5 μ M, 1 μ M, 2 μ M, 3 μ M, 4 μ M, 5 μ M, 6 μ M, 7 μ M, 8 μ M, 9 μ M, 10 μ M.
At present, methods such as an enzyme-linked digestion method, a tissue block culture method, a tissue block enzyme digestion method and the like are commonly used in a preparation method of dental pulp stem cells, although dental pulp stem cells can be obtained by various methods, the dental pulp stem cells are easy to pollute in preparation and culture due to various types of cells in oral cavities, the separated and extracted dental pulp stem cells are few, the cells grow slowly, and the dental pulp stem cells are difficult to prepare due to cell loss caused by the fact that the dental pulp mesenchymal stem cells are not easy to adhere to the wall. In addition, the existing cell cryopreservation liquid inevitably causes certain damage to dental pulp stem cells in the process of freezing the dental pulp stem cells, so that the activity of the stem cells cannot be ensured, and the cell proliferation rate is low. The clinical utilization of dental pulp stem cells is seriously affected by the above factors. The inventor finds that after the phenethylisothiocyanate or the resveratrol and the frozen dental pulp stem cells are added into the common frozen stock solution and recovered, the proliferation capacity of the dental pulp stem cells is remarkably improved, and the separation and culture efficiency of the dental pulp stem cells can be greatly improved. The dental pulp stem cells can be better suitable for clinical application. This is clearly of practical significance.
In some embodiments, the cryopreservation solution contains phenylethyl isothiocyanate at a concentration of 10 μ M; the concentration of the resveratrol is 5 mu M.
In some embodiments, the DMSO concentration is 10 v/v% and the FBS concentration is 90 v/v%.
In some embodiments, the invention provides a method for cryopreserving stem cells, wherein dental pulp stem cells are cryopreserved by using the stem cell cryopreserving liquid.
In some embodiments, the cryopreservation method comprises the steps of (1) mixing dental pulp stem cells with the stem cell cryopreservation solution; and (2) carrying out programmed cooling to-80 ℃, and then transferring to liquid nitrogen for freezing and storing.
In some embodiments, the cryopreservation method comprises the steps of: (1) preparation of frozen stock solution: preparing the stem cell freezing solution; (2) preparation of cell suspension: centrifugally washing the dental pulp stem cells, mixing the collected dental pulp stem cells with the cryopreservation liquid to obtain a cell suspension, and placing the cell suspension in a sterile cryopreservation tube; (3) freezing: carrying out programmed cooling on the freezing tube to 75-85 ℃, and then transferring to liquid nitrogen for freezing preservation; (4) cell recovery: taking out the cryopreservation tube, rapidly placing in 37-40 deg.C water bath for 1-3 min, oscillating until the cell suspension is completely melted, centrifuging, washing with PBS to remove the cryopreservation solution, and obtaining the revived cell.
In some embodiments, the invention provides a use of the stem cell cryopreservation liquid in dental pulp stem cell cryopreservation resuscitation.
In some embodiments, the invention provides a kit for cryopreserving stem cells, which comprises the stem cell cryopreserving liquid, wherein the stem cells are dental pulp stem cells.
Drawings
FIG. 1 shows the MTT method for detecting the cell proliferation after the recovery of cells frozen in the frozen stock solution of each group of cells.
FIG. 2 shows the proliferation of cells in each group measured by the EdU method.
Figure 3 shows the proliferative capacity of the DPSCs of each group, p <0.05, as measured by EdU method.
Detailed Description
The technical solutions of the present invention are further illustrated by the following specific examples, which do not represent limitations to the scope of the present invention. Insubstantial modifications and adaptations of the present invention by others of the concepts fall within the scope of the invention.
Herein, peitc (phenyl isothiocyanate) refers to phenethylisothiocyanate.
Herein Resveratrol refers to Resveratrol.
Herein, DPSC refers to dental pulp stem cells.
Herein, t-butyl refers to t-Butylhydroquinone (t-Butylhydroquinone).
Example 1 isolation and culture of dental pulp Stem cells
The dental pulp tissue is buffered with phosphate containing antibiotic (Penicillin-Streptomycin)Washing with PBS, cutting dental pulp tissue pieces with ophthalmic scissors, adding 3 mL Collagenase and Dispase mixture (collagen/Dispase) in 37 deg.C water bath, digesting for 45 min-1 h, adding complete culture solution (containing 10% FBS, 100U/mL penicillin, 100 μ g/mL streptomycin, 2 mM L-glutamine and 10 mM vitamin C Phosphate) to stop digestion, centrifuging at 1000 rpm for 5 min, discarding supernatant, resuspending the obtained precipitate and tissue with 10% fetal bovine serum-containing alpha-MEM culture solution, inoculating into cell culture flask, resuspending at 37 deg.C, 5% CO 5%2Culturing in a constant-temperature incubator, changing the liquid for 72 h, changing the liquid once a week later, and changing the liquid once every three days after the cells migrate out. When the cells grow to more than 80% of the fusion degree, the cells are digested and passaged. P6 passage cells were used for this experiment.
Example 2 formulation and preparation of dental pulp Stem cell cryopreservation solution
DMSO and FBS were mixed so that the DMSO content in the mixture was 10 v/v% and the FBS content was 90 v/v%, and PEITC 0.1. mu.M was added thereto and stored at 4 ℃ until use.
Test example 1
Mixing DMSO and FBS to make the content of DMSO in the mixture be 10 v/v% and the content of FBS be 90 v/v%, adding resveratrol 0.1 μ M, and storing at 4 deg.C.
EXAMPLE 3 formulation and preparation of dental pulp Stem cell cryopreservation solution
DMSO and FBS were mixed so that the DMSO content in the mixture was 10 v/v% and the FBS content was 90 v/v%, and PEITC 0.5. mu.M was added thereto and stored at 4 ℃ until use.
Example 4 formulation and preparation of dental pulp Stem cell cryopreservation solution
DMSO and FBS were mixed so that the DMSO content in the mixture was 10 v/v% and the FBS content was 90 v/v%, and PEITC 1. mu.M was added thereto and stored at 4 ℃ until use.
EXAMPLE 5 formulation and preparation of dental pulp Stem cell cryopreservation solution
DMSO and FBS were mixed so that the DMSO content in the mixture was 10 v/v% and the FBS content was 90 v/v%, and PEITC 5. mu.M was added thereto and stored at 4 ℃ until use.
EXAMPLE 6 formulation and preparation of dental pulp Stem cell cryopreservation solution
DMSO and FBS were mixed so that the DMSO content in the mixture was 10 v/v% and the FBS content was 90 v/v%, and PEITC 10. mu.M was added thereto and stored at 4 ℃ until use.
Test example 2 recipe and preparation method of dental pulp stem cell cryopreservation solution
DMSO and FBS were mixed so that the DMSO content in the mixture was 10 v/v% and the FBS content was 90 v/v%, and resveratrol 10. mu.M was added thereto.
Comparative example 1
DMSO and FBS were mixed so that the DMSO content in the mixture was 10 v/v% and the FBS content was 90 v/v%, and the mixture was stored at 4 ℃ until use.
Comparative example 2
DMSO and FBS were mixed so that the DMSO content in the mixture was 10 v/v% and the FBS content was 90 v/v%, and t-Butylhydroquinone (t-Butylhydroquinone) was added thereto at 1. mu.M and stored at 4 ℃ until use.
Example 7 cryopreservation of dental pulp Stem cells
(1) Taking the P6 generation dental pulp stem cells described in the embodiment 1, discarding the old culture medium when the cell fusion degree reaches 80-90%, cleaning the culture dish by PBS, adding a pancreatin mixed solution containing EDTA for digestion for 1-2 minutes, adding a complete culture solution to stop digestion, and blowing and beating the cells into a single cell suspension to obtain the P6 generation dental pulp stem cells.
(2) A small amount of cell suspension is taken for cell counting, and the rest suspension is centrifuged at 1200 rpm/min for 5 min to collect cells. According to the counting results, the cell freezing medium of example 2-example 6, test examples 1-2 and comparative examples 1-2, which had resuspended cells, was added to adjust the cell density to 1X 106each/mL, dispensed into a cryopreservation tube, and 1.5 mL was added to each tube.
(3) And (4) putting the freezing tube into a box of a program cooling instrument, cooling by a conventional program, and transferring to liquid nitrogen for freezing.
The above cryopreservation procedure is a conventional procedure.
Test example 3 evaluation of cryopreservation Effect of frozen stock solution
1. Cell survival rate after cell recovery
The frozen dental pulp stem cells of each group obtained in example 7 were collected and thawed, and then centrifuged and washed with PBS to remove the frozen stock solution. Counting is carried out, and the cell survival rate is determined. The results are shown in Table 1.
TABLE 1
2. Cell proliferation after cell recovery
(1) The frozen dental pulp stem cells of each group obtained in example 7 were collected and thawed, and then centrifuged and washed with PBS to remove the frozen stock solution.
(2) After resuspension, the ratio was 1X 103The density of each well was inoculated in a 96-well plate, and the medium was an α -MEM medium containing 10% fetal bovine serum, which was allowed to grow adherent for 24 hours.
(3) MTT (sigma) reagent was added for 3 hours and the reaction was stopped by adding the reagent according to the manufacturer's instructions.
(4) And detecting the absorbance value.
The results of examples 2 to 6, test examples 1 to 2 and comparative example 1 are shown in Table 2 and FIG. 1.
TABLE 2
The results show that freezing the dental pulp stem cells with the freezing medium containing PEITC can significantly promote the proliferation ability of the recovered dental pulp stem cells. The effect is even better than that of adding resveratrol.
In addition, the results of comparing the cell lysates of example 4, comparative example 1, and comparative example 2 are shown in fig. 2 and 3.
The detection method of fig. 2 is an EdU method, and includes the following steps:
1) cell cultureCulturing: taking cells in logarithmic growth phase at a rate of 1X 10 per well3Cells were seeded in 96-well plates and allowed to grow adherently to a stage of approximately 60-70% cell confluence;
2) adding an EdU reagent for labeling: the cells were cultured in a cell culture medium at 1000: 1 (reagent A) to prepare an appropriate amount of 50. mu.M EdU medium; staining and counting were performed according to the kit manufacturer (Abcam) instructions.
Claims (8)
1. The stem cell cryopreservation liquid is characterized by comprising a basic cryopreservation liquid and added with phenethylisothiocyanate, wherein the concentration of the phenethylisothiocyanate in the stem cell cryopreservation liquid is 1-10 mu M, and the basic cryopreservation liquid comprises 5v/v% -10 v/v% of DMSO and 90 v/v% -95v/v% of FBS; the stem cells are dental pulp stem cells.
2. The stem cell cryopreservation solution of claim 1 wherein the concentration of DMSO is 10 v/v% and the concentration of FBS is 90 v/v%.
3. The stem cell cryopreservation solution of claim 1 wherein the concentration of phenethylisothiocyanate is 10 μ M.
4. A method for cryopreserving a stem cell, which comprises cryopreserving a dental pulp stem cell using the stem cell cryopreserving liquid according to any one of claims 1 to 3.
5. The method of cryopreserving stem cells according to claim 4, comprising the steps of:
(1) mixing dental pulp stem cells with the stem cell cryopreservation solution of any one of claims 1 to 3; and
(2) the temperature is reduced to-80 ℃ by the program, and then the mixture is transferred to liquid nitrogen for freezing storage.
6. The method of cryopreserving stem cells according to claim 4, wherein the method comprises the steps of:
(1) preparation of frozen stock solution: preparing a frozen stock of stem cells according to any one of claims 1 to 3;
(2) preparation of cell suspension: centrifugally washing the dental pulp stem cells, mixing the collected dental pulp stem cells with the cryopreservation liquid to obtain a cell suspension, and placing the cell suspension in a sterile cryopreservation tube;
(3) freezing: carrying out programmed cooling on the freezing tube to-75-85 ℃, and then transferring to liquid nitrogen for freezing and storing;
(4) cell recovery: taking out the cryopreservation tube, rapidly placing in 37-40 deg.C water bath for 1-3 min, oscillating until the cell suspension is completely melted, centrifuging, washing with PBS to remove the cryopreservation solution, and obtaining the revived cell.
7. Use of the stem cell cryopreservation solution of any one of claims 1 to 3 in the recovery of dental pulp stem cell cryopreservation.
8. A kit for cryopreserving stem cells, comprising the stem cell cryopreserving liquid according to any one of claims 1 to 3, wherein the stem cells are dental pulp stem cells.
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Effective date of registration: 20220712 Address after: A102-3, building 9 (Building 7), No. 6, Nanjiang Second Road, Zhujiang street, Nansha District, Guangzhou, Guangdong 511466 Patentee after: Guangzhou Qianqi Kuncheng Biotechnology Co.,Ltd. Address before: 510000 Room 501, 5th floor, building 1, No. 6, Nanjiang 2nd Road, Zhujiang street, Nansha District, Guangzhou City, Guangdong Province Patentee before: Medical micro cell biotechnology (Guangzhou) Co.,Ltd. |
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