Cell freezing medium as well as preparation method and application thereof

Abstract

The invention discloses a cell freezing medium as well as a preparation method and application thereof, and belongs to the technical field of biomedicine. The cell freezing medium disclosed by the invention comprises the following components in parts by volume. The preparation method of the cell freezing medium disclosed by the invention comprises the following step of uniformly mixing the components in proportion. The invention also discloses an application of the cell cryopreservation liquid in cryopreservation of cells. The cell cryopreservation liquid disclosed by the invention has good cryoprotection capability, high cell survival rate, high safety and high cryopreserved cell density, cells cryopreserved by adopting the cryopreservation liquid can be used without cleaning, and the osteogenesis and cartilage formation capability of mesenchymal stem cells can be enhanced.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to a cell freezing solution and a preparation method and application thereof. Background Art

[0002] Cell freezing solution is a solution used to protect cells for long-term storage under low temperature conditions. It ensures that cells can maintain a high survival rate after thawing by preventing damage to the cell membrane caused by ice crystal formation. The main components of cell freezing solution usually include basal culture medium, cryoprotectant, serum or serum substitutes and other auxiliary components. Dimethyl sulfoxide (DMSO), as the most commonly used cryoprotectant, can lower the freezing point of the solution and reduce the formation of ice crystals, thereby reducing the mechanical damage of ice crystals to the cell membrane. However, higher concentrations of DMSO are toxic to certain cells and will affect the survival rate and function of cells.

[0003] In addition, when cells are thawed and used after freezing, they usually need to be washed to remove the cryoprotectant and other components in the freezing solution that may be harmful to the cells, but there are risks of complex operations, possible cell loss, and introduction of new contamination.

[0004] Therefore, providing a cell freezing solution which not only has good cryoprotection ability and high cell survival rate, but also is highly safe, and the frozen cells can be used without washing, has become an urgent problem to be solved by those skilled in the art. Summary of the invention

[0005] One of the purposes of the present invention is to provide a cell freezing solution with good cryoprotection ability, high cell survival rate and high safety, and the frozen cells can be used without washing.

[0006] The second object of the present invention is to provide a method for preparing the cell freezing solution.

[0007] The third object of the present invention is to provide an application of the cell freezing solution.

[0008] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0009] In a first aspect, the present invention provides a cell freezing solution, which comprises the following components by volume: 8-12 parts of human albumin, 35-45 parts of sodium chloride injection, 45-55 portions of CS10 cell cryopreservation solution.

[0010] In some embodiments of the present invention, the cell freezing solution comprises the following components by volume: 9-11 parts of human albumin, 37-43 parts of sodium chloride injection, 47-52 copies of CS10 cell cryopreservation solution.

[0011] In some embodiments of the present invention, the cell freezing solution comprises the following components by volume: 10 parts of human albumin, 40 parts of sodium chloride injection, 50 copies of CS10 cell cryopreservation solution.

[0012] In some embodiments of the present invention, the specification of the human albumin is 20wt%; the sodium chloride content in the sodium chloride injection is 9wt%.

[0013] In a second aspect, the present invention discloses a method for preparing a cell freezing solution, comprising the following steps: taking human albumin, sodium chloride injection and CS10 cell freezing solution is obtained by mixing evenly according to the proportion.

[0014] In a third aspect, the present invention discloses an application of the above-mentioned cell freezing solution, wherein the application is application in freezing cells.

[0015] In some embodiments of the present invention, the application is application in cryopreservation of stem cells.

[0016] In some embodiments of the present invention, the stem cells include dental follicle mesenchymal stem cells (DFSCs), dental pulp mesenchymal stem cells, adipose mesenchymal stem cells and umbilical cord mesenchymal stem cells.

[0017] In some embodiments of the present invention, the cell density during freezing is 1×10 7 ~7×10 7 Pieces / mL.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The cell cryopreservation solution of the present invention has good cryoprotective ability, high cell survival rate, high safety, high cryopreserved cell density, and the cells cryopreserved by the cryopreservation solution can be used without washing. In addition, the applicant surprisingly found that the cryopreservation solution of the present invention can not only maintain the multidirectional differentiation potential of mesenchymal stem cells, but also enhance the osteogenic and chondrogenic abilities of mesenchymal stem cells compared with fresh cells and commercial cryopreservation solution groups.

[0020] The cryopreservation solution and the cryopreservation solution mixed with cells of the present invention will not cause severe inflammatory response in animals, have high safety, and can promote periodontal tissue regeneration, and can be used without cleaning. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Attached Figure 1 The figure is the result of investigating the number of living cells and cell survival rate in Experimental Example 1;

[0022] Attached Figure 2 This is a graph showing the results of investigating the effective cell recovery rate in Experimental Example 1;

[0023] Attached Figure 3 The figure is the result of investigating the number of living cells and the cell survival rate of Experimental Example 2;

[0024] Attached Figure 4 This is a graph showing the results of investigating the proliferation of DFSCs in Experimental Example 4;

[0025] Attached Figure 5 The results of the investigation of the scratch healing of DFSCs in Experimental Example 4 are shown in Figure A, where Figure A is a micrograph of the scratch healing of each group, and Figure B is a statistical result of the scratch healing of each group;

[0026] Attached Figure 6 The β-galactosidase staining result diagram of DFSCs in Experimental Example 4, wherein Figure A is the staining result diagram of each group, and Figure B is the statistical result diagram of the staining situation of each group;

[0027] Attached Figure 7 The results of the cloning of DFSCs in Experimental Example 4 are shown in Figure A, where Figure A is a result of cloning in each group, the lower figure in Figure A is a partial enlarged view of the upper figure, and Figure B is a statistical result of the number of cell clones in each group;

[0028] Attached Figure 8 This is a graph showing the results of investigating the expression levels of Oct3 / 4, P16 and P21 of the effective surviving cells in Experimental Example 5;

[0029] Attached Fig. 9 The results of alizarin red staining for osteogenic differentiation potential investigation in Experimental Example 6 are shown, wherein Figure A is a macroscopic and microscopic image of alizarin red staining after 21 days of osteogenic differentiation induction, and Figure B is a quantitative result of alizarin red staining after 21 days of osteogenic differentiation induction;

[0030] Attached Fig.10 The results of RT-PCR analysis of osteogenic differentiation potential in Experimental Example 6 are shown in Figure A, where Figure A shows the expression of early osteogenic differentiation-related genes in cells collected from each group after 7 days of osteogenic differentiation induction and RT-PCR analysis, Figure B shows the expression of mid- and late-stage osteogenic differentiation-related genes in cells collected from each group after 14 days of osteogenic differentiation induction and RT-PCR analysis, and Figure C shows the expression of mid- and late-stage osteogenic differentiation-related genes in cells collected from each group after 21 days of osteogenic differentiation induction and RT-PCR analysis;

[0031] Attached Fig.11 The ALP color development results of the osteogenic differentiation potential investigation of Experimental Example 6, wherein Figure A is a macroscopic and microscopic ALP color development image after 3 days of osteogenic differentiation induction, Figure B is a semi-quantitative analysis image of ALP color development after 3 days of osteogenic differentiation induction, Figure C is a macroscopic and microscopic ALP color development image after 7 days of osteogenic differentiation induction, and Figure D is a semi-quantitative analysis image of ALP color development after 7 days of osteogenic differentiation induction;

[0032] Attached Fig.12 Figures are the results of the adipogenic differentiation potential investigation of Experimental Example 6, wherein Figure A is a micrograph of Oil Red O staining of each group after 21 days of adipogenic differentiation induction culture, Figure B is a graph of the expression of adipogenic differentiation-related genes analyzed by RT-PCR by collecting cells from each group after 7 days of adipogenic induction, and Figure C is a graph of the expression of adipogenic differentiation-related genes analyzed by RT-PCR by collecting cells from each group after 21 days of adipogenic induction;

[0033] Attached Fig.13 These are the results of the investigation of the chondrogenic differentiation potential of Experimental Example 6, wherein Figure A shows the gross appearance of the chondrogenic spheres formed after 28 days of chondrogenic differentiation induction culture of each component and the micrographs of the sections stained with Alcian blue, Figure B shows the expression of chondrogenic-related genes in each group of cells collected for 7 days after chondrogenic induction and analyzed by RT-PCR, and Figure C shows the expression of chondrogenic-related genes in each group of cells collected for 21 days after chondrogenic induction and analyzed by RT-PCR. DETAILED DESCRIPTION

[0034] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0035] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in the field can be used. The reagents and other instruments used without specifying the manufacturer are all conventional reagent products that can be purchased on the market.

[0036] The dental follicle mesenchymal stem cells described in the embodiment of the present invention are human dental follicle mesenchymal stem cells MSC, which are derived from the China Center for Type Culture Collection, with a preservation number of CCTCC NO: C2024384, a preservation time of December 6, 2024, and a preservation address of No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0037] The dental pulp mesenchymal stem cells described in the embodiments of the present invention are obtained by the following method:

[0038] a) Rinse clinically discarded wisdom teeth repeatedly with saline to remove blood, dirt and impurities on the surface;

[0039] b) On a sterile operating table, use surgical forceps or other tools to carefully open the pulp cavity of the tooth and expose the pulp tissue.

[0040] c) Use sterile micro forceps to remove the pulp tissue intact from the pulp cavity and place it in a sterile culture dish containing physiological saline.

[0041] d) The cleaned pulp tissue was transferred to a 15 mL centrifuge tube after being chopped up, and 2 mL of collagenase I with a final concentration of 0.25% was added, and the tube was placed in a constant temperature oscillator at 37° C. and 150 rpm for digestion for 30 minutes.

[0042] e) After digestion is complete, add complete medium twice the volume of the digestion solution to terminate digestion and obtain a tissue and cell suspension;

[0043] f) Transfer the tissue and cell suspension to a centrifuge tube, centrifuge at 400 g for 5 minutes, and discard the supernatant;

[0044] g) Resuspend the precipitate with complete medium and inoculate it evenly in a culture bottle, place it in a 37°C, 5% CO2 incubator, and completely replace the medium every three days;

[0045] h) When the primary cell confluence reached 80%, the cells were digested with recombinant trypsin and centrifuged to collect the cells. 2 , inoculated in a culture bottle;

[0046] i) When the number reaches 90%, a new round of digestion, harvesting and passage is performed, and the above steps are repeated to obtain P5 dental pulp mesenchymal stem cells.

[0047] The adipose-derived mesenchymal stem cells described in the embodiments of the present invention are obtained by the following method:

[0048] a) Place clinically discarded adipose tissue in a sterile container and repeatedly rinse with saline or buffer to remove blood, tissue debris and other impurities until the rinse fluid becomes clear;

[0049] b) The washed adipose tissue was transferred to a 50 mL centrifuge tube, and collagenase I with a final concentration of 0.25% was added, and the tube was placed in a constant temperature oscillator at 37° C. and 150 rpm for digestion for 30 minutes.

[0050] c) After digestion is completed, complete medium with a volume twice that of the digestion solution is added to terminate the digestion, and the digested cell suspension is filtered through a cell sieve to remove undigested tissue blocks and larger fragments to obtain a single cell suspension;

[0051] d) Transfer the single cell suspension to a centrifuge tube, centrifuge at 400 g for 5 minutes, and discard the supernatant;

[0052] e) resuspend the cell pellet with complete medium and inoculate the culture flask evenly, place the flask in a 37°C, 5% CO2 incubator, and completely replace the medium every three days;

[0053] f) When the primary cell confluence reached 80%, the cells were digested with recombinant trypsin and centrifuged to collect the cells.2 , inoculated in a culture bottle;

[0054] g) When the number of cells reaches 90%, a new round of digestion, harvesting and passage is performed, and the above steps are repeated to obtain P5 adipose-derived mesenchymal stem cells. The umbilical cord mesenchymal stem cells described in the embodiment of the present invention are obtained by the following method:

[0055] a) Clean the clinically discarded umbilical cord and cut it into 2-3 cm segments, cut the umbilical cord along the vein with sterile surgical scissors, remove the artery and vein with sterile forceps, and peel off the Wharton's jelly tissue;

[0056] b) Use sterile ophthalmic scissors to cut the Wharton's jelly of the umbilical cord into a minced meat shape;

[0057] c) Mix 10 mL of complete medium evenly for each gram of minced meat tissue, inoculate evenly in a culture bottle, place in a 37°C 5% CO2 incubator, and completely change the medium every three days. When the primary cell confluence reaches 40%, digest with recombinant trypsin, centrifuge and collect the cells, and count them at 5000 cells / cm 2 , inoculated in a culture bottle;

[0058] d) When the cell proliferation rate reaches 90%, a new round of digestion, harvesting and passage is performed, and the above steps are repeated to obtain P5 umbilical cord mesenchymal stem cells.

[0059] The subculture medium described in the embodiment of the present invention is prepared by the following method: 1.0 mL of vitamin C injection (2 mL: 0.5 g) is mixed evenly with 500 mL of serum-free cell culture additive to obtain a mixed solution; 25 mL of the mixed solution is mixed evenly with 500 mL of MEMα culture medium to obtain a subculture medium; the serum-free cell culture additive is UltraGRO TM -Advanced; In some embodiments of the present invention, the MEMα medium contains nucleosides but does not contain phenol red.

[0060] The embodiments of the present invention CS10 is provided by BioLife Solutions, Inc. CS10 cell cryopreservation medium.

[0061] Example 1

[0062] This embodiment discloses the composition and preparation method of the cell freezing solution of the present invention. The cell freezing solution in this embodiment is composed of the following components: human albumin 10v%, sodium chloride injection 40v%, CS10 cell freezing solution 50v%.

[0063] The preparation method is as follows: human serum albumin, sodium chloride injection, CS10 cell freezing solution is obtained by mixing evenly according to the proportion.

[0064] Example 2

[0065] This embodiment discloses the composition and preparation method of the cell freezing solution of the present invention. The cell freezing solution in this embodiment is composed of the following components: human albumin 9v%, sodium chloride injection 41v%, CS10 cell freezing solution 50v%.

[0066] The preparation method is as follows: human serum albumin, sodium chloride injection, CS10 cell freezing solution is obtained by mixing evenly according to the proportion.

[0067] Example 3

[0068] This embodiment discloses the composition and preparation method of the cell freezing solution of the present invention. The cell freezing solution in this embodiment is composed of the following components: human albumin 11v%, sodium chloride injection 37v%, CS10 cell cryopreservation solution 52v%.

[0069] The preparation method is as follows: human serum albumin, sodium chloride injection, CS10 cell freezing solution is obtained by mixing evenly according to the proportion.

[0070] Example 4

[0071] This embodiment discloses the composition and preparation method of the cell freezing solution of the present invention. The cell freezing solution in this embodiment is composed of the following components: human albumin 12v%, sodium chloride injection 43v%, CS10 cell freezing solution 45v%.

[0072] The preparation method is as follows: human serum albumin, sodium chloride injection, CS10 cell freezing solution is obtained by mixing evenly according to the proportion.

[0073] Example 5

[0074] This embodiment discloses the composition and preparation method of the cell freezing solution of the present invention. The cell freezing solution in this embodiment is composed of the following components: human albumin 8v%, sodium chloride injection 45v%, CS10 cell cryopreservation solution 47v%.

[0075] The preparation method is as follows: human serum albumin, sodium chloride injection, CS10 cell freezing solution is obtained by mixing evenly according to the proportion.

[0076] Test Example 1

[0077] This test example investigates the freezing effects of different freezing solutions on cells. The cell freezing solutions used in this test example are shown in Table 1. The preparation methods of each freezing solution are: taking the formula raw materials respectively, mixing them evenly according to the proportion.

[0078] Table 1 Cryopreservation solution formulas of Experimental Example 1

[0079]

[0080] 1. The dental follicle stem cells were cryopreserved for one month using different cell cryopreservation solutions in Table 1, and then the dental follicle stem cells were revived to calculate the number of living cells and cell viability.

[0081] Specifically, after the dental follicle stem cells were digested and counted, different freezing solutions in Table 1 were used at 5.00×10 6 The samples were packaged in the specifications of individual cells / branch. After programmed cooling, the dental follicle stem cells were frozen in vapor phase liquid nitrogen (-176°C) for one month, and then revived (taken out, placed in a water bath preheated at 37°C, and incubated for 1 minute 40 seconds to 2 minutes), sampled for counting, and the cell viability was calculated. The programmed cooling steps are: wait for the temperature to drop to 4°C; cool down at 1.0°C / min until the sample probe temperature reaches -4°C; cool down at 25.0°C / min until the chamber probe temperature reaches -40°C; cool down at 10.0°C / min until the chamber probe temperature reaches -12°C; cool down at 1.0°C / min until the chamber probe temperature reaches -40°C; cool down at 10.0°C / min until the chamber probe temperature reaches -90°C±5°C.

[0082] The results are shown in Table 2 and Figure 1 shown.

[0083] Table 2 Results of viable cell count and cell survival rate in Experimental Example 1

[0084] Experimental Group Control group 1 Control group 2 Control group 3 Control group 4 Number of living cells (10E7) 4.49±0.85 4.38±0.47 4.49±0.28 3.44±0.33* 3.68±0.54 Cell viability (%) 90.98±1.64 90.81±0.67 91.41±1.01 61.94±5.72* 77.06±1.98*

[0085] Table 2 and Figure 1 The * in the figure indicates that there is a significant difference compared with the experimental group at p≤0.05.

[0086] The results showed that there was a significant difference in the number of resuscitated cells between the experimental group and control group 3; there was a significant difference in cell viability between the experimental group and control groups 3 and 4.

[0087] 2. Resuspend the revived dental follicle stem cells in the culture medium at 2.00 × 10 6 pcs / 75cm 2 Nunc TM EasYFlask TMIn the cell culture bottle, 5% CO2, 37℃ culture for 20 hours, digest and count, record the number of effective resuscitated cells, and calculate the effective cell recovery rate. The calculation formula for the effective cell recovery rate is:

[0088] Cell effective recovery rate (%) = number of effectively recovered cells / number of inoculated cells × 100%.

[0089] The results are shown in Table 3 and Appendix Figure 2 shown.

[0090] Table 3 Results of investigation on effective cell recovery rate of Experimental Example 1

[0091]

[0092] Table 3 and Appendix Figure 2 * indicates p≤0.05 compared with the experimental group, ** indicates p≤0.01 compared with the experimental group, and *** indicates p≤0.001 compared with the experimental group.

[0093] The above results showed that the number of effectively recovered cells and the effective cell recovery rate in the experimental group were better than those in the control groups, and the differences were statistically significant.

[0094] Test Example 2

[0095] This test example compares the freezing effect of the freezing solution of the present invention with that of the freezing solution containing 5% DMSO.

[0096] In this test example, the cryopreservation solution of the present invention is the cryopreservation solution of the experimental group in Table 1. The cryopreservation solution and the cryopreservation solution containing 5% DMSO were used to cryopreserve dental follicle stem cells for one month according to the method of Test Example 1, and then the dental follicle stem cells were revived to calculate the number of living cells and cell viability. The results are shown in Table 4 and Attached Figure 3 shown.

[0097] The preparation method of the cryopreservation solution containing 5% DMSO in this test example is as follows: 1.0 mL of vitamin C injection (2 mL: 0.5 g) and 500 mL of serum-free cell culture supplement (UltraGRO TM -Advanced) to obtain a mixed solution; 25 mL of the mixed solution was mixed evenly with 500 mL of MEMα culture medium (containing nucleosides but not phenol red), and then DMSO was added and mixed evenly to make its volume content 5%, thereby obtaining a freezing solution containing 5% DMSO.

[0098] Table 4 Results of viable cell count and cell survival rate in Experimental Example 2

[0099] Experimental Group Freezing medium containing 5% DMSO Cell number (10E7) 4.91±0.18 4.48±0.09* Cell viability (%) 91.05±0.47 86.85±1.85*

[0100] Table 4 and Appendix Figure 3In the table, * indicates p≤0.05% compared with the experimental group, which is statistically different.

[0101] The above results show that the viability and number of cells revived after cryopreservation in the experimental group of the present invention are superior to those in the cryopreservation solution containing 5% DMSO, and the difference is statistically significant.

[0102] Test Example 3

[0103] This test example investigates the freezing effect of the freezing solution of the present invention on different types of cells and different cell densities. The freezing solution used in this test is prepared according to the method of Example 1.

[0104] Dental follicle mesenchymal stem cells, dental pulp mesenchymal stem cells, adipose mesenchymal stem cells and umbilical cord mesenchymal stem cells were obtained and expanded to a certain amount, and the cells were harvested; 1×10 7 / mL, 3×10 7 / mL, 5×10 7 / mL, 7×10 7 Cryopreserved cells were frozen at a density of 1 mL / mL and a specification of 1 mL / vial. After one month of freezing in liquid nitrogen, the cells were taken out for revival and counted, and the cell viability and viable cell density were calculated to calculate the cell recovery rate. The cell recovery operation was the same as in Experiment 1. The calculation formula for the cell recovery rate was: the number of viable cells after revival / the number of cells before freezing × 100%

[0105] Table 5 Cell protection and recovery results of different types and densities of cryopreservation solutions of the present invention

[0106]

[0107] The above results show that the cryoprotectant of the present invention has a viability of more than 90% after resuscitation of dental follicle mesenchymal stem cells, umbilical cord mesenchymal stem cells, dental pulp mesenchymal stem cells and adipose mesenchymal stem cells, and a cell recovery rate of more than 85%. There is no significant difference in the viability and recovery rate between different densities, indicating that the cryoprotectant of the present invention can effectively protect a variety of mesenchymal stem cell types at a high density.

[0108] Test Example 4

[0109] This test example investigates the effect of the cryopreservation solution of the present invention on maintaining the basic biological characteristics of mesenchymal stem cells. The cryopreservation solution used in this test was prepared according to the method of Example 1, with dental follicle mesenchymal stem cells as the object of investigation, and a commercial cryopreservation solution was used as a control. The commercial cryopreservation solution described in this test example was provided by BioLife Solutions, Inc., USA. CS10 cell cryopreservation medium. 1. Comparison of the proliferation of P5 generation DFSCs in each group

[0110] 1.1 Experimental Grouping: P4 generation DFSCs and P5 generation DFSCs were respectively taken and cryopreserved for 1 month using the cryopreservation solution of Example 1 according to the method of Experimental Example 1. P5 generation DFSCs were also taken and cryopreserved for 1 month using the commercial cryopreservation solution according to the method of Experimental Example 1.

[0111] Fresh dental follicle stem cells group (DFSCs-F): P4 generation cryopreserved DFSCs of the present invention were revived and centrifuged according to the method of Example 1, resuspended in the passage medium, and centrifuged at 5000 / cm 2 The cells were inoculated into T75 cells at a density of 2.00 × 10 and cultured in an incubator at 37°C and 5% CO2 for 96 h. The cells were collected by digestion and centrifugation, resuspended in subculture medium, and cultured again at 2.00 × 10 6 pcs / 75cm 2 The cells were inoculated at a density of 100 μg / mL and inoculated into new T75 cells, placed in a 37°C 5% CO2 incubator, and allowed to adhere within 12-24 hours. The cells were harvested after digestion and centrifugation and recorded as DFSCs-F.

[0112] The P5 generation cryopreserved DFSCs of the present invention and the P5 generation commercial cryopreserved DFSCs were directly revived, washed and centrifuged, resuspended in the subculture medium, and then diluted to 2.00×10 6 pcs / 75cm 2 The cells were inoculated at a density of 100 μg / mL into T75 and adhered to the wall for 12 h to 24 h. The cells were then digested and centrifuged to collect the cells. Among them, the P5 generation cryopreservation solution DFSCs of the present invention was recorded as DFSCs-C1, and the P5 generation cryopreservation commercial cryopreservation solution DFSCs was recorded as DFSCs-C2.

[0113] 1.2 DFSCs-C1, DFSCs-C2 and fresh dental follicle stem cells (DFSCs-F) were subjected to proliferation experiments at the same time. The details are as follows:

[0114] 100ul of each group of cell suspension resuspended in the subculture medium was added to each well of the 96-well plate, 2000 cells per well, and cultured in an incubator at 37°C and 5% CO2. The medium was replaced every 2 days. The proliferation was detected using the CCK8 kit, the absorbance was detected at 450nm, and statistical analysis was performed using GraphPad Prism statistical software.

[0115] The results are attached Figure 4 The results showed that the CCK8 proliferation experiment showed that the OD value of the DFSCs group (DFSCs-C1) of the present invention was better than that of the other two groups on the 5th and 6th days, and the difference was statistically significant. There was no significant statistical difference among the three groups at other time points.

[0116] 2. Comparison of scratch healing of P5 DFSCs in each group

[0117] 2.1 Experimental grouping: the same as the experimental grouping under the item “Comparison of proliferation of P5 generation DFSCs in each group” in this experimental example.

[0118] 2.2 The cells in each group were cultured at a cell density of 5x10 5 Each well was inoculated with 100 cells / well in a 12-well plate. When the cells were fully grown, a 1mL pipette tip was used to draw a line perpendicular to the well plate, and the old culture medium was discarded. The cells were gently rinsed two or three times with PBS until the cells were clean, and fresh culture medium was added. A photo was taken at 0h under a microscope as a control. After culturing in a 37℃, 5% CO2 incubator for 13h, the cells were taken out, the width of the scratch at the same position was observed under a microscope, and photos were taken. ImageJ software was used to analyze the migration area of ​​the scratch experiment. The results are shown in the attached figure. Figure 5 The results showed that 13 hours after scratching, there was no significant difference in the migration area of ​​DFSCs among the fresh dental follicle stem cells group (DFSCs-F), the DFSCs group of the present invention (DFSCs-C1), and the DFSCs group of the commercialized cryopreservation solution (DFSCs-C2), and the scratch test results showed no significant statistical difference.

[0119] 3. β-galactosidase staining results of P5 DFSCs in each group

[0120] 3.1 Experimental grouping: the same as the experimental grouping under the item “Comparison of proliferation of P5 generation DFSCs in each group” in this experimental example.

[0121] 3.2 Cells in each group were plated at 5000 cells / cm 2 Inoculate into a 6-well plate and add subculture medium to culture until the cell confluence reaches 70%. Aspirate the medium, gently rinse the cells 2-3 times with PBS buffer, then add an appropriate amount of fixative 4% paraformaldehyde and fix at room temperature for 15-30 minutes. After fixation, rinse with PBS 3 times, 5 minutes each time. Perform β-galactosidase staining according to the instructions of the β-galactosidase staining kit (Beijing Solebold Technology Co., Ltd., Cat. No. G1580), observe the cell staining under a microscope, β-galactosidase positive cells (senescent cells) will appear blue, and the number and distribution of blue cells can be analyzed and counted using GraphPad Prism statistical software.

[0122] The results are attached Figure 6 The results showed that there was no significant statistical difference in the results of β-galactosidase staining among the fresh dental follicle stem cells group (DFSCs-F), the DFSCs group (DFSCs-C1) of the present invention, and the DFSCs group (DFSCs-C2) of the commercialized DFSCs group.

[0123] 4. Comparison of clone formation of P5 DFSCs in each group

[0124] 4.1 Experimental grouping: the same as the experimental grouping under the item “Comparison of proliferation of P5 generation DFSCs in each group” in this experimental example.

[0125] 4.2 Inoculate cells at 500 cells / 60 mm culture dish, add 3 mL of subculture medium, and culture at 37°C, 5% CO2. Terminate the culture after clone colonies are formed (about 12 days, the number of cells in each colony is >50), fix with 4% paraformaldehyde for 15 min, and observe colony formation by crystal violet staining.

[0126] The results are attached Figure 7 As shown. The results showed that the cloning statistical results showed that the cloning ability of the cryopreservation solution DFSCs group (DFSCs-C1) of the present invention was better than that of the fresh dental follicle stem cell group (DFSCs-F) and the commercial cryopreservation solution DFSCs group (DFSCs-C2), and the difference was statistically significant. The above results show that the cryopreservation solution of the present invention can maintain the basic biological characteristics of mesenchymal stem cells, such as proliferation, migration, cloning, cell senescence, etc.

[0127] Test Example 5

[0128] This test example investigates the effect of the cryopreservation solution of the present invention on the expression of Oct3 / 4, P16 and P21 in the effective surviving cells after the resuscitation of mesenchymal stem cells. The cryopreservation solution used in this test was prepared according to the method of Example 1, with dental follicle mesenchymal stem cells as the object of investigation, and a commercial cryopreservation solution as a control.

[0129] 1. Experimental Grouping

[0130] P5 DFSCs were taken and cryopreserved for 1 month using the cryopreservation solution of Example 1 and the commercial cryopreservation solution according to the method of Experimental Example 1. The two P5 frozen dental follicle stem cells were taken out from liquid nitrogen, directly revived and cultured, and digested and collected within 24 hours. The specific method was the same as that of Experimental Example 4.

[0131] The collected cells were used for polymerase chain reaction (RT-PCR), wherein the P5 generation cryopreservation solution DFSCs group of the present invention was recorded as DFSCs-C1, and the P5 generation cryopreservation solution DFSCs group of commercial cryopreservation solution was recorded as DFSCs-C2.

[0132] Fresh P5 dental follicle stem cells were directly subcultured and digested and collected within 24 hours for RT-PCR, which were recorded as DFSCs-F.

[0133] 2. RT-PCR detection The P5 dental follicle stem cells of each group were subjected to real-time polymerase chain reaction (RT-PCR) to detect the expression of Oct3 / 4, P16 and P21. The detection methods of Oct3 / 4, P16 and P21 are existing technologies. The total RNA was extracted using an RNA extraction kit, and the reverse transcription kit was used for reverse transcription of RNA. The specific operations were carried out according to the instructions of the kit.

[0134] The results are attached Figure 8 The results showed that there were certain differences in the expression of cell cycle-related genes P16 and P21 and multidirectional differentiation potential-related transcription factors Oct3 / 4 among the three groups, especially in the cells that effectively survived in the two groups of frozen dental follicle stem cell injections, the expression of Oct3 / 4 was significantly upregulated, and the expression of P16 and P21 was reduced, indicating that these effectively surviving cells may be a group of younger stem cells with stronger multidirectional differentiation potential. The cryopreservation solution of the present invention can enhance the self-renewal and differentiation potential of dental follicle stem cells.

[0135] Figure 8 In the table, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns indicates no significant difference.

[0136] Test Example 6

[0137] This test example investigates the multidirectional differentiation potential of the cryopreservation solution of the present invention on the effective surviving cells after the resuscitation of mesenchymal stem cells. The cryopreservation solution used in this experiment was prepared according to the method of Example 1, with dental follicle mesenchymal stem cells as the object of investigation, and a commercial cryopreservation solution was used as a control. The human-related stem cell osteogenic induction differentiation kit (item number HUXXC-90021), human-related stem cell adipogenic induction differentiation kit (item number HUXXC-90031) and human-related stem cell chondrogenic induction differentiation kit (item number HUXXC-90041) described in this experiment are all provided by Saiye (Guangzhou) Biotechnology Co., Ltd.; the alkaline phosphatase colorimetric kit (item number C3250S) is provided by Shanghai Biyuntian Biotechnology Co., Ltd.

[0138] 1. Experimental Grouping

[0139] P5 DFSCs were taken and cryopreserved for 1 month using the cryopreservation solution of Example 1 and the commercial cryopreservation solution according to the method of Experimental Example 1. The two P5 frozen dental follicle stem cells were taken out from liquid nitrogen, directly revived and cultured, and digested and collected within 24 hours. The specific method was the same as that of Experimental Example 4.

[0140] The collected cells were used to investigate their multidirectional differentiation potential, wherein the P5 generation DFSCs group cryopreserved in the present invention was designated as DFSCs-C1, and the P5 generation DFSCs group cryopreserved in commercial cryopreserved in the commercial cryopreserved solution was designated as DFSCs-C2.

[0141] Fresh P5 dental follicle stem cells were directly subcultured and digested within 24 hours for investigation of multidirectional differentiation potential, which were recorded as DFSCs-F.

[0142] 2. Investigation of multidirectional differentiation potential

[0143] 2.1 Investigation of osteogenic differentiation potential

[0144] Prepare osteoinduction differentiation medium according to the instructions of the human stem cell osteoinduction differentiation kit, store at 4°C, and use within one month. 4 viable cells / cm 2 The cells were seeded in a 12-well plate at a density of 1 mL of culture medium per well, and the plates were cultured in an incubator at 37°C, 5% CO2, and saturated humidity. When the cell confluence reached 70%, 1 mL of osteogenic differentiation induction medium was replaced, and fresh osteogenic differentiation induction medium was used every 3 days. After 7, 14, and 21 days of induction, the cells in each group were collected for real-time polymerase chain reaction (RT-PCR) to detect the expression of osteogenic-related genes (ALP, RUNX2, OCN, BSP). In addition, the samples collected after 3, 7, and 21 days of induction were fixed with 4% paraformaldehyde for 15 minutes, and the 3- and 7-day samples were stained with an alkaline phosphatase colorimetric kit to evaluate the alkaline phosphatase activity or the 21-day samples were stained with Alizarin Red to observe calcium nodules. The results are shown in the attached figure. Fig. 9 ~Attached Fig.11 The results showed that the frozen dental follicle stem cells (DFSCs-C1) prepared by the freezing solution of the present invention had excellent osteogenic differentiation ability, and a large number of calcium nodules were produced after 21 days of osteogenic induction culture, and Alizarin red staining was strongly positive ( Fig. 9 A, B). RT-PCR results showed that at 7 days of osteogenic induction, the expression of early osteogenic-related genes ALP and RUNX2 in the frozen dental follicle stem cells (DFSCs-C1) prepared by the freezing solution of the present invention was better than that in the other two groups; at 14 days and 21 days of osteogenic induction, the expression of mid- and late-stage osteogenic-related genes OCN and BSP in the frozen dental follicle stem cells (DFSCs-C1 group) prepared by the freezing solution of the present invention was better than that in the other two groups, and the differences were statistically significant ( Fig.10 In addition, the ALP staining results showed that the ALP activity of the frozen dental follicle stem cells (DFSCs-C1) prepared by the freezing solution of the present invention was superior to that of the other two groups ( Fig.11A~D), and the difference is statistically significant. This shows that the cryopreservation solution of the present invention can enhance the osteogenic differentiation ability of dental follicle stem cells, and is superior to the frozen dental follicle stem cells prepared by the same method using the commercial cryopreservation solution.

[0145] Figures 9 to 11 In the table, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns indicates no significant difference.

[0146] 2.2 Investigation of adipogenic differentiation potential

[0147] According to the instructions of the human stem cell adipogenic differentiation kit, the adipogenic differentiation medium A and B were prepared and stored at 4°C. They were used within one month. 4 viable cells / cm 2 The cells were inoculated in a 12-well plate at a density of 1 mL of SLCPCA complete medium to each well, and the plates were cultured in an incubator at 37°C, 5% CO2, and saturated humidity. When the cell confluence reached 100%, 1 mL of adipogenic differentiation induction medium A was replaced. After 3 days of induction, the medium was replaced with adipogenic differentiation induction medium B. After 1 day of induction, the medium B was removed and replaced with medium A for induction. The medium A and medium B were used alternately. After 7 and 21 days of induction, the cells in each group were collected for real-time polymerase chain reaction (RT-PCR) to detect the expression of adipogenic-related genes (Adiponectin, PPARγ). In addition, the samples collected 21 days after induction were fixed with 4% paraformaldehyde for 15 minutes, and the formation of lipid droplets was observed by Oil Red O staining. The results are shown in the attached figure. Fig.12 The results showed that the expression of adipogenic related genes Adiponectin and PPARγ in dental follicle stem cells (DFSCs-C1) prepared by the cryopreservation solution of the present invention was lower than that in the other two groups ( Fig.12 B, C), indicating that the adipogenic differentiation ability of dental follicle stem cells (DFSCs-C1) prepared by the cryopreservation solution of the present invention is reduced, but it can still retain a good adipogenic differentiation ability to a certain extent. After 21 days of adipogenic induction, obvious lipid droplets can be seen in the cytoplasm, and Oil Red O staining is positive ( Fig.12 A).

[0148] Fig.12 **p<0.01, ***p<0.001, ns indicates no significant difference.

[0149] 2.3 Investigation of chondrogenic differentiation potential

[0150] Prepare chondrogenic differentiation premix and complete medium according to the instructions of the human stem cell chondrogenic differentiation kit, store at 4°C, and use within one month. 5 The P5 generation dental follicle stem cells of each group to be induced that survived effectively were transferred to a 15 mL centrifuge tube, centrifuged at 20°C, 250 g for 4 min, and the supernatant was removed; 0.5 mL of chondrogenic differentiation premix was added for resuspending, and the cells were centrifuged at 20°C, 150 g for 5 min; the steps were repeated to wash the cells again; 0.5 mL of chondrogenic differentiation complete medium was added for resuspending, and the cells were centrifuged at 20°C, 150 g for 5 min; the cap of the centrifuge tube was loosened to facilitate gas exchange, and the tube was placed upright in an incubator at 37°C, 5% CO2, and saturated humidity for culture; after 24 hours, the cells began to aggregate, and the bottom of the centrifuge tube was gently tapped to suspend the chondrocytes in the liquid. Every 2 to 3 days, 0.5 mL of fresh chondrogenic differentiation complete medium was used, and the bottom of the tube was gently tapped after each change of the medium. After 7 and 21 days of induction, the cells of each group were collected for real-time polymerase chain reaction (RT-PCR) to detect the expression of chondrogenic-related genes (YWHAZ, ACAN, MIA, COL10A1). In addition, continuous induction for 28 days can form cartilage balls with a diameter of about 1 mm, which were fixed with 4% paraformaldehyde for 30 min, dehydrated with gradient alcohol, embedded in paraffin, and sliced ​​into 4 μm sections and stained with Alcian blue for observation. The results are shown in the attached figure. Fig.13 As shown. The results showed that at 7 days of induction, the expression of chondrogenic related genes YWHAZ, ACAN, MIA, and COL10A1 in the frozen dental follicle stem cells (DFSCs-C1) prepared by the freezing solution of the present invention was significantly higher than that in the fresh cell group, among which the expression of the MIA gene was significantly better than that in the commercial preparation group (DFSCs-C2). There was no significant difference among the three at 21 days of induction; it shows that the freezing solution of the present invention can enhance the chondrogenic differentiation ability of dental follicle stem cells at an early stage. After 28 days of chondrogenic induction culture, all three can form smooth chondrocytes, which are blue in color after Alecin blue staining, indicating the internal acidic mucopolysaccharide in the cartilage tissue. Fig.13 In the table, *p<0.05, ***p<0.001, ns indicates no significant difference.

[0151] Finally, it should be noted that the above embodiments are only preferred embodiments of the present invention to illustrate the technical solution of the present invention, but not to limit it, and certainly not to limit the patent scope of the present invention. Any changes or modifications that are made to the main design concept and spirit of the present invention without any substantial meaning, and the technical problems they solve are still consistent with the present invention, should be included in the protection scope of the present invention; in addition, the direct or indirect application of the technical solution of the present invention in other related technical fields is also included in the patent protection scope of the present invention.

Claims

1. A cell freezing solution, characterized in that: The composition comprises the following components by volume: 8-12 parts of human albumin, 35-45 parts of sodium chloride injection, 45-55 portions of CS10 cell cryopreservation solution.

2. A cell freezing solution according to claim 1, characterized in that: The composition comprises the following components by volume: 9-11 parts of human albumin, 37-43 parts of sodium chloride injection, 47-52 copies of CS10 cell cryopreservation solution.

3. A cell freezing solution according to claim 1, characterized in that: The composition includes the following components by volume: 10 parts of human albumin, 40 parts of sodium chloride injection, 50 copies of CS10 cell cryopreservation solution.

4. A cell freezing solution according to any one of claims 1 to 3, characterized in that: The specification of human albumin is 20wt%; the sodium chloride content in the sodium chloride injection is 9wt%.

5. A method for preparing a cell freezing solution according to any one of claims 1 to 4, characterized in that: Take human albumin, sodium chloride injection and CS10 cell freezing solution is obtained by mixing evenly according to the proportion.

6. The use of the cell freezing solution according to any one of claims 1 to 4, characterized in that: The application is application in cryopreserved cells.

7. The use according to claim 6, characterized in that: The application is application in cryopreservation of stem cells.

8. The use according to claim 7, characterized in that: The stem cells include dental follicle mesenchymal stem cells (DFSCs), dental pulp mesenchymal stem cells, adipose mesenchymal stem cells and umbilical cord mesenchymal stem cells.

9. The use according to any one of claims 6 to 8, characterized in that: The cell density when freezing cells was 1×10 7 ~7×10 7 Pieces / mL.

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