Adipose mesenchymal stem cell cryopreservation protection solution and cryopreservation method

Abstract

The present invention belongs to the field of cell culture technology, and specifically relates to a cryopreservation protection solution and a cryopreservation method for adipose mesenchymal stem cells. The cryopreservation protection solution for adipose mesenchymal stem cells includes a basal liquid, a nutrient agent and an antifreeze agent; the basal liquid is: a culture medium; the nutrient agent is: transferrin, linoleic acid, cholesterol; the antifreeze agent is: hydroxyethyl starch, bergenia lactone, geraniol. The cryopreservation protection solution provided by the present invention adds a nutrient agent and an antifreeze agent to the basal liquid culture medium, especially the added antifreeze agents hydroxyethyl starch, bergenia lactone, and geraniol, which can effectively reduce the damage caused to cells during the cryopreservation process, improve the activity of frozen cells after recovery, and effectively ensure that the adipose mesenchymal stem cells after cryopreservation can still be used in scientific research or clinical practice. The present invention also provides a cryopreservation method for adipose mesenchymal stem cells, which is simple in process and easy to operate.

Description

Technical Field

[0001] The present invention belongs to the technical field of stem cells, and in particular relates to a cryopreservation protective solution and a cryopreservation method for adipose mesenchymal stem cells. Background Art

[0002] Mesenchymal stem cells are a type of multipotent stem cells that have all the common properties of stem cells, namely, self-renewal and multidirectional differentiation capabilities. Mesenchymal stem cells can be isolated from umbilical cord blood, bone marrow, and fat, and have unlimited replication potential and the ability to differentiate into mature mesodermal tissue cells in vitro. Obtaining mesenchymal stem cells from adipose tissue is a source of adult stem cells.

[0003] Adipose-derived mesenchymal stem cells have strong self-renewal ability and multidirectional differentiation potential. They proliferate quickly in vitro, have a low death rate, and have strong differentiation ability. Adipose-derived mesenchymal stem cells are less restricted by ethics and are the most promising regenerative source stem cells for transplantation. Human adipose-derived mesenchymal stem cells have been successfully induced to differentiate into mesodermal cells such as osteoblasts, chondrocytes, skeletal muscle cells, and liver cells under different conditions. They can also differentiate into other germ layers. The potential of adipose-derived mesenchymal stem cells for clinical treatment and regenerative medicine has been widely concerned by stem cell researchers. Therefore, the preservation of adipose-derived mesenchymal stem cells is of great significance.

[0004] At present, in order to prolong the storage time of adipose-derived mesenchymal stem cells, adipose-derived mesenchymal stem cells are mostly stored in liquid nitrogen after adding cryopreservation fluid. Dimethyl sulfoxide and fetal bovine serum albumin are added to the cryopreservation fluid to reduce the damage to the cells during the cryopreservation process. However, dimethyl sulfoxide has certain cytotoxicity and is not conducive to cell recovery. 1,2-propylene glycol and glycerol are also used to replace dimethyl sulfoxide and added to the cryopreservation fluid, but the preservation effect is not as good as dimethyl sulfoxide, and the cell activity after recovery is not ideal. Therefore, it is necessary to improve the cryopreservation fluid of adipose-derived mesenchymal stem cells to improve the preservation effect of adipose-derived mesenchymal stem cells. Summary of the invention

[0005] In order to solve the above problems, the purpose of the present invention is to provide a cryopreservation protective solution for adipose-derived mesenchymal stem cells, which does not contain DMSO and animal serum and has a good cryopreservation effect.

[0006] Another object of the present invention is to provide a method for cryopreservation of adipose-derived mesenchymal stem cells, which uses the cryopreservation protection solution provided by the present invention and is easy to operate.

[0007] In order to achieve the above object, the first technical solution adopted by the present invention is:

[0008] A cryopreservation protective solution for adipose mesenchymal stem cells comprises a base solution, a nutrient agent and an antifreeze agent; the base solution is a culture medium; the nutrient agent is transferrin, linoleic acid and cholesterol; and the antifreeze agent is hydroxyethyl starch, bergenia lactone and geraniol.

[0009] Furthermore, the culture medium is DMEM / F12 culture medium.

[0010] Furthermore, the final concentrations of the nutrients in the culture medium are: transferrin 5.5-10 μg / mL, linoleic acid 0.1-0.5 μg / mL, and cholesterol 1.5-5.5 μg / mL.

[0011] Furthermore, the final concentrations of the antifreeze agents in the culture medium are: 4.5-8.0 μg / mL of hydroxyethyl starch, 0.5-1.0 μg / mL of bergenia lactone, and 2.5-7.5 μg / mL of geraniol.

[0012] In order to achieve the above object, the second technical solution adopted by the present invention is:

[0013] A method for freezing adipose-derived mesenchymal stem cells comprises freezing adipose-derived mesenchymal stem cells using the above-mentioned cryopreservation protection solution.

[0014] Specifically, the freezing method of adipose-derived mesenchymal stem cells comprises resuspending the adipose-derived mesenchymal stem cells in a cryopreservation protection solution and then dispensing them into cryopreservation tubes, placing them in a programmed cooling device for cooling, and the cooling process is as follows: cooling to 4°C at 1-2°C / min, then cooling to -15°C at 15-20°C / min, then cooling to -40°C at 2-3°C / min, and finally cooling to -80°C at 10-15°C / min, and transferring them to liquid nitrogen after storing them for 2 hours.

[0015] Preferably, the density of adipose-derived mesenchymal stem cells in the cryopreservation protection solution is: 1-8×10 7 Pieces / mL.

[0016] Preferably, the adipose-derived mesenchymal stem cells are P1-P9 generation adipose-derived mesenchymal stem cells.

[0017] The method of the present invention has the following advantages: the present invention provides a cryopreservation protection solution for adipose mesenchymal stem cells, wherein nutrients and antifreeze agents are added to a basal liquid culture medium, and in particular, the added antifreeze agents hydroxyethyl starch, bergenia lactone, and geraniol can effectively reduce the damage to cells caused by the cryopreservation process, improve the activity of the frozen cells after recovery, and effectively ensure that the adipose mesenchymal stem cells after cryopreservation can still be used in scientific research or clinical practice. The cryopreservation protection solution of the present invention does not add DMSO and animal serum, and the components are safe.

[0018] The present invention also provides a method for freezing adipose-derived mesenchymal stem cells, which has a simple process and is easy to operate. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The morphological observation results of the adherence of adipose-derived mesenchymal stem cells at generation 3;

[0020] Figure 2 The morphological observation results of the adherence of adipose-derived mesenchymal stem cells at generation 4;

[0021] Figure 3 The morphological observation results of the adherence of adipose-derived mesenchymal stem cells at generation 5;

[0022] Figure 4 The adhesion rates of adipose-derived mesenchymal stem cells frozen for 6 months and 12 months in Example 1 and Comparative Examples 1 to 5 are shown. DETAILED DESCRIPTION

[0023] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto. The biological materials used in the following examples can all be obtained from commercial channels.

[0024] The preparation method of adipose mesenchymal stem cells used in Examples 1-3 is as follows: aseptically take the fat extract from liposuction surgery, repeatedly rinse with PBS containing penicillin and streptomycin to remove residual blood. Add an equal volume of preheated 0.5% type I collagenase to the washed fat tissue, put it in a 37°C constant temperature shaker, digest it at a shaking speed of 80 r / min for 1 hour, centrifuge it at 400×g for 10 minutes at 4°C, discard the upper liquid and incompletely digested fat tissue, resuspend the precipitate product with complete culture medium, filter it into a new centrifuge tube with a 200-mesh cell sieve, centrifuge it at 400×g for 10 minutes at 4°C, discard the supernatant, suspend it with DMEM / F12 culture medium containing 10% FBS by volume, inoculate it in a 10 cm culture dish, and culture it in a 37°C, 5% CO2 incubator; after 48 hours, aspirate the suspended cell liquid and replace it with new culture medium; change the medium once every 3 days, and subculture it when the cells reach a confluence of more than 80% after 7 days of culture. The P3, P4, and P5 cells obtained by subculture were taken and the adhesion and growth of adipose-derived mesenchymal stem cells were observed using a phase contrast microscope and photographed. Figure 1-3 shown.

[0025] Figure 1-3 The morphology of adipose-derived mesenchymal stem cells at generations P3, P4, and P5, respectively. Figure 1-3 It can be seen that adipose mesenchymal cells grow in a long spindle shape, have good polarity, smooth contours, and good three-dimensional sense. The above results indicate that the morphology of adipose mesenchymal stem cells extracted and separated by the present invention conforms to the long spindle shape characteristics of mesenchymal stem cells.

[0026] Example 1

[0027] A cryopreservation protection solution for adipose mesenchymal stem cells, comprising a base solution, a nutrient agent and an antifreeze agent; the base solution is: DMEM / F12 culture medium; the nutrient agent is: transferrin, linoleic acid, cholesterol; the antifreeze agent is: hydroxyethyl starch, bergenia lactone, geraniol;

[0028] The final concentrations of the above nutrients in the culture medium are: transferrin 5.5 μg / mL, linoleic acid 0.1 μg / mL, cholesterol 1.5 μg / mL;

[0029] The final concentrations of the above antifreeze agents in the culture medium are: 4.5 μg / mL hydroxyethyl starch, 0.5 μg / mL bergenia lactone, and 2.5 μg / mL geraniol.

[0030] A method for cryopreservation of adipose-derived mesenchymal stem cells, comprising resuspending P3 adipose-derived mesenchymal stem cells in a cryopreservation protection solution, wherein the density of the adipose-derived mesenchymal stem cells in the cryopreservation protection solution is: 1×10 7 / mL, dispense into cryopreservation tubes, and put into a programmed cooling instrument for cooling. The cooling process is as follows: cool to 4°C at 1°C / min, then cool to -15°C at 15°C / min, then cool to -40°C at 2°C / min, and finally cool to -80°C at 10°C / min. After storing for 2 hours, transfer to liquid nitrogen.

[0031] Example 2

[0032] A cryopreservation protection solution for adipose mesenchymal stem cells, comprising a base solution, a nutrient agent and an antifreeze agent; the base solution is: DMEM / F12 culture medium; the nutrient agent is: transferrin, linoleic acid, cholesterol; the antifreeze agent is: hydroxyethyl starch, bergenia lactone, geraniol;

[0033] The final concentrations of the above nutrients in the culture medium are: transferrin 8.5 μg / mL, linoleic acid 0.3 μg / mL, cholesterol 3.5 μg / mL;

[0034] The final concentrations of the above antifreeze agents in the culture medium are: 6 μg / mL hydroxyethyl starch, 0.8 μg / mL bergenia lactone, and 5 μg / mL geraniol.

[0035] A method for cryopreservation of adipose-derived mesenchymal stem cells, comprising resuspending P4 adipose-derived mesenchymal stem cells in a cryopreservation protection solution, wherein the density of the adipose-derived mesenchymal stem cells in the cryopreservation protection solution is: 5×10 7 / mL, dispense into cryopreservation tubes, and put into a programmed cooling instrument for cooling. The cooling process is as follows: cool to 4°C at 2°C / min, then cool to -15°C at 20°C / min, then cool to -40°C at 2°C / min, and finally cool to -80°C at 15°C / min. After storing for 2 hours, transfer to liquid nitrogen.

[0036] Example 3

[0037] A cryopreservation protection solution for adipose mesenchymal stem cells, comprising a base solution, a nutrient agent and an antifreeze agent; the base solution is: DMEM / F12 culture medium; the nutrient agent is: transferrin, linoleic acid, cholesterol; the antifreeze agent is: hydroxyethyl starch, bergenia lactone, geraniol;

[0038] The final concentrations of the above nutrients in the culture medium are: transferrin 10 μg / mL, linoleic acid 0.5 μg / mL, cholesterol 5.5 μg / mL;

[0039] The final concentrations of the above antifreeze agents in the culture medium are: hydroxyethyl starch 8.0 μg / mL, bergenia lactone 1.0 μg / mL, and geraniol 7.5 μg / mL.

[0040] A method for cryopreservation of adipose-derived mesenchymal stem cells, comprising resuspending P5 adipose-derived mesenchymal stem cells in a cryopreservation protection solution, wherein the density of the adipose-derived mesenchymal stem cells in the cryopreservation protection solution is: 8×10 7 / mL, dispense into cryopreservation tubes, and put into a programmed cooling instrument for cooling. The cooling process is as follows: cool to 4°C at 2°C / min, then cool to -15°C at 15°C / min, then cool to -40°C at 3°C / min, and finally cool to -80°C at 10°C / min. After storing for 2 hours, transfer to liquid nitrogen.

[0041] Comparative Example 1

[0042] Comparative Example 1 provides a cryopreservation protection solution and a cryopreservation method for adipose-derived mesenchymal stem cells, which differs from Example 1 in that bergenia lactone is not added to the cryoprotectant, and the rest is the same as Example 1.

[0043] Comparative Example 2

[0044] Comparative Example 2 provides an adipose-derived mesenchymal stem cell cryopreservation protection solution and a cryopreservation method, which differs from Example 1 in that geraniol is not added to the antifreeze agent, and the rest is the same as Example 1.

[0045] Comparative Example 3

[0046] Comparative Example 3 provides a cryopreservation protection solution and a cryopreservation method for adipose-derived mesenchymal stem cells, which differs from Example 1 in that bergenia lactone and geraniol are not added to the cryoprotectant, and the rest is the same as Example 1.

[0047] Comparative Example 4

[0048] Comparative Example 4 provides a cryopreservation protection solution and a cryopreservation method for adipose-derived mesenchymal stem cells, which differs from Example 1 in that geraniol is not added to the antifreeze agent, the dosage of bergenia lactone is adjusted to 3 μg / mL, and the rest is the same as Example 1.

[0049] Comparative Example 5

[0050] Comparative Example 5 provides a cryopreservation protection solution and a cryopreservation method for adipose-derived mesenchymal stem cells, which differs from Example 1 in that: bergenia lactone is not added to the antifreeze agent, the dosage of geraniol is adjusted to 3 μg / mL, and the rest is the same as Example 1.

[0051] Test Example 1

[0052] Detection of cell viability: Before freezing, the survival rates of the adipose-derived mesenchymal stem cells in Examples 1 to 3 were detected by trypan blue staining. After freezing for 6 months and 12 months, samples were taken and placed in a 37°C water bath for resuscitation. Three tubes were taken each time, and the cell viability was detected by trypan blue staining, and the average value was taken. The trypan blue staining process was as follows: the cell suspension was mixed with an equal amount of 0.4% trypan blue, and the number of live cells was detected using a cell counter. Cell viability = number of live cells / total number of cells × 100%. The results are shown in Table 1:

[0053] Table 1

[0054]

[0055] It can be seen from Table 1 that the cell survival rate of the adipose-derived mesenchymal stem cells in Examples 1 to 3 decreases less with the extension of storage time, and the cell survival rate is above 90% after 12 months of storage. In Comparative Examples 1 to 5, one or both of bergenia lactone and geraniol were omitted, and the survival rate of the cells after recovery decreased to varying degrees. It can be seen from Comparative Examples 1 and 2 that after omitting bergenia lactone, the cell survival rate decreases more significantly with the extension of storage time, indicating that bergenia lactone is effective in extending the storage time of cells, and geraniol can reduce the damage to cells during the cooling process. The two components work synergistically to improve the survival rate of adipose-derived mesenchymal stem cells after cryopreservation.

[0056] Test Example 2

[0057] Detection of cell adhesion rate: The cells frozen for 6 months and 12 months in Example 1 and Comparative Examples 1 to 5 were revived in a 37°C water bath, centrifuged to remove the protective solution, and diluted with DMEM / F12 medium to adjust the cell density in the medium to 1×10 4 / mL, take 1mL and inoculate it into the culture plate, culture it for 3h, add TrypLE to digest the cells and then add culture medium to terminate the digestion, remove the supernatant after centrifugation, resuspend it in DMEM / F12 culture medium, use a cell counter to count the cells, count the number of cells after adhesion, and calculate the cell adhesion rate, cell adhesion rate = number of cells after adhesion / 1×10 4 ×100%, the result is Figure 4 shown.

[0058] Depend on Figure 4 It can be seen that the adipose mesenchymal stem cells in Example 1 have a high adhesion rate after cryopreservation, indicating that the cell activity is good. In Comparative Examples 1 to 5, the composition of the cryopreservation protection solution was adjusted, and the cell adhesion rate decreased to varying degrees, indicating that the cell activity was not as good as that of the adipose mesenchymal stem cells in Example 1. It can be seen that the cryopreservation preservation solution provided by the present invention can effectively maintain the activity of adipose mesenchymal stem cells and reduce the damage to the cells caused by the cryopreservation process.

[0059] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them. The basic principles and main features of the present invention have been described above with specific implementation schemes. On the basis of the present invention, some modifications or replacements may be made thereto, but these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of protection claimed by the present invention.

Claims

1. A cryopreservation protective solution for adipose-derived mesenchymal stem cells, characterized in that: It includes a base liquid, a nutrient agent and an antifreeze agent; the base liquid is a culture medium; the nutrient agent is transferrin, linoleic acid, and cholesterol; the antifreeze agent is hydroxyethyl starch, bergenia lactone, and geraniol; The final concentrations of the nutrients in the culture medium are: transferrin 5.5-10 μg / mL, linoleic acid 0.1-0.5 μg / mL, cholesterol 1.5-5.5 μg / mL; The final concentration of the antifreeze agent in the culture medium is: 4.5-8.0 μg / mL of hydroxyethyl starch, 0.5-1.0 μg / mL of bergenia lactone, and 2.5-7.5 μg / mL of geraniol; The culture medium is DMEM / F12 culture medium.

2. A method for cryopreservation of adipose-derived mesenchymal stem cells, characterized in that: Adipose-derived mesenchymal stem cells are cryopreserved using the cryopreservation protection solution described in claim 1.

3. The cryopreservation method of adipose-derived mesenchymal stem cells according to claim 2, characterized in that: The method comprises resuspending the adipose-derived mesenchymal stem cells in cryopreservation protection solution, dividing the suspension into cryopreservation tubes, and placing the tubes into a programmed cooling apparatus for cooling. The cooling process is as follows: cooling to 4°C at 1-2°C / min, then cooling to -15°C at 15-20°C / min, then cooling to -40°C at 2-3°C / min, and finally cooling to -80°C at 10-15°C / min. The tubes are stored for 2 hours and then transferred to liquid nitrogen.

4. The cryopreservation method of adipose-derived mesenchymal stem cells according to claim 2, characterized in that: The density of adipose-derived mesenchymal stem cells in the cryopreservation protection solution is: 1-8×10 7 Pieces / mL.

5. The method for cryopreservation of adipose-derived mesenchymal stem cells according to claim 2, characterized in that: The adipose-derived mesenchymal stem cells are P1-P9 generation adipose-derived mesenchymal stem cells.

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