CN111727961B - Dental pulp stem cell cryopreservation liquid and cryopreservation method thereof - Google Patents
Dental pulp stem cell cryopreservation liquid and cryopreservation method thereof Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
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- A—HUMAN NECESSITIES
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Abstract
The invention belongs to the field of biomedicine, and relates to dental pulp stem cell cryopreservation liquid and a cryopreservation method thereof. The stem cell cryopreservation liquid comprises a basic cryopreservation liquid and is added with resveratrol, the concentration of the resveratrol in the stem cell cryopreservation liquid is 0.1-10 mu M, the basic cryopreservation liquid comprises 5-10 v/v% of DMSO and 90-95 v/v% of FBS, and 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 biomedicine, and relates to dental pulp stem cell cryopreservation liquid and a cryopreservation method thereof.
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 stem cell cryopreservation solution capable of promoting cell proliferation after cryopreservation.
In some embodiments, the invention provides a stem cell cryopreservation solution which can obviously promote proliferation of dental pulp stem cells after cryopreservation. In some embodiments, the present invention provides a stem cell cryopreservation solution comprising resveratrol.
In some embodiments, the invention provides a stem cell cryopreservation solution, the stem cell cryopreservation solution comprises a base cryopreservation solution and is added with resveratrol, the concentration of the resveratrol in the stem cell cryopreservation solution is 0.1-10 mu M, the base cryopreservation solution comprises 5v/v% -10 v/v% of DMSO and 90 v/v% -95v/v% of FBS, and the stem cells are dental pulp stem cells.
In some embodiments, the stem cell cryopreservation solution comprises the following components: 5-10 v/v% of DMSO (dimethyl sulfoxide), 90-95 v/v% of FBS (fiber glass substrate), 0.1-10 mu M of resveratrol is contained in the stem cell freezing solution, and the stem cells are dental pulp stem cells.
In some embodiments, the stem cell cryopreserved contains phenethylisothiocyanate.
In some embodiments, the stem cell cryopreserved contains 0.1 to 10 μ M phenethylisothiocyanate.
In some embodiments, the stem cell frozen stock solution contains phenethylisothiocyanate and resveratrol in a molar ratio of 0.02-10: 1.
in some embodiments, the stem cell frozen stock solution contains phenethylisothiocyanate and resveratrol in a molar ratio of 0.2-2: 1.
in some embodiments, the stem cell 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 stem cell cryopreserved contains 0.01 to 30 μ M phenethylisothiocyanate.
In some embodiments, the stem cell cryopreserved contains 0.05 to 20 μ M phenethylisothiocyanate.
In some embodiments, the stem cell cryopreserved contains 0.1 to 20 μ M phenethylisothiocyanate.
In some embodiments, the stem cell cryopreserved contains 0.5-20 μ M phenethylisothiocyanate.
In some embodiments, the stem cell cryopreserved contains 0.5 to 10 μ M phenethylisothiocyanate.
In some embodiments, the stem cell cryopreserved contains 1-15 μ M phenethylisothiocyanate.
In some embodiments, the stem cell cryopreserved contains 1-10 μ M phenethylisothiocyanate.
In some embodiments, the stem cell cryopreserved contains 5-15 μ M phenethylisothiocyanate.
In some embodiments, the stem cell cryopreserved contains 5-10 μ M phenethylisothiocyanate.
In some embodiments, the stem cell lysate 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 stem cell cryopreservation solution further comprises 0.01-30 μ M resveratrol.
In some embodiments, the stem cell cryopreservation solution comprises 0.1-30 μ M resveratrol.
In some embodiments, the stem cell cryopreservation solution comprises 0.1-20 μ M resveratrol.
In some embodiments, the stem cell cryopreservation solution comprises 0.1-15 μ M resveratrol.
In some embodiments, the stem cell cryopreservation solution comprises 0.1-10 μ M resveratrol.
In some embodiments, the stem cell cryopreservation solution comprises 0.5-10 μ M resveratrol.
In some embodiments, the stem cell cryopreservation solution comprises 1-10 μ M resveratrol.
In some embodiments, the stem cell cryopreservation solution comprises 5-10 μ M resveratrol.
In some embodiments, the stem cell 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.
In some embodiments, the stem cell cryopreserving contains 1-10 μ M phenethylisothiocyanate and 5-10 μ M resveratrol.
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 stem cell cryopreserving contains phenethylisothiocyanate at a concentration of 10 μ M; the concentration of the resveratrol is 5 mu M.
In some embodiments, the stem cell cryopreservation solution contains DMSO at a concentration of 10 v/v%, and the FBS at a concentration of 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) preparing a stem cell freezing medium: 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 stem cell 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.
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.
FIG. 4 is a diagram showing the MTT method for detecting the cell proliferation after the recovery of the cells cryopreserved in the two groups of cell cryopreserved solutions in comparative example 3.
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
Cleaning dental pulp tissue with Phosphate Buffered Saline (PBS) containing antibiotic (penicilin-Streptomycin), cutting dental pulp tissue pieces into pieces with ophthalmic scissors, adding 3 mL of Collagenase and Dispase mixture (Collagenase/Dispase) in 37 deg.C water bath, digesting for 45 min-1 h, adding complete culture solution (containing 10% FBS, 100U/mL Penicillin, 100. mu.g/mL Streptomycin, 2 mM L-glutamine and 10 mM vitamin C Phosphate) to terminate digestion, centrifuging at 1000 rpm for 5 min, discarding supernatant, and collecting precipitate and tissueThe suspension of 10% fetal bovine serum-containing alpha-MEM culture medium was inoculated into a cell culture flask and incubated at 37 ℃ with 5% CO2Culturing 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.
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.
Test example 2
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 0.5 μ M, and storing at 4 deg.C.
Test example 3
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 1 μ M, and storing at 4 deg.C.
Test example 4
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 5 μ M, and storing at 4 deg.C for use.
Test example 5 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.
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.
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
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 5 μ M and PEITC 0.1 μ M, and storing at 4 deg.C.
EXAMPLE 5 formulation and preparation of dental pulp Stem cell cryopreservation solution
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 5 μ M and PEITC 0.5 μ M, and storing at 4 deg.C.
EXAMPLE 6 formulation and preparation of dental pulp Stem cell cryopreservation solution
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 5 μ M and PEITC 1 μ M, and storing at 4 deg.C.
Example 7 formulation and preparation of dental pulp Stem cell cryopreservation solution
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 5 μ M and PEITC 5 μ M, and storing at 4 deg.C.
EXAMPLE 8 formulation and preparation of dental pulp Stem cell cryopreservation solution
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 5 μ M and PEITC 10 μ M, and storing at 4 deg.C.
Example 9 formulation and preparation of dental pulp Stem cell cryopreservation solution
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 PEITC 1 μ M, and storing at 4 deg.C.
Example 10 formulation and preparation of dental pulp Stem cell cryopreservation solution
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 0.5 μ M and PEITC 1 μ M, and storing at 4 deg.C.
EXAMPLE 11 formulation and preparation of dental pulp Stem cell cryopreservation solution
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 1 μ M and PEITC 1 μ M, and storing at 4 deg.C.
EXAMPLE 12 formulation and preparation of dental pulp Stem cell cryopreservation solution
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 10 μ M and PEITC 1 μ M, and storing at 4 deg.C.
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 13 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 solutions of examples 2 to 12, test examples 1 to 5 and comparative examples 1 to 2 were added to resuspend the cells, respectively, 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 6 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 13 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
| Frozen stock solution | Number of cells | Cell survival rate |
| Example 2 frozen stock solution | 1.026×106 | 66.9% |
| Example 3 frozen stock solution | 0.957×106 | 68.5% |
| Example 4 frozen stock solution | 0.986×106 | 69.6% |
| Example 5 frozen stock solution | 0.990×106 | 71.3% |
| Example 6 frozen stock solution | 0.977×106 | 79.6% |
| Example 7 frozen stock solution | 1.032×106 | 82.2% |
| Example 8 frozen stock solution | 0.993×106 | 82.5% |
| Example 9 frozen stock solution | 0.975×106 | 73.4% |
| Example 10 frozen stock solution | 0.988×106 | 74.7% |
| Example 11 frozen stock solution | 0.949×106 | 75.6% |
| Example 12 frozen stock solution | 0.945×106 | 81.2% |
| Test example 1 | 0.953×106 | 66.2% |
| Test example 2 | 0.979×106 | 65.9% |
| Test example 3 | 1.035×106 | 67.2% |
| Test example 4 | 0.979×106 | 67.1% |
| Test example 5 | 1.105×106 | 68.1% |
| Comparative example 1 | 0.986×106 | 65.2% |
| Comparative example 2 | 0.991×106 | 67.4% |
2. Cell proliferation after cell recovery
(1) The frozen dental pulp stem cells of each group obtained in example 13 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 12, test examples 1 to 5 and comparative example 1 are shown in Table 2 and FIG. 1.
TABLE 2
The results show that the proliferation capacity of the recovered dental pulp stem cells can be obviously promoted by freezing the dental pulp stem cells by using the freezing stock solution containing resveratrol. Meanwhile, the frozen stock solution containing the PEITC and the resveratrol has a synergistic effect on cell proliferation.
In addition, the results of comparing the cell lysates of test 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 culture: 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.
Comparative example 3
The MTT method was used to examine the effect of cryopreserving cells in a cryopreserved medium containing phenethylisothiocyanate (1. mu.M) and tert-butylhydroquinone (5. mu.M), and the other conditions were the same as those in the example 6. Cell proliferation is shown in table 3 and figure 4.
The survival of the other two groups of cells is shown in Table 3. As can be seen from Table 3, phenethylisothiocyanate (1. mu.M) + tert-butylhydroquinone (5. mu.M) was less effective than the composition of example 6.
TABLE 3
Claims (8)
1. The stem cell cryopreservation solution is characterized by comprising a basic cryopreservation solution and resveratrol, wherein the resveratrol is added into the basic cryopreservation solution, the concentration of the resveratrol in the stem cell cryopreservation solution is 0.1-10 mu M, the basic cryopreservation solution comprises 5v/v% -10 v/v% of DMSO and 90 v/v% -95v/v% of FBS, and the stem cells are dental pulp stem cells;
the stem cell frozen stock solution also contains 0.1-10 mu M of phenethylisothiocyanate; the molar ratio of the phenethylisothiocyanate to the resveratrol is 0.2-2: 1.
2. the stem cell cryopreservation solution of claim 1, wherein the concentration of phenethylisothiocyanate is 1-10 μ Μ and the concentration of resveratrol is 5-10 μ Μ.
3. The stem cell cryopreservation solution of claim 1 wherein the concentration of phenethylisothiocyanate is 10 μ Μ; the concentration of the resveratrol is 5 mu M.
4. 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%.
5. A method for cryopreserving a stem cell, comprising cryopreserving a dental pulp stem cell with the stem cell cryopreserving liquid according to any one of claims 1 to 4.
6. The method of cryopreserving stem cells according to claim 5, comprising the steps of:
(1) mixing stem cells with the cryopreservation solution; and
(2) the temperature is reduced to-80 ℃ by the program, and then the mixture is transferred to liquid nitrogen for freezing storage.
7. The method of cryopreserving stem cells according to claim 5, 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 4;
(2) preparation of cell suspension: centrifuging and washing the stem cells, mixing the collected stem cells with the frozen stock solution to obtain a cell suspension, and placing the cell suspension in a sterile frozen stock tube;
(3) freezing: and (4) carrying out programmed temperature reduction on the freezing tube to-75-85 ℃, and then transferring to liquid nitrogen for freezing preservation.
8. A kit for cryopreserving stem cells, comprising the stem cell cryopreserving solution according to any one of claims 1 to 4.
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