阅读说明：本技术 一种亚全能间充质干细胞分泌素的制备方法 (Preparation method of sub-totipotent mesenchymal stem cell secretin ) 是由 王彬 林思恩 李刚 于 2019-09-12 设计创作，主要内容包括：本申请提供一种亚全能间充质干细胞分泌素的制备方法,包括间充质干细胞预培养、间充质干细胞的大规模培养、间充质干细胞分泌素原液的制备和间充质干细胞分泌素冻干粉的制备。本申请的一种亚全能间充质干细胞分泌素的制备方法,简单易行、快捷方便、易于大规模连续生产,该方法不添加任何生物因子,即可实现间充质干细胞的快速增殖,而且仅通过细胞培养液的更换方式就能高效、连续生产间充质干细胞分泌素,大大降低了生产成本；该间充质干细胞分泌素活性成分较高,具有显著提高皮肤细胞和骨细胞增殖的能力。(The application provides a preparation method of a sub-totipotent mesenchymal stem cell secretin, which comprises the steps of mesenchymal stem cell pre-culture, large-scale culture of mesenchymal stem cells, preparation of a mesenchymal stem cell secretin stock solution and preparation of mesenchymal stem cell secretin freeze-dried powder. The preparation method of the sub-totipotent mesenchymal stem cell secretin is simple, feasible, rapid and convenient, and easy for large-scale continuous production, the method can realize rapid proliferation of the mesenchymal stem cells without adding any biological factor, and the mesenchymal stem cell secretin can be efficiently and continuously produced only by changing the cell culture solution, so that the production cost is greatly reduced; the mesenchymal stem cell secretin has high active ingredient, and has the capability of remarkably improving the proliferation of skin cells and bone cells.)

1. A preparation method of a sub-totipotent mesenchymal stem cell secretin is characterized by comprising the following steps:

1) Mesenchymal stem cell pre-culture

Inoculating the mesenchymal stem cells into an Eagle culture medium, and culturing to obtain a mesenchymal stem cell suspension;

2) Large-scale culture of mesenchymal stem cells

Inoculating the mesenchymal stem cell suspension obtained in the step 1) into a mesenchymal stem cell culture medium liquid, uniformly mixing, transferring into a bioreactor, standing for 2h, stirring at the speed of 60rpm/min, and culturing for 7-14 days at 37 ℃, 5% of CO ₂ and 5-20% of O ₂ to obtain a mesenchymal stem cell culture medium liquid;

3) Preparation of mesenchymal stem cell secretin stock solution

Filtering the mesenchymal stem cell base solution obtained in the step 2) to obtain mesenchymal stem cells, washing the mesenchymal stem cells for 3 times by using a phosphate buffer solution, adding 100mL of a serum-free base culture medium, continuously culturing for 24 hours, and collecting the culture medium to obtain a mesenchymal stem cell secretin stock solution;

4) preparation of mesenchymal stem cell secretin freeze-dried powder

Filtering the stock solution of the mesenchymal stem cell secretin obtained in the step 3) by a cell filter with the aperture of 70 mu m to obtain filtrate, then centrifuging the filtrate at the speed of 1500g for 10 minutes, taking supernatant, and freeze-drying the supernatant to obtain freeze-dried powder, namely the mesenchymal stem cell secretin.

2. The method for preparing a sub-totipotent mesenchymal stem cell secretin according to claim 1, wherein the mesenchymal stem cell is derived from one or more of bone marrow, fat, skin, umbilical cord blood, umbilical cord and placenta.

3. the method for preparing a sub-totipotent mesenchymal stem cell secretin according to claim 2, wherein the Eagle medium used in the step 1) is a modified Eagle medium supplemented with 10% fetal bovine serum and 1% antibiotics, i.e., a DMEM medium.

4. the method for preparing a sub-totipotent mesenchymal stem cell secretin according to claim 3, wherein in the step 1), the mesenchymal stem cell is pre-cultured by using a monolayer culture, the inoculation amount of the mesenchymal stem cell is 0.5 ten thousand/cm ² during the pre-culture, and the mesenchymal stem cell secretin is cultured for 2-3 generations at 37 ℃.

5. The method for preparing a sub-totipotent mesenchymal stem cell secretin according to claim 4, wherein the mesenchymal stem cell density of the mesenchymal stem cell suspension obtained in the pre-culture of the step 1) is 10 ten thousand per 100mL to 1000 ten thousand per 100 mL.

6. The method for preparing a sub-totipotent mesenchymal stem cell secretin according to claim 5, wherein the mesenchymal stem cell density of the mesenchymal stem cell suspension obtained in the pre-culture of the step 1) is 100 ten thousand per 100 mL.

7. the method for preparing a sub-totipotent mesenchymal stem cell secretin according to claim 6, wherein the mesenchymal stem cell culture medium liquid of step 2) is a sterile microbial carrier and the ratio of the cell culture medium to the total mesenchymal stem cell secretin is 3 g: 100mL of the carrier base solution.

8. the method of claim 7, wherein the biological microcarrier is one of polycaprolactone, polylactic acid-glycolic acid copolymer, hydrogel, sodium alginate, chitin or polystyrene.

9. the method for preparing a sub-totipotent mesenchymal stem cell secretin according to claim 8, wherein the serum-free basal medium in step 3) is a serum-free α -minimum essential medium, i.e., α -MEM medium.

Technical Field

The invention relates to the technical field of biology, in particular to a preparation method of a sub-totipotent mesenchymal stem cell secretin.

Background

Mesenchymal Stem Cells (MSCs) are stem cells with strong proliferative capacity and multipotentiality, have all the commonalities of stem cells, namely, self-renewal and multipotentiality, and are the most clinically used stem cells at present. The combined application of the mesenchymal stem cells and the hematopoietic stem cells can improve the success rate of transplantation and accelerate hematopoietic reconstruction. 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 medicine is safe and has no adverse reaction.

Mesenchymal stem cells are present not only in the bone marrow but also in skeletal muscle, periosteum and trabecula. It has wide tissue types, so it is of great clinical application value.

At present, mesenchymal stem cells are the focus of research of various large medicine enterprises. The FDA has approved nearly 60 clinical trials in the united states, mainly involving several aspects.

1. Hematopoietic stem cell transplantation: enhancing hematopoietic function; facilitating the engraftment of a hematopoietic stem cell graft; treating graft versus host disease.

2. Repairing tissue damage: bone, cartilage, joint damage, heart damage; liver damage; spinal cord injury and neurological diseases.

3. autoimmune diseases: systemic lupus erythematosus, scleroderma, inflammatory enteritis, etc.

4. As a vector for gene therapy.

Among them, the treatment of graft versus host disease, Crohn's disease, has already entered the third clinical stage in the United states. In China, mesenchymal stem cells are used for treating some clinical refractory diseases, such as spinal cord injury, cerebral palsy, amyotrophic lateral sclerosis, systemic lupus erythematosus, systemic sclerosis, Crohn's disease, stroke, diabetes, diabetic foot, liver cirrhosis and the like, and the mesenchymal stem cells have obvious curative effects on the treatment of the diseases according to preliminary clinical reports.

Therefore, the production and preparation of the mesenchymal stem cell and the secretin thereof are very important, and whether the mesenchymal stem cell secretin with less impurities, high activity and lasting effectiveness can be provided is a key factor for the success of the clinical tests.

At present, the sources of mesenchymal stem cells are various, such as bone marrow, fat, synovium, bone, muscle, lung, liver, pancreas and other tissues, and the mesenchymal stem cells are separated and prepared from amniotic fluid and umbilical cord blood, and the most used mesenchymal stem cells are bone marrow-derived mesenchymal stem cells.

However, regardless of the starting material, mesenchymal stem cells are generally present in low amounts in the source and usually require in vitro culture. At present, there are many ways for culturing mesenchymal stem cells in vitro, but usually only laboratory-scale culture cannot achieve the purpose of large-scale continuous in vitro culture, so that industrial application cannot be realized, and particularly dressing application with large dose cannot be realized.

Disclosure of Invention

The present application is provided to solve the above-mentioned technical problems.

The technical scheme adopted by the application is as follows: a preparation method of a sub-totipotent mesenchymal stem cell secretin is characterized by comprising the following steps:

1) Mesenchymal stem cell pre-culture

inoculating the mesenchymal stem cells into an Eagle culture medium, and culturing to obtain a mesenchymal stem cell suspension;

2) Large-scale culture of mesenchymal stem cells

Inoculating the mesenchymal stem cell suspension obtained in the step 1) into a mesenchymal stem cell culture medium liquid, uniformly mixing, transferring into a bioreactor, standing for 2h, stirring at the speed of 60rpm/min, and culturing for 7-14 days at 37 ℃, 5% of CO ₂ and 5-20% of O ₂ to obtain a mesenchymal stem cell culture medium liquid;

3) Preparation of mesenchymal stem cell secretin stock solution

Filtering the mesenchymal stem cell base solution obtained in the step 2) to obtain mesenchymal stem cells, washing the mesenchymal stem cells for 3 times by using a phosphate buffer solution, adding 100mL of a serum-free base culture medium, continuously culturing for 24 hours, and collecting the culture medium to obtain a mesenchymal stem cell secretin stock solution;

4) Preparation of mesenchymal stem cell secretin freeze-dried powder

filtering the stock solution of the mesenchymal stem cell secretin obtained in the step 3) by a cell filter with the aperture of 70 mu m to obtain filtrate, then centrifuging the filtrate at the speed of 1500g for 10 minutes, taking supernatant, and freeze-drying the supernatant to obtain freeze-dried powder, namely the mesenchymal stem cell secretin.

Further, the mesenchymal stem cells are derived from one or more of umbilical cord blood, umbilical cord, placenta and bone tissue of human fetus which voluntarily terminates pregnancy.

Further, the Eagle medium used in the step 1) is a modified Eagle medium supplemented with 10% fetal bovine serum and 1% antibiotics, i.e., a DMEM medium.

Further, in the step 1), the mesenchymal stem cells are pre-cultured by adopting monolayer culture, the inoculation amount of the mesenchymal stem cells is 0.5 ten thousand/cm ² during pre-culture, and the mesenchymal stem cells are cultured for 2-3 generations at the temperature of 37 ℃.

Further, the density of the mesenchymal stem cells in the mesenchymal stem cell suspension obtained by the pre-culture in the step 1) is 10 ten thousand/100 mL-1000 ten thousand/100 mL.

Further, the density of the mesenchymal stem cells in the mesenchymal stem cell suspension obtained by the pre-culture in the step 1) is 100 ten thousand per 100 mL.

further, the mesenchymal stem cell culture medium liquid in the step 2) is a sterile microbial carrier and a cell culture medium, wherein the ratio of the sterile microbial carrier to the cell culture medium is 3 g: 100mL of the carrier base solution.

Further, the biological micro-carrier is one of polycaprolactone, polylactic acid-glycolic acid copolymer, hydrogel, sodium alginate, chitin or polystyrene.

Further, the serum-free basal medium in the step 3) is a serum-free alpha-minimum essential medium, namely alpha-MEM medium.

The application has the advantages and positive effects that: the preparation method of the sub-totipotent mesenchymal stem cell secretin is simple, feasible, rapid and convenient, and easy for large-scale continuous production, the method can realize rapid proliferation of the mesenchymal stem cells without adding any biological factor, and the mesenchymal stem cell secretin can be efficiently and continuously produced only by changing the cell culture solution, so that the production cost is greatly reduced; the mesenchymal stem cell secretin has high active ingredient, and has the capability of remarkably improving the proliferation of skin cells and bone cells.

In addition to the technical problems addressed by the present application, the technical features constituting the technical solutions, and the advantages brought by the technical features of the technical solutions described above, other technical problems solved by the present application, other technical features included in the technical solutions, and advantages brought by the technical features will be further described in detail below with reference to the accompanying drawings.

Drawings

FIG. 1 is a fluorescent photograph under an inverted fluorescent microscope after mass-culturing cells of example 1, example 2 and example 3;

Fig. 2 is a graph of activity of mesenchymal stem cell secretin-treated AMSCs obtained in example 1, example 2 and example 3, and a blank control, tested for cell proliferative capacity using alamar blue kit on the first, third and fifth days after treatment;

Fig. 3 is a graph of activity of HFB after treatment in testing cell proliferative capacity using alamar blue kit, of mesenchymal stem cell secretin-treated AMSC obtained in example 1, example 2 and example 3, and a blank control;

fig. 4 is a graph of optical density of human malignant melanoma cells, skin squamous cell carcinoma cells co-injected with mesenchymal stem cell secretin, and a blank control, IVIS 200 in vivo imaging system.

Detailed Description

The present application will be described in further detail with reference to examples. It is to be understood that the specific embodiments described herein are merely illustrative of the relevant invention and not restrictive of the invention.

It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict. The present application will be described in detail with reference to examples.