Adipose-derived mesenchymal stem cell cryopreservation solution and application thereof

Abstract

The present invention belongs to the field of biotechnology, and specifically relates to a kind of adipose mesenchymal stem cell cryopreservation solution and its application. The above-mentioned adipose mesenchymal stem cell cryopreservation solution is composed of a culture medium and a cryoprotectant, and the composition of the cryoprotectant is: Exendin-4, sofalcone, rosmarinic acid, glycerol, and the weight ratio of each component of Exendin-4, sofalcone, rosmarinic acid, and glycerol in the cryoprotectant is 1:0.8~1.7:0.4~1.2:50~70. The present invention has been found through experiments that Exendin-4, sofalcone, and rosmarinic acid can make adipose mesenchymal stem cells have good viability, adhesion rate and proliferation ability after cryopreservation and recovery.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to an adipose mesenchymal stem cell cryopreservation solution and an application thereof. Background Art

[0002] Mesenchymal stem cells are a branch of the stem cell field. They are easy to separate and prepare, have multiple differentiation potentials, support hematopoiesis, promote hematopoietic stem cell engraftment, and regulate the body's immune response. Therefore, they have attracted much attention in the stem cell field. Currently, mesenchymal stem cells can be separated and prepared from fat, bone marrow, umbilical cord, etc. Among them, adipose-derived mesenchymal stem cells have broad application prospects in fields such as tissue engineering due to their rich sources and content, easy cultivation, safe transplantation, and no ethical disputes.

[0003] Liquid nitrogen cryopreservation is one of the commonly used technical means for preserving adipose-derived mesenchymal stem cells in clinical practice. It uses the characteristic that the metabolic activity of cells is almost stagnant under extremely low temperature conditions to achieve the purpose of long-term cell preservation. However, under extremely low temperature conditions, the water in cells and tissues is very easy to form ice crystals, which may cause cell dehydration, protein denaturation, and disorder of the internal spatial structure of cells. If there are too many ice crystals, cell death may also occur. Therefore, the use of cryoprotectants is crucial. At present, the protective agents used usually contain dimethyl sulfoxide, serum protein, etc. Dimethyl sulfoxide is a good cell cryopreservation protective agent, but it is also a reagent that is very toxic to cells and is greatly limited in dosage. Serum protein, due to its animal origin, greatly increases the possibility of cells being infected by viruses and is expensive. In view of the above problems, it is urgent to improve the composition of adipose-derived mesenchymal stem cell cryopreservation solution. Summary of the invention

[0004] The first object of the present invention is to provide a fat-derived mesenchymal stem cell cryopreservation solution, which has simple ingredients and can improve the viability, adhesion rate and proliferation ability of the adipose-derived mesenchymal stem cells after cryopreservation and resuscitation.

[0005] The second objective of the present invention is to provide an application of adipose-derived mesenchymal stem cell cryopreservation solution.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is:

[0007] A fat mesenchymal stem cell cryopreservation solution is composed of a culture medium and a cryoprotectant. The cryoprotectant is composed of Exendin-4, sofalcone, rosmarinic acid and glycerol. The weight ratio of Exendin-4, sofalcone, rosmarinic acid and glycerol in the cryoprotectant is 1:0.8-1.7:0.4-1.2:50-70.

[0008] Furthermore, the adipose-derived mesenchymal stem cell cryopreservation solution is composed of the following parts by weight of culture medium and cryoprotectant: 120-150 parts of culture medium and 5-20 parts of cryoprotectant.

[0009] Furthermore, the weight ratio of Exendin-4, sofalcone, rosmarinic acid and glycerol in the cryoprotectant is 1:1.1:0.7:62. Furthermore, the culture medium is α-MEM serum-free culture medium.

[0010] The use of the above-mentioned adipose-derived mesenchymal stem cell freezing solution comprises the following steps: mixing adipose-derived mesenchymal stem cells with the above-mentioned adipose-derived mesenchymal stem cell freezing solution to prepare an adipose-derived mesenchymal stem cell suspension, followed by programmed cooling for pre-freezing, and transferring the pre-freezing to liquid nitrogen for storage.

[0011] Furthermore, the density of the adipose-derived mesenchymal stem cells in the adipose-derived mesenchymal stem cell freezing solution is 1-8×10 7 cells / mL.

[0012] Furthermore, the programmed cooling pre-freezing process is specifically: pre-freezing at 2~4°C for 0.5~1h, pre-freezing at -20~-25°C for 1.5-2.5h, and then cooling to -75~-80°C at a rate of 1-5°C / min for pre-freezing for 4-5h.

[0013] Compared with the prior art, the beneficial effects of the present invention are mainly:

[0014] The recovery viability, adhesion rate and proliferation ability of adipose-derived mesenchymal stem cells after cryopreservation using the cryopreservation solution of the present invention were tested and it was found that Exendin-4, sofalcone and rosmarinic acid can improve the viability, adhesion rate and proliferation ability of adipose-derived mesenchymal stem cells after cryopreservation and recovery.

[0015] The cryopreservation method of adipose mesenchymal stem cells provided by the present invention has simple steps and is easy to implement. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The growth status and morphological characteristics of adipose-derived mesenchymal stem cells after resuscitation in Example 1-3;

[0017] Figure 2 The results of the ratio of infection-rejecting cells in adipose-derived mesenchymal stem cells after resuscitation in Example 1-3 and Comparative Example 1-5 are shown in FIG.

[0018] Figure 3 The graph shows the adhesion rate of adipose-derived mesenchymal stem cells after resuscitation in Examples 1-3 and Comparative Examples 1-5. DETAILED DESCRIPTION

[0019] The technical scheme of the present invention is further described below in conjunction with specific embodiments. However, it should be understood by those skilled in the art that the following examples are only used to illustrate the present invention and should not be regarded as limiting the present invention. The specific conditions not specified in the examples are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used, unless otherwise specified, are conventional products obtained through commercial channels.

[0020] 1. Implementation

[0021] Example 1

[0022] Example 1 provides an adipose-derived mesenchymal stem cell cryopreservation solution, which is composed of 131 parts of α-MEM serum-free culture medium and 12 parts of cryoprotectant. The cryoprotectant is composed of Exendin-4, Sofalcone, Rosmarinic acid, and Glycerol, and the weight ratio of Exendin-4, Sofalcone, Rosmarinic acid, and Glycerol in the cryoprotectant is 1:1.1:0.7:62.

[0023] Example 1 also provides an application of adipose-derived mesenchymal stem cell cryopreservation solution, and the specific application process is as follows:

[0024] Obtaining P4 generation adipose-derived mesenchymal stem cells: washing adipose tissue with saline at a volume ratio of 1:1 between adipose tissue and saline, aliquoting the washed adipose tissue into centrifuge tubes, adding 0.25% type I collagenase at a volume ratio of 3:1 between adipose and type I collagenase, digesting for 25 min, obtaining adipose-derived mesenchymal stem cell precipitates after centrifugation, resuspending the adipose-derived mesenchymal stem cell precipitates with complete medium of α-MEM+10% FBS, the density of adipose-derived mesenchymal stem cell precipitates in complete medium of α-MEM+10% FBS is 1×104 cells / mL, culturing at 37°C and 5% CO2 until the cell confluence reaches 90%, digesting with 0.25% trypsin, passage at a ratio of 1:3, obtaining P4 generation adipose-derived mesenchymal stem cells for later use;

[0025] The obtained P4 adipose-derived mesenchymal stem cells were mixed with the above-mentioned adipose-derived mesenchymal stem cell freezing solution, the density of the adipose-derived mesenchymal stem cells in the adipose-derived mesenchymal stem cell freezing solution was 1×107 cells / mL, and an adipose-derived mesenchymal stem cell suspension was prepared. The adipose-derived mesenchymal stem cell suspension was then dispensed into 2 mL cryopreservation tubes, pre-frozen at 3°C ​​for 1 h, pre-frozen at -22°C for 2 h, and then cooled to -78°C at a rate of 2°C / min for pre-freezing for 4.2 h. After the pre-freezing was completed, it was transferred to liquid nitrogen for storage.

[0026] Example 2

[0027] Example 2 provides an adipose-derived mesenchymal stem cell cryopreservation solution, which is composed of 120 parts of α-MEM serum-free culture medium and 20 parts of cryoprotectant. The cryoprotectant is composed of Exendin-4, Sofalcone, Rosmarinic acid, and Glycerol, and the weight ratio of Exendin-4, Sofalcone, Rosmarinic acid, and Glycerol in the cryoprotectant is 1:0.8:1.2:70.

[0028] Example 2 also provides an application of adipose-derived mesenchymal stem cell cryopreservation solution, and the specific application process is as follows:

[0029] The process of obtaining P4 adipose-derived mesenchymal stem cells is the same as in Example 1;

[0030] The obtained P4 adipose-derived mesenchymal stem cells were mixed with the above-mentioned adipose-derived mesenchymal stem cell freezing solution, the density of adipose-derived mesenchymal stem cells in the adipose-derived mesenchymal stem cell freezing solution was 3×107 cells / mL, and an adipose-derived mesenchymal stem cell suspension was prepared. The adipose-derived mesenchymal stem cell suspension was dispensed into 2 mL cryopreservation tubes, pre-frozen at 2°C for 1 h, pre-frozen at -20°C for 2.5 h, and then cooled to -75°C at a rate of 1°C / min for pre-freezing for 5 h. After the pre-freezing was completed, it was transferred to liquid nitrogen for storage.

[0031] Example 3

[0032] Example 3 provides an adipose-derived mesenchymal stem cell cryopreservation solution, which is composed of 150 parts of α-MEM serum-free culture medium and 5 parts of cryoprotectant. The cryoprotectant is composed of Exendin-4, Sofalcone, Rosmarinic acid, and Glycerol, and the weight ratio of Exendin-4, Sofalcone, Rosmarinic acid, and Glycerol in the cryoprotectant is 1:1.7:0.4:50.

[0033] Example 3 also provides an application of adipose-derived mesenchymal stem cell cryopreservation solution, and the specific application process is as follows:

[0034] The process of obtaining P4 adipose-derived mesenchymal stem cells is the same as in Example 1;

[0035] The obtained P4 generation adipose-derived mesenchymal stem cells were added to the above-mentioned adipose-derived mesenchymal stem cell freezing solution and mixed. The density of adipose-derived mesenchymal stem cells in the adipose-derived mesenchymal stem cell freezing solution was 6×107 cells / mL, and an adipose-derived mesenchymal stem cell suspension was prepared. The adipose-derived mesenchymal stem cell suspension was divided into 2 mL cryopreservation tubes, pre-frozen at 4°C for 0.5 h, pre-frozen at -25°C for 1.5 h, and then cooled to -80°C at a rate of 5°C / min for pre-freezing for 4 h. After pre-freezing, it was transferred to liquid nitrogen for storage.

[0036] (II) Comparative Example

[0037] Comparative Example 1

[0038] The difference between Comparative Example 1 and Example 1 is that Exendin-4 is missing from the cryoprotectant, and the rest is the same as Example 1.

[0039] Comparative Example 2

[0040] The difference between Comparative Example 2 and Example 1 is that the cryoprotectant lacks sofalcone, and the rest is the same as Example 1.

[0041] Comparative Example 3

[0042] The difference between Comparative Example 3 and Example 1 is that Exendin-4 and Sofalcone are missing from the cryoprotectant, and the rest is the same as Example 1.

[0043] Comparative Example 4

[0044] The difference between Comparative Example 4 and Example 1 is that the cryoprotectant lacks rosmarinic acid, and the rest is the same as Example 1.

[0045] Comparative Example 5

[0046] The difference between Comparative Example 5 and Example 1 is that Exendin-4 and Sofalcone are missing from the cryoprotectant, DMSO with a volume fraction of 10% is added, and the rest is consistent with Example 1.

[0047] (III) Experimental examples

[0048] Experimental Example 1

[0049] The growth state and morphological characteristics of the resuscitated adipose-derived mesenchymal stem cells of Example 1-3 were observed. The detailed experimental process is as follows:

[0050] (1) Resuscitation of adipose-derived mesenchymal stem cells

[0051] Take the cryopreserved tubes stored for 12 months in Examples 1-3, and immediately place the cryopreserved tubes of each experimental group in a 38°C water bath until the cryopreservation solution is completely thawed, then transfer to a 15 mL centrifuge tube, add an equal volume of α-MEM medium, centrifuge at 1000 rpm for 5 min, discard the supernatant, add α-MEM medium to the cell pellet, adjust the concentration of adipose mesenchymal stem cells to 1×104 cells / mL, and obtain the revived adipose mesenchymal stem cells in each group for later use.

[0052] (2) Microscopic examination

[0053] The revived adipose-derived mesenchymal stem cells in each group were placed under a microscope (the magnification of the microscope was 10 times) to observe the growth status and morphological characteristics of the adipose-derived mesenchymal stem cells in each group. The results are shown in the attached figure. Figure 1 shown.

[0054] Figure 1The figure is a diagram showing the growth status and morphological characteristics of adipose-derived mesenchymal stem cells after resuscitation in Example 1-3. Figure 1 AC are the growth status and morphological characteristics of adipose-derived mesenchymal stem cells after resuscitation in Examples 1-3. Figure 1 It can be seen that the adipose-derived mesenchymal stem cells all appear to be elongated and fibrous after being cryopreserved and revived in the cryopreservation solution of the present invention.

[0055] Experimental Example 2

[0056] The viability of the resuscitated adipose-derived mesenchymal stem cells of Examples 1-3 and Comparative Examples 1-5 was tested. The detailed experimental process is as follows:

[0057] (1) Resuscitation of adipose-derived mesenchymal stem cells

[0058] Take the cryopreserved tubes of Examples 1-3 and Comparative Examples 1-5 that were stored for 6 months and 12 months, respectively, and immediately place the cryopreserved tubes of each experimental group in a 38°C water bath until the cryopreserved solution is completely thawed, and then transfer them to a 15 mL centrifuge tube, add an equal volume of α-MEM medium, centrifuge at 1000 rpm for 5 min, discard the supernatant, add α-MEM medium to the cell pellet, adjust the concentration of adipose mesenchymal stem cells to 1×104 cells / mL, and obtain the revived adipose mesenchymal stem cells in each group for later use.

[0059] (2) Trypan blue exclusion method

[0060] The trypan blue exclusion method was used to detect the viability of adipose-derived mesenchymal stem cells after recovery in each group, and the ratio of cells excluding staining was calculated. The ratio of cells excluding staining was used to characterize the viability of cells after recovery. The results are shown in the attached figure. Figure 2 shown.

[0061] Figure 2 The results of the ratio of cells refusing to be infected in the resuscitated adipose-derived mesenchymal stem cells of Examples 1-3 and Comparative Examples 1-5 are shown in FIG. Figure 2 It can be seen that the ratio of infection-resistant cells in the adipose-derived mesenchymal stem cells after resuscitation in Examples 1-3 and Comparative Example 5 is high, and there is no significant difference; the ratio of infection-resistant cells in the adipose-derived mesenchymal stem cells after resuscitation in Comparative Example 3 is the lowest, and the ratio of infection-resistant cells in the adipose-derived mesenchymal stem cells after resuscitation in Comparative Example 2 is the second highest. The above results show that Exendin-4, sofalcone, and rosmarinic acid can make the adipose-derived mesenchymal stem cells have a good viability after cryopreservation and resuscitation.

[0062] Experimental Example 3

[0063] The adhesion rate of resuscitated adipose-derived mesenchymal stem cells of Examples 1-3 and Comparative Examples 1-5 was tested. The detailed experimental process is as follows:

[0064] (1) The recovery of adipose-derived mesenchymal stem cells was the same as in Experimental Example 2.

[0065] (2) Adhesion test

[0066] α-MEM + 10% FBS complete medium was added to a 25 cm2 culture flask in advance. The revived cells of each group were inoculated in a 25 cm2 culture flask with 1 × 106 cells in each flask. The cells were cultured in a 37°C, 5% CO2 incubator for 4 h. The cells were digested with TrypLE enzyme and diluted with α-MEM basal medium to terminate the digestion process. The number of cells after adhesion was counted. The cell adhesion rate = (number of cells after adhesion / total number of inoculated cells) × 100%. The results are shown in the attached figure. Figure 3 shown.

[0067] Figure 3 The results of the adhesion rate of adipose-derived mesenchymal stem cells after resuscitation in Examples 1-3 and Comparative Examples 1-5 are shown in FIG. Figure 3 It can be seen that the cell adhesion rate of adipose mesenchymal stem cells frozen using the cryopreservation solution of the present invention after 6 months of cryopreservation and recovery is more than 92.84%, which is not significantly different from Comparative Example 5 and is significantly better than Comparative Examples 1-4. The cell adhesion rate of adipose mesenchymal stem cells frozen using the cryopreservation solution of the present invention after 12 months of cryopreservation and recovery is still more than 90.47%. The above results show that the cryopreservation solution of the present invention has the best cryopreservation effect on adipose mesenchymal stem cells.

[0068] Experimental Example 4

[0069] The proliferation of adipose-derived mesenchymal stem cells after resuscitation in Examples 1-3 and Comparative Examples 1-5 was tested. The detailed experimental process is as follows:

[0070] (1) Resuscitation of adipose-derived mesenchymal stem cells

[0071] The cryopreserved tubes of Examples 1-3 and Comparative Examples 1-5 that had been stored for 6 months were used for cell resuscitation. The specific resuscitation process was the same as that of Experimental Example 2.

[0072] (2) CCK-8 assay

[0073] The unfrozen P4 adipose-derived mesenchymal stem cells (control group) obtained in Example 1 and the cells after resuscitation in the above groups were inoculated in a 24-well plate. The culture medium was α-MEM+10% FBS complete culture medium. The cell seeding density was 1×104 cells / well. The plates were cultured in a 37°C, 5% CO2 incubator for 48 h. CCK-8 reagent was added to each well and incubated at 37°C for 2 h. The absorbance of each group at a wavelength of 450 nm was detected on an ELISA reader. The proliferation ability of the resuscitated adipose-derived mesenchymal stem cells was characterized by the relative proliferation rate. The relative proliferation rate = (OD value of each experimental group / OD value of the control group) × 100%. The results are shown in Table 1.

[0074] Table 1 Proliferation of adipose-derived mesenchymal stem cells after resuscitation in Examples 1-3 and Comparative Examples 1-5

[0075]

[0076] Table 1 shows the proliferation of adipose-derived mesenchymal stem cells after thawing for 6 months in Examples 1-3 and Comparative Examples 1-5. It can be seen from Table 1 that the cryopreservation solution of the present invention can better maintain the good proliferation ability of adipose-derived mesenchymal stem cells after cryopreservation.

[0077] 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 solution of adipose-derived mesenchymal stem cells, characterized in that: The invention is composed of a culture medium and a cryoprotectant, wherein the cryoprotectant is composed of Exendin-4, sofalcone, rosmarinic acid and glycerol, and the weight proportion of Exendin-4, sofalcone, rosmarinic acid and glycerol in the cryoprotectant is 1:0.8-1.7:0.4-1.2:50-70.

2. The adipose-derived mesenchymal stem cell cryopreservation solution according to claim 1, characterized in that: The adipose-derived mesenchymal stem cell cryopreservation solution is composed of the following weight parts of culture medium and cryopreservation protective agent: 120-150 parts of culture medium and 5-20 parts of cryopreservation protective agent.

3. The adipose-derived mesenchymal stem cell cryopreservation solution according to claim 1, characterized in that: The weight ratio of Exendin-4, sofalcone, rosmarinic acid and glycerol in the cryoprotectant is 1:1.1:0.7:

62.

4. The adipose-derived mesenchymal stem cell cryopreservation solution according to claim 1, characterized in that: The culture medium is α-MEM serum-free culture medium.

5. The use of the adipose-derived mesenchymal stem cell cryopreservation solution according to any one of claims 1 to 4, characterized in that: The following steps are involved: The adipose-derived mesenchymal stem cells are mixed with an adipose-derived mesenchymal stem cell freezing solution to prepare an adipose-derived mesenchymal stem cell suspension, which is then pre-frozen by programmed cooling and transferred to liquid nitrogen for storage after the pre-freezing is completed.

6. The use of the adipose-derived mesenchymal stem cell cryopreservation solution as claimed in claim 5, characterized in that: The density of the adipose-derived mesenchymal stem cells in the adipose-derived mesenchymal stem cell freezing solution is 1-8×10 7 cells / mL.

7. The use of the adipose-derived mesenchymal stem cell cryopreservation solution as claimed in claim 5, characterized in that: The programmed cooling pre-freezing process is specifically: pre-freezing at 2-4°C for 0.5-1h, pre-freezing at -20-25°C for 1.5-2.5h, and then cooling to -75-80°C at a rate of 1-5°C / min for 4-5h.

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