Cryopreservation liquid for mesenchymal stem cells

Abstract

The invention relates to the technical field of stem cell cryopreservation, and particularly provides a cryopreservation solution for mesenchymal stem cells. The cryopreservation liquid for the mesenchymal stem cells is prepared from the following components in percentage by mass: 17 to 28 percent of traditional Chinese medicine extract, 35 percent of trehalose, 32 percent of ethylene glycol and the balance of adipose-derived mesenchymal stem cell culture medium. The invention aims at a cryopreservation method of directly putting into liquid nitrogen at-196 DEG C; extracellular macromolecular trehalose is used as a non-permeable protective agent, permeable membrane micromolecular ethylene glycol is used as a permeable protective agent, and formation of ice crystals in cells is avoided while cell dehydration is promoted and cell membranes are stabilized through a permeation effect; furthermore, the traditional Chinese medicine extracts are matched, so that the cell viability is improved, and low-temperature damage and possible damage of each protective agent to cells are reduced.

Description

Technical Field

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

[0002] Mesenchymal stem cells are a type of multipotent stem cells derived from the mesoderm, which are widely present in various tissues of the human body. These unique cells have the ability of self-renewal and multidirectional differentiation potential, and can differentiate into various tissue cells under specific conditions, such as osteoblasts, chondrocytes and adipocytes. Adipose-derived mesenchymal stem cells show similar biological characteristics to traditional bone marrow mesenchymal stem cells, but are easier to obtain and expand, and have low immunogenicity, so adipose-derived mesenchymal stem cells have become a hot application direction.

[0003] At present, most of the mesenchymal stem cells used in clinical practice are cryopreserved after in vitro culture and then revived for transplantation. Efficient cryopreservation of mesenchymal stem cells is a necessary condition for maintaining cell activity during transplantation. Cryopreservation technology is often used to store cells in -196°C liquid nitrogen at low temperatures. Generally, gradient cooling is used, but the process is cumbersome and requires instruments such as programmed cooling devices, which is not conducive to the promotion of mesenchymal stem cell cryopreservation technology. The new vitrification freezing technology achieves the simple operation of mixing mesenchymal stem cells with cryopreservation fluid and directly immersing them in liquid nitrogen through the synergistic effect of permeable and non-permeable protective agents. However, there is still much room for improvement in the activity of cells after resuscitation.

[0004] Therefore, it is of great practical significance to provide a cell freezing solution that has high activity of adipose-derived mesenchymal stem cells after thawing. Summary of the invention

[0005] In order to solve the above problems, the present invention aims to provide a cryopreservation solution for mesenchymal stem cells.

[0006] The present invention provides a cryopreservation solution for mesenchymal stem cells, wherein the cryopreservation solution for mesenchymal stem cells is composed of the following components: 17-28% by weight of a Chinese herbal medicine extract, 35% by weight of trehalose, 32% by weight of ethylene glycol, and the balance of adipose-derived mesenchymal stem cell culture medium; The extraction method of the Chinese medicine extract is: Take 13-21% of epimedium, 11-18% of safflower, 8-16% of rhodiola rosea, 8-15% of salvia miltiorrhiza, 6-12% of coptis chinensis and make up the rest of the mass fraction with pure water, cut the epimedium, safflower, rhodiola rosea, salvia miltiorrhiza and coptis chinensis into pieces and pass through a 20-mesh sieve, add them into a hammer mill with a rotation speed of 6000 r / min and a discharge fineness of 200 mesh at a feeding rate of 10 kg / h, and grind them until fine materials are obtained, add 85% ethanol with a mass of 4 times the mass of the fine materials, immerse them at 35°C for 24 hours, and then filter them through 400 mesh to obtain an extract; distill the extract and the pure water at 69°C and a vacuum degree of 800 mbar under reduced pressure until no liquid flows out continuously, collect the remaining liquid and filter it through a 0.22 μm film to obtain the Chinese medicine extract.

[0007] Furthermore, the mass fraction of the epimedium is 18%.

[0008] Furthermore, the mass fraction of safflower is 15%.

[0009] Furthermore, the mass fraction of the Rhodiola rosea is 13%.

[0010] Furthermore, the mass fraction of the Salvia miltiorrhiza is 12%.

[0011] Furthermore, the mass fraction of the Coptis chinensis is 9%.

[0012] Furthermore, the mass fraction of the traditional Chinese medicine extract is 23%.

[0013] The present invention can bring the following beneficial effects: The present invention is directed to a freezing method of directly placing cells into -196°C liquid nitrogen; using extracellular macromolecular trehalose as a non-permeable protective agent and membrane-permeable small molecule ethylene glycol as a permeable protective agent, promoting cell dehydration and stabilizing cell membranes through an osmotic effect while avoiding formation of ice crystals in cells; further using Chinese herbal extracts to improve cell activity, reduce low-temperature damage and possible damage to cells by various protective agents.

[0014] Epimedium promotes cell proliferation, safflower protects cell membrane, and rhodiola improves cell metabolism. Epimedium, safflower and rhodiola work together to protect cell activity and repair cell damage; Salvia miltiorrhiza and Coptis chinensis have the effects of antioxidant damage, maintaining the stability of the intracellular environment and inhibiting cell apoptosis. The two work together to reduce low temperature damage and possible damage to cells by various protective agents; Epimedium, safflower, rhodiola, Salvia miltiorrhiza and Coptis chinensis work together to improve cell activity, reduce low temperature damage and possible damage to cells by various protective agents.

[0015] Trehalose, ethylene glycol and Chinese herbal extracts are combined to form a unique antifreeze-protection system of the present invention, which effectively prevents the formation of ice crystals and can also protect the integrity and function of cells to the greatest extent, thereby improving cell activity. DETAILED DESCRIPTION

[0016] In order to more clearly explain the overall concept of the present invention, the overall scheme of the present invention is described in detail in the form of embodiments below; in the following description, a large number of specific details are given to provide a more thorough understanding of the present invention; however, it is obvious to those skilled in the art that the present invention can be implemented without one or more of these details; in other examples, in order to avoid confusion with the present invention, some technical features known in the art are not described.

[0017] In the present invention: trehalose was purchased from Jiangsu Yanke Bioengineering Co., Ltd., with a molecular weight of 378.3 and a CAS number of 6138-23-4; ethylene glycol was purchased from Shandong Longyu Chemical Co., Ltd., with a CAS number of 107-21-1; adipose-derived mesenchymal stem cell culture medium, adipose-derived mesenchymal stem cells and umbilical cord mesenchymal stem cells were all purchased from Hefei Wanwu Biological Co., Ltd., with the item numbers of Delf-16278, Delf-16277 (human primary) and Delf-10770 (human primary), respectively; epimedium, safflower, rhodiola rosea, salvia miltiorrhiza and coptis chinensis were all purchased from Yuan'an County Rutanghui Trading Co., Ltd., with a water content of 5%. The above-mentioned traditional Chinese medicines all meet the standards of the "Quality Management Standard for the Production of Chinese Medicinal Materials" and the "Chinese Pharmacopoeia"; a hammer mill was purchased from Guangzhou Xiangming Machinery Equipment Co., Ltd., model DX-20; a vacuum distiller was purchased from Zhengzhou Yarong Instrument Co., Ltd., model RE-2000E; and a TST-BLGL membrane filter was used for membrane filtration.

[0018] Unless otherwise specified, all raw material components in the following examples can be purchased through commercial channels, the experimental instruments used are conventional laboratory experimental instruments, and the performance testing methods are the testing methods known in the art.

[0019] The preferred implementation is as follows: Embodiment 1: The cryopreservation solution for mesenchymal stem cells was prepared by the following method: Extraction of Chinese herbal medicine extracts: 18% of epimedium, 15% of safflower, 13% of rhodiola rosea, 12% of salvia miltiorrhiza, 9% of coptis chinensis and the rest of the mass fraction of pure water are taken, and the epimedium, safflower, rhodiola rosea, salvia miltiorrhiza and coptis chinensis are chopped and passed through a 20-mesh sieve, and then added into a hammer mill with a rotation speed of 6000r / min and a discharge fineness of 200 mesh at a feeding rate of 10kg / h to obtain fine materials, and 85% ethanol with a mass of 4 times the mass of the fine materials is added, immersed at 35°C for 24h, and then filtered through 400 mesh to obtain an extract; the extract and the pure water are distilled under reduced pressure at 69°C and a vacuum degree of 800mbar until no liquid flows out continuously, and the remaining liquid is collected and filtered through a 0.22μm film to obtain the Chinese herbal medicine extract.

[0020] 23% by weight of a Chinese herbal medicine extract, 35% by weight of trehalose, 32% by weight of ethylene glycol, and adipose-derived mesenchymal stem cell culture medium are mixed evenly to obtain a cryopreservation solution for mesenchymal stem cells; Embodiment 2-13: The only difference between Example 2 and Example 1 is that the mass fraction of Epimedium is 13%; The only difference between Example 3 and Example 1 is that the mass fraction of Epimedium is 21%; The only difference between Example 4 and Example 1 is that the mass fraction of safflower is 11%; The only difference between Example 5 and Example 1 is that the mass fraction of safflower is 18%; The only difference between Example 6 and Example 1 is that the mass fraction of Rhodiola rosea is 8%; The only difference between Example 7 and Example 1 is that the mass fraction of Rhodiola rosea is 16%; The only difference between Example 8 and Example 1 is that the mass fraction of Salvia miltiorrhiza is 8%; The only difference between Example 9 and Example 1 is that the mass fraction of Salvia miltiorrhiza is 15%; The only difference between Example 10 and Example 1 is that the mass fraction of Coptis chinensis is 6%; The only difference between Example 11 and Example 1 is that the mass fraction of Coptis chinensis is 12%; The only difference between Example 12 and Example 1 is that the mass fraction of the Chinese herbal medicine extract is 17%; The only difference between Example 13 and Example 1 is that the mass fraction of the Chinese herbal medicine extract is 28%.

[0021] Comparative Examples 1-22: The difference between Comparative Example 1 and Example 1 is that the mass fraction of Epimedium is 6%; The difference between Comparative Example 2 and Example 1 is that the mass fraction of Epimedium is 35%; The only difference between Comparative Example 3 and Example 1 is that the mass fraction of safflower is 5%; The only difference between Comparative Example 4 and Example 1 is that the mass fraction of safflower is 32%; The only difference between Comparative Example 5 and Example 1 is that the mass fraction of Rhodiola rosea is 4%; The only difference between Comparative Example 6 and Example 1 is that the mass fraction of Rhodiola rosea is 30%; The only difference between Comparative Example 7 and Example 1 is that the mass fraction of Salvia miltiorrhiza is 4%; The only difference between Comparative Example 8 and Example 1 is that the mass fraction of Salvia miltiorrhiza is 30%; The only difference between Comparative Example 9 and Example 1 is that the mass fraction of Coptis chinensis is 3%; The difference between Comparative Example 10 and Example 1 is that the mass fraction of Coptis chinensis is 25%; The only difference between Comparative Example 11 and Example 1 is that the mass fraction of the Chinese herbal medicine extract is 8%; The only difference between Comparative Example 12 and Example 1 is that the mass fraction of the Chinese herbal medicine extract is 33%; The difference between Comparative Example 13 and Example 1 is that the temperature of vacuum distillation is 55°C; The difference between Comparative Example 14 and Example 1 is that the temperature of vacuum distillation is 85°C; The difference between Comparative Example 15 and Example 1 is that Epimedium is replaced by Carthamus tinctorius, Rhodiola rosea, Salvia miltiorrhiza, and Coptis chinensis of the same mass ratio as that of Example 1; The difference between Comparative Example 16 and Example 1 is that safflower is replaced with epimedium, rhodiola rosea, salvia miltiorrhiza, and coptis chinensis of the same mass ratio as that of Example 1; The difference between Comparative Example 17 and Example 1 is that Rhodiola rosea is replaced with Epimedium, Carthamus tinctorius, Salvia miltiorrhiza and Coptis chinensis of the same mass ratio as that of Example 1; The difference between Comparative Example 18 and Example 1 is that Salvia miltiorrhiza is replaced by Epimedium, Carthamus tinctorius, Rhodiola rosea and Coptis chinensis of the same mass ratio as that of Example 1; The difference between Comparative Example 19 and Example 1 is that Coptis chinensis is replaced with Epimedium, Carthamus tinctorius, Rhodiola rosea and Salvia miltiorrhiza in the same mass ratio as that in Example 1; The difference between Comparative Example 20 and Example 1 is that the Chinese herbal medicine extract is replaced with pure water of equal mass; The only difference between Comparative Example 21 and Example 1 is that the cryopreservation solution for mesenchymal stem cells is replaced with pure water of equal mass; The only difference between Comparative Example 22 and Example 1 is that the later experiments were conducted on umbilical cord mesenchymal stem cells Delf-10770.

[0022] Adipose-derived mesenchymal stem cells were tested using survival rate and proliferation ability as cell activity standards. Two groups of mesenchymal stem cell cryopreservation solution samples numbered 1 and 2 were prepared for each example, and each group contained three samples. Adipose-derived mesenchymal stem cells were mixed with each cryopreservation solution sample for mesenchymal stem cells at 25°C to obtain each cryopreservation system. The final number density of adipose-derived mesenchymal stem cells in each cryopreservation system was 6×10 5 / mL; transfer each cryopreservation system into 5ml cryopreservation tubes, let stand for 20min, then transfer each cryopreservation tube into -196℃ liquid nitrogen to complete the cryopreservation, and resuscitate and prepare for testing after 30 days; Survival rate test: Take out the cryopreservation tube of group 1 of each example, suck out the cell suspension with a pipette, add it to a centrifuge tube and add more than 10 times of adipose-derived mesenchymal stem cell culture medium, mix well; centrifuge at 1000rpm for 5 minutes; discard the supernatant, add adipose-derived mesenchymal stem cell culture medium to resuspend the cells, add 2 drops of trypan blue dye for every 1ml of cell suspension, dye at room temperature for 4 minutes, take a drop of cell suspension from the stained cell material and place it on a glass slide, add a coverslip and observe under a high-power microscope; dead cells are light blue and swollen, dull; living cells are not colored and maintain normal morphology and are shiny; count the number of live and dead cells, and calculate the cell viability according to the formula: cell viability (%) = number of unstained cells / total number of observed cells × 100%.

[0023] Proliferation capacity test: Take out the cryopreserved tubes of group 2 of each example, resuscitate them, add them to the centrifuge tubes and add 10 times more adipose-derived mesenchymal stem cell culture medium, mix well; centrifuge at 1000 rpm for 5 min, and rinse with PBS buffer to remove the cryopreservation solution. Resuspend and dilute at 1×10 3 The cells were seeded in a 96-well plate at a density of 100 cells / well, and an appropriate amount of adipose-derived mesenchymal stem cell culture medium was added to allow them to adhere to the plate and grow. The time for the cell proliferation rate to reach 1.5 was calculated up to 200 h.

[0024] The survival rate and proliferation ability test results of each example are shown in Table 1. In Table 1, the survival rate test result is the average of the results of Group 1 of each example, and the proliferation ability test result is the average of the results of Group 2 of each example, and both are retained to the integer.

[0025] Table 1: Test results of survival rate and proliferation ability of each example:

[0026] From the data in Table 1, it can be seen that compared with other examples, the cell survival rate of the adipose-derived mesenchymal stem cells of the present invention, especially Example 1, is higher and the time for the cell proliferation rate to reach 1.5 is shorter. The survival rate and proliferation ability of the adipose-derived mesenchymal stem cells after cryopreservation and resuscitation of the present invention, especially Example 1, are higher, and further, the cell activity is also higher. It should be noted that Comparative Example 22, the present invention is a preferred experiment for adipose-derived mesenchymal stem cells, and the embodiments of the present invention have better effects on adipose-derived mesenchymal stem cells than other types of stem cells.

[0027] The above description is only an embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modification, equivalent substitution, improvement, etc. made within the spirit and principle of the present invention should be included in the scope of the claims of the present invention.

Claims

1. A cryopreservation solution for mesenchymal stem cells, characterized in that: The cryopreservation solution for mesenchymal stem cells consists of the following components: 17-28% by weight of a Chinese herbal medicine extract, 35% by weight of trehalose, 32% by weight of ethylene glycol, and the balance of adipose-derived mesenchymal stem cell culture medium; The extraction method of the Chinese medicine extract is: Take 13-21% of epimedium, 11-18% of safflower, 8-16% of rhodiola rosea, 8-15% of salvia miltiorrhiza, 6-12% of coptis chinensis and make up the rest of the mass fraction with pure water, cut the epimedium, safflower, rhodiola rosea, salvia miltiorrhiza and coptis chinensis into pieces and pass through a 20-mesh sieve, add them into a hammer mill with a rotation speed of 6000 r / min and a discharge fineness of 200 mesh at a feeding rate of 10 kg / h, and grind them until fine materials are obtained, add 85% ethanol with a mass of 4 times the mass of the fine materials, immerse them at 35°C for 24 hours, and then filter them through 400 mesh to obtain an extract; distill the extract and the pure water at 69°C and a vacuum degree of 800 mbar under reduced pressure until no liquid flows out continuously, collect the remaining liquid and filter it through a 0.22 μm film to obtain the Chinese medicine extract.

2. The cryopreservation solution for mesenchymal stem cells according to claim 1, characterized in that: The mass fraction of the epimedium is 18%.

3. The cryopreservation solution for mesenchymal stem cells according to claim 1, characterized in that: The mass fraction of the safflower is 15%.

4. The cryopreservation solution for mesenchymal stem cells according to claim 1, characterized in that: The mass fraction of the Rhodiola rosea is 13%.

5. The cryopreservation solution for mesenchymal stem cells according to claim 1, characterized in that: The mass fraction of the salvia miltiorrhiza is 12%.

6. The cryopreservation solution for mesenchymal stem cells according to claim 1, characterized in that: The mass fraction of the coptis root is 9%.

7. The cryopreservation solution for mesenchymal stem cells according to claim 1, characterized in that: The mass fraction of the traditional Chinese medicine extract is 23%.

Images