阅读说明：本技术 一种间充质干细胞的冻存液及冻存方法 (Cryopreservation solution and cryopreservation method for mesenchymal stem cells ) 是由 刘彦彦 雷童 李晓梅 王治世 李颖娴 杜宏武 于 2021-02-26 设计创作，主要内容包括：本发明涉及生物技术领域,具体涉及一种间充质干细胞的冻存液及冻存方法,该冻存液在避免添加动物血清和二甲基亚砜同时,还能使冻存的间充质干细胞有良好的复苏后活率,使该冻存液在临床实际应用方面更具价值；冻存液的配方包括：甘油的体积百分含量为10-20%,条件培养基的体积百分含量为18-40%,人血白蛋白的体积百分含量为40-65%,海藻糖的体积百分含量为1-5%,甘氨酸的体积百分含量为1-2%。(The invention relates to the technical field of biology, in particular to a cryopreservation solution and a cryopreservation method for mesenchymal stem cells, wherein the cryopreservation solution can avoid adding animal serum and dimethyl sulfoxide, and can also enable the cryopreserved mesenchymal stem cells to have good survival rate after recovery, so that the cryopreservation solution has higher value in the aspect of clinical practical application; the formula of the frozen stock solution comprises: the volume percentage of the glycerol is 10-20%, the volume percentage of the conditioned medium is 18-40%, the volume percentage of the human serum albumin is 40-65%, the volume percentage of the trehalose is 1-5%, and the volume percentage of the glycine is 1-2%.)

1. The cryopreservation solution for the mesenchymal stem cells is characterized by comprising the following components in percentage by weight: the volume percentage of the glycerol is 10-20%, the volume percentage of the conditioned medium is 18-40%, the volume percentage of the human serum albumin is 40-65%, the volume percentage of the trehalose is 1-5%, and the volume percentage of the glycine is 1-2%.

2. The mesenchymal stem cell cryopreservation solution of claim 1, wherein the conditioned medium is obtained by the following steps: taking the supernatant of the normally cultured cells, centrifuging for 15 minutes at 2000 rpm, taking the supernatant after centrifugation, filtering through a 0.22 micron filter membrane to obtain a conditioned medium, and standing at 4 ℃ for later use.

3. The mesenchymal stem cell cryopreservation solution of claim 1, wherein the formulation of the cryopreservation solution comprises: the volume percentage of glycerol is 10%, the volume percentage of conditioned medium is 38%, the volume percentage of human serum albumin is 50%, the volume percentage of trehalose is 1%, and the volume percentage of glycine is 1%.

4. A cryopreservation method of mesenchymal stem cells, which is characterized in that the cryopreservation solution of any one of claims 1 to 3 is added into the mesenchymal stem cells, and the mixture is frozen and preserved after being mixed uniformly.

5. The cryopreservation method of mesenchymal stem cells according to claim 4, wherein the specific cryopreservation method comprises the following steps: and (3) uniformly mixing the frozen stock solution and the stem cells, subpackaging the mixture into a frozen stock tube, transferring the frozen stock solution into a programmed cooling box which is restored to room temperature in advance, cooling overnight in an ultralow temperature refrigerator at minus 80 ℃ for stabilization, and transferring the frozen stock solution into liquid nitrogen for long-term storage.

Technical Field

The invention relates to the technical field of biology, in particular to a cryopreservation solution and a cryopreservation method for mesenchymal stem cells.

Background

Mesenchymal Stem Cells (MSCs) are pluripotent stem cells that can be highly proliferated and multi-lineage differentiated, and are potential clinical materials for regenerative medicine. MSC has all the commonalities of stem cells, namely self-renewal and multi-directional differentiation capacity, and is also widely applied in clinical application at present, for example, when the MSC is combined with hematopoietic stem cells, the success rate of transplantation can be effectively improved, and hematopoietic reconstruction is accelerated. After a patient receives a large dose of chemotherapy, the mesenchymal stem cells and the hematopoietic stem cells are input together, so that the recovery time of the blood cells of the patient can be obviously accelerated, and the chemotherapy is safe and has no adverse reaction.

Mesenchymal stem cells are found not only in bone marrow but also in fat, umbilical cord and other different sources, including exfoliated deciduous tooth pulp stem cells (SHED) which have shown good prospects in recent clinical applications, and have also been identified as a new source of mesenchymal stem cells.

MSCs are generally highly proliferative in cell culture, and can expand large numbers of cells in a short passage after primary extraction. However, despite much experience in the art, large scale expansion and culture of MSCs has been through the process of cryopreservation and recovery, which results in inevitable cell damage and death. At present, the clinical demand for MSC stem cells increases, and the research on stem cell cryopreservation and resuscitation draws more and more attention. As a seed cell for tissue engineering research and potential clinical application, the MSC can show obvious senescence or apoptosis after excessive passage, and is easy to undergo spontaneous differentiation after long-term in vitro culture, lose the multi-differentiation potential, reduce the proliferation and adhesion capacity and increase the apoptosis rate, so that the cryopreservation of the MSC is an important link of the current application research.

In order to protect stem cells, the currently used cell freezing solution or the commercially available cell freezing solution is usually composed of a mixture of dimethyl sulfoxide (DMSO) and Fetal Bovine Serum (FBS). Although conventional formulations can ensure a high survival rate of cells before and after cryopreservation, both animal-derived serum and DMSO have significant clinical limitations. As a high nutrient component of animal origin, FBS is a natural culture medium component, belongs to a heterologous substance, has complex components, has risks of introducing pollution and allergens, and is not suitable for clinical application. In addition, there are large differences in FBS from place to place and from batch to batch, which is disadvantageous for stable culture and expansion of cells. The action mechanism of DMSO is to penetrate cell membrane to enter into cell in the process of cooling, and reduce the electrolyte concentration in unfrozen solution inside and outside the cell, thereby protecting the cell from being damaged by high-concentration electrolyte, and simultaneously, the water in the cell can not be excessively exosmosed, and the cell is prevented from being excessively dehydrated and shrunk. However, DMSO is a highly toxic chemical reagent, which can cause nausea, headache, vomiting and even shock in human body, and is strictly controlled below 1% in conventional cell culture. However, in order to protect cells, the proportion of DMSO in the freezing medium is as high as 10%, although DMSO can play a role in protection under the condition of low-temperature freezing, the cells are subjected to adverse environments once being rewarming in the recovery process, and the adverse environments are also a main reason for cell death in the processes of cell freezing and recovery. Therefore, it is very necessary to develop a new frozen stock solution free of FBS and DMSO.

In the process of cell cryopreservation, the cryopreservation solution only needs to meet 2 conditions: one is that the components of the frozen stock solution can not form ice crystal damaged cells when the ambient temperature is from 37 ℃ to-196 ℃ in liquid nitrogen preservation; another condition is to provide the required nutrients for the cells. Thus, it is only necessary to search for a material satisfying the above conditions to develop a new frozen stock solution. Glycerol, which can prevent water from forming ice crystals in a low-temperature environment, is used for strain preservation. The human serum albumin can provide nutrition for cells, maintain normal osmotic pressure of cells and keep integrity of cell membranes besides the conditioned medium can provide basic inorganic salts, water, amino acids, lipids, growth factors and the like necessary for cell survival. Among the cryoprotectants, the most common of the sugars and amino acids are trehalose and glycine, respectively. Trehalose can form enough gaps or hydrogen bonds with proteins in a low-temperature environment to form a local hydrophobic environment, so that the force suffered by cells in a freezing storage process is resisted. The glycine plays a role in maintaining the pH of the solution in the freezing storage process, and can inhibit the change of the pH when the temperature changes so as to protect the protein. The novel freezing preservation protective solution is formed by combining glycerol, a conditioned medium, human serum albumin, trehalose and glycine, and all substances in the formula play a synergistic effect, so that the freezing preservation solution can be prevented from forming ice crystals at a low temperature to damage cells, nutrition can be provided, pH can be maintained, and proteins can be protected, so that the cells can keep a high survival rate in a low-temperature environment.

Disclosure of Invention

In order to solve the above technical problems, an object of the present invention is to provide a cryopreservation solution for mesenchymal stem cells, which can make the cryopreserved stem cells have a high cryopreservation and revival rate, and at the same time, avoid introducing animal serum and dimethyl sulfoxide into the cryopreservation solution, so that the cryopreservation solution has a higher clinical value.

The invention also aims to provide a cryopreservation method of mesenchymal stem cells, which has the characteristic of high recovery rate, and the survival rate of the cells recovered from the cryopreserved stem cells is over 90 percent.

The invention discloses a mesenchymal stem cell cryopreservation solution, which comprises the following components in parts by weight: the volume percentage of the glycerol is 10-20%, the volume percentage of the conditioned medium is 18-40%, the volume percentage of the human serum albumin is 40-65%, the volume percentage of the trehalose is 1-5%, and the volume percentage of the glycine is 1-2%.

The invention relates to a cryopreservation solution of mesenchymal stem cells, which comprises the following steps: taking the supernatant of the normally cultured cells, centrifuging for 15 minutes at 2000 rpm, taking the supernatant after centrifugation, filtering through a 0.22 micron filter membrane to obtain a conditioned medium, and standing at 4 ℃ for later use.

The invention discloses a mesenchymal stem cell cryopreservation solution, which comprises the following components in parts by weight: the volume percentage of glycerol is 10%, the volume percentage of conditioned medium is 38%, the volume percentage of human serum albumin is 50%, the volume percentage of trehalose is 1%, and the volume percentage of glycine is 1%.

The cryopreservation method of the mesenchymal stem cells comprises the steps of adding the cryopreservation solution into the mesenchymal stem cells, and performing cryopreservation after uniformly mixing.

The invention discloses a cryopreservation method of mesenchymal stem cells, which comprises the following specific steps: and (3) uniformly mixing the frozen stock solution and the stem cells, subpackaging the mixture into a frozen stock tube, transferring the frozen stock solution into a programmed cooling box which is restored to room temperature in advance, cooling overnight in an ultralow temperature refrigerator at minus 80 ℃ for stabilization, and transferring the frozen stock solution into liquid nitrogen for long-term storage.

Compared with the prior art, the invention has the beneficial effects that: the invention provides a cryopreservation solution and a cryopreservation method for mesenchymal stem cells, which have the characteristic of high recovery rate, and the survival rate of the cells recovered from the cryopreserved stem cells is over 90 percent; the invention is suitable for clinical application, and the formula has no FBS and DMSO, is non-toxic to cells and has no heat source substances; the invention has simple components and low cost, the formula is all common reagents, and the formula can be finished without expensive cost; the invention provides a high-efficiency frozen stock solution formula for expanded culture and clinical application of mesenchymal stem cells, and is suitable for popularization and application.

Drawings

FIG. 1 shows the growth state of stem cells after cryopreservation recovery using the cryopreservation solutions of comparative example 1 and examples 1 to 9;

FIG. 2 is a graph of the expression rate of a stem cell surface marker using comparative example 1;

FIG. 3 is a graph of the expression rate of stem cell surface markers using example 2;

FIG. 4 is a graph of the expression rate of stem cell surface markers using example 6;

FIG. 5 is a graph of the expression rate of stem cell surface markers using example 8.

Detailed Description

The following detailed description of embodiments of the present invention is provided in connection with the accompanying drawings and examples. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

The invention provides a mesenchymal stem cell cryopreservation solution and a cryopreservation method, which can effectively avoid the defect that the stem cells are adversely affected by using conventional fetal calf serum and dimethyl sulfoxide cryopreservation solution.

The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1:

a first part: cell culture, cryopreservation and recovery

1) Stem cells were seeded in dishes and cultured in α -MEM medium containing 10% FBS and 1% penicillin and streptomycin. After the cell fusion rate reaches 80%, taking out stem cells which are in a good state and are fully paved at the bottom from the incubator, removing supernatant, washing for 3 times by using PBS, and adding pancreatin for digestion;

2) digesting the dispersed stem cells, centrifuging at 1000rpm for 5min, removing the supernatant, adding a culture medium for resuspension, centrifuging again at 1000rpm for 5min, and removing the upper PBS;

3) preparing the frozen stock solution. The preparation is carried out according to the total volume of 10mL and the following proportion: 10% of glycerol, 40% of conditioned medium and 50% of human serum albumin;

4) the cells are respectively added into a centrifuge tube according to certain cell quantity, and then the cells are transferred into a freezing tube. Placing the freezing tube in a gradient cooling box, standing overnight at-80 ℃, and then transferring to a liquid nitrogen tank;

5) after freezing for several days, taking out the cells for resuscitation and culture;

a second part: and (3) morphological observation:

taking out the culture dish from the incubator 24h after the cells are recovered, placing the culture dish under an inverted microscope for observation and photographing for recording;

and a third part: detection of cell viability and cell Activity assays

1) Detecting cell viability: rapidly thawing the recovered cells, adding the cells into a culture medium to dilute DMSO or glycerol, centrifuging to remove supernatant, and adding a fresh culture medium to carry out resuspension;

2) taking out a few cells, diluting by a certain multiple, adding trypan blue for dyeing for 1-2 min;

3) stained cells were dropped into a cell counting plate for counting. Cell viability and relative viability for the examples were calculated as follows:

cell viability = 1-number of stained cells/total number of cells

Relative survival = experimental survival/control survival

4) The cells were cultured until the confluency reached more than 80%, after digestion, 100 μ l was seeded at about 10^4cells/mL in 96-well plates, 5 wells, after 48h, CCK8 was added and incubated for 1h, and the OD was measured at 450 nm.

5) Calculating the relative proliferation rate of the cells by the following method:

relative proliferation rate = cell OD value/comparative cell OD value

The fourth part: detection of cell surface markers by flow cytometry

1) Taking the stem cells cultured in the third part of the step and having the fusion rate of more than 80%, digesting and collecting the stem cells according to the cell amount of 0.5-1.0 x 10^ 6.

2) Labeling the flow tube according to the sample type, and adding the cells to the tube according to the label, respectively.

3) Add 2mL of room temperature PBS to the flow tube.

4) 1200rpm at ambient temperature for 5 min.

5) The supernatant was discarded and 100uL of room temperature PBS was added to resuspend the cell pellet.

6) Labeled by adding CD34, CD45, CD90, CD105 and HLA-DR with fluorescent labels, respectively. The labeling condition is incubation for 25-30min at 2-8 ℃.

7) After incubation was complete, PBS was added to each flow tube for washing, followed by centrifugation at 1200rpm for 5min at 4 ℃. Finally, resuspend with 300. mu.L PBS, and test on the machine.

Comparative example 1:

the procedure and test items of this comparative example were not different from those of example 1, except that in the formulation of the frozen stock solution, in this comparative example, the formulation used was a conventional frozen stock solution of stem cells, i.e., 90% FBS and 10% DMSO.

Example 2:

the procedure and test items of this example are not different from those of example 1, but only in the formulation of the frozen stock solution, in this example, the formulations are 10% glycerol, 38% conditioned medium, 50% human serum albumin, 1% trehalose, and 1% glycine.

Example 3:

the procedure and test items of this example are not different from those of example 1, but only in the formulation of the frozen stock solution, in this example, 10% of glycerol, 33% of conditioned medium, 50% of human serum albumin, 5% of trehalose, and 2% of glycine are used.

Example 4:

the procedure and test items of this example are not different from those of example 1, but only in the formulation of the frozen stock solution, in this example, the formulations are 20% glycerol, 30% conditioned medium and 50% human serum albumin.

Example 5:

the procedure and test items of this example are not different from those of example 1, but only in the formulation of the frozen stock solution, in this example, 20% of glycerol, 28% of conditioned medium, 50% of human serum albumin, 1% of trehalose, and 1% of glycine are used.

Example 6:

the procedure and test items of this example are not different from those of example 1, but only in the formulation of the frozen stock solution, in this example, 20% of glycerol, 23% of conditioned medium, 50% of human serum albumin, 5% of trehalose, and 2% of glycine are used.

Example 7:

the procedure and test items in this example are not different from those in example 1, but only in the formulation of the frozen stock solution, in this example, 15% of glycerol, 20% of conditioned medium, and 65% of human serum albumin are used.

Example 8:

the procedure and test items of this example are not different from those of example 1, but only in the formulation of the frozen stock solution, in this example, 15% of glycerol, 18% of conditioned medium, 65% of human serum albumin, 1% of trehalose, and 1% of glycine are used.

Example 9:

the procedure and test items of this example are not different from those of example 1, but only in the formulation of the frozen stock solution, in this example, the formulations used are 20% glycerol, 38% conditioned medium, 40% human serum albumin, 1% trehalose, and 1% glycine.

The concrete result is as follows:

the growth state and morphological characteristics of stem cells are detected, as shown in figure 1, in the frozen stock solution of the invention, after frozen storage, the growth of the recovered cells is not different from that of the traditional frozen stock solution, and the cells are in adherent growth under a microscope and are in a slender fibroblast shape.

After the stem cells are cultured and cryopreserved and recovered, the survival rate of the cells is firstly detected by staining necrotic cells, and the results are shown in the following table:

group of	Example 1	Example 2	Example 3	Example 4	Example 5
						Survival rate	89.37%	95.24%	93.68%	90.39%	92.83%
Group of	Example 6	Example 7	Example 8	Example 9	Comparative example
						Survival rate	96.06%	82.84%	94.67%	86.64%	95.07%

As can be seen from the survival rate, after all stem cells are frozen and recovered, the survival rate of the cells is higher than 80%, and the survival rate of most stem cells adopting the invention is higher than 90%;

the relative viability of the cells was then examined and the results are shown in the following table:

group of	Example 1	Example 2	Example 3	Example 4	Example 5
						Relative survival rate	94.00%	100.18%	98.54%	95.08%	97.64%
Group of	Example 6	Example 7	Example 8	Example 9	Comparative example
						Relative survival rate	101.04%	87.14%	99.58%	91.13%	100.00%

From the relative survival rates, it was possible to analyze whether the survival rates in the different examples were higher or lower than those in the comparative examples, and the results showed that examples 2 and 6 and example 8 exhibited similar or even higher survival rates than those in the comparative examples.

Subsequently, in order to observe the effect of the cells on the proliferative capacity of the cells through the cryopreservation process described in the present invention, we examined the proliferative state of the cells using CCK-8. Results the following table shows that the cryopreservation solution of the invention can promote stem cell proliferation compared to the conventional cryopreservation solution containing FBS and DMSO, and the formulation of the invention described in example 2 is the optimal proliferation promoting combination.

Experimental group	Example 1	Example 2	Example 3	Example 4	Example 5
						Relative increment rate	107.93%	118.05%	104.18%	103.68%	106.70%
Experimental group	Example 6	Example 7	Example 8	Example 9	Comparative example
						Relative increment rate	94.99%	88.76%	99.57%	89.05%	100.00%

Next, combining the above results, examples 2,6 and 8 showed more significant beneficial effects on stem cells, and we examined immunophenotypes of the corresponding cells by flow cytometry, and the results are shown in fig. 2-5. The results showed that the cultured stem cells all showed positive expression of markers CD73, CD90 and CD105, and negative expression of CD14, CD19, CD34, CD45 and HLA-DR. As shown in the following table:

	comparative example	Example 2	Example 6	Example 8
					CD14	0.06%	0.00%	0.02%	0.00%
CD19	0.08%	0.00%	0.04%	0.00%
					CD34	0.04%	0.01%	0.00%	0.00%
CD45	1.64%	2.11%	2.27%	1.71%
					HLA-DR	0.01%	0.00%	0.00%	0.00%
CD90	99.99%	99.96%	100.00%	99.96%
					CD73	98.82%	99.19%	99.26%	99.05%
CD105	98.70%	98.95%	98.35%	99.10%

The comparative examples show negative expression of less than 2% for CD14, CD19, CD34, CD45 and HLA-DR, while the positive markers CD73, CD90 and CD105 are greater than 98%. The positive rates of examples 2,6, and 8 were all low for the negative markers, similar to the comparative examples. In addition, examples 2,6, and 8 all exhibited similar or even higher marker positivity for positive markers than the comparative examples.