阅读说明：本技术 干细胞提取液及其制备方法与应用 (Stem cell extracting solution and preparation method and application thereof ) 是由 董健伸 徐汝强 孙永沛 程洪斌 王晓东 徐俊 于 2020-07-15 设计创作，主要内容包括：本发明公开了干细胞提取液及其制备方法与应用,其制备方法包括以下步骤：将原代干细胞接种于培养基中,原代培养,隔天换液,当原代干细胞生长至80～90%时,去除培养基,剩余的干细胞经过漂洗后与生理盐水混合,诱导培养后进行离心分离,得到上清液；然后将上清液过滤后,得到干细胞提取液。本发明公开了干细胞提取液及其制备方法与应用,没有外源性动物成分而更安全,得到的提取液呈现丰富的细胞因子,可以直接作为药物制剂,或者冻存后与辅料混合配置药物。(The invention discloses a stem cell extracting solution, a preparation method and application thereof, wherein the preparation method comprises the following steps: inoculating the primary stem cells into a culture medium, carrying out primary culture, changing the culture medium every other day, removing the culture medium when the primary stem cells grow to 80-90%, rinsing the rest stem cells, mixing with normal saline, carrying out induction culture, and carrying out centrifugal separation to obtain a supernatant; then filtering the supernatant to obtain the stem cell extracting solution. The invention discloses a stem cell extracting solution, a preparation method and application thereof, wherein the stem cell extracting solution is safer without exogenous animal components, and the obtained extracting solution presents rich cell factors and can be directly used as a medicinal preparation or mixed with auxiliary materials to prepare a medicament after being frozen.)

1. The stem cell extracting solution is characterized in that the preparation method of the stem cell extracting solution comprises the following steps: inoculating the primary stem cells into a culture medium, carrying out primary culture, changing the culture medium every other day, removing the culture medium when the primary stem cells grow to 80-90%, rinsing the rest stem cells, mixing with normal saline, carrying out induction culture, and carrying out centrifugal separation to obtain a supernatant; then filtering the supernatant to obtain the stem cell extracting solution.

2. The stem cell extract solution as claimed in claim 1, wherein the stem cells are human umbilical cord mesenchymal stem cells.

3. The stem cell extract solution according to claim 1, wherein the primary stem cells are seeded in the culture medium at a density of 10⁵～10⁷Per T150 bottles; the culture medium is serum-free MSC culture medium.

4. The stem cell extract solution according to claim 1, wherein the stem cell extract solution is rinsed with a physiological saline solution.

5. The stem cell extract liquid according to claim 1, wherein the induction culture is carried out at 37 ℃ and 5% CO₂Is carried out in an incubator; the time for induction culture is 48-96 hours.

6. The stem cell extract solution according to claim 1, wherein the centrifugation is performed at 1500rpm for 10 minutes; the filtration membrane was 0.1 micron.

7. The stem cell extract solution according to claim 1, wherein the amount of the physiological saline used in the induction culture is 20 to 30mL/T150 flask.

8. The stem cell extract solution according to claim 1, wherein vitamin E is added during induction culture.

9. The preparation method of the stem cell extracting solution is characterized by comprising the following steps of: inoculating the primary stem cells into a culture medium, carrying out primary culture, changing the culture medium every other day, removing the culture medium when the primary stem cells grow to 80-90%, rinsing the rest stem cells, mixing with normal saline, carrying out induction culture, and carrying out centrifugal separation to obtain a supernatant; then filtering the supernatant to obtain the stem cell extracting solution.

10. Use of the stem cell extract of claim 1 for the preparation of a medicament.

Technical Field

The invention belongs to the technical field of cell culture, and particularly relates to a stem cell extracting solution as well as a preparation method and application thereof.

Background

In the actual working scene of cells, most molecules (such as proteins) in the cells are very large and cannot directly pass through the membrane structure in the cells, so that basic life processes such as energy conversion, information recognition and transmission, substance transportation and the like between cells need to be carried out at any moment. In recent years, the development of science and technology is changing day by day, and as one of vesicles, exosomes participate in important regulation of cell activities, and are exposing at the head corner in the aspect of treatment of various difficult and complicated diseases. The exosome is a lipid bilayer membranous vesicle which is generated by various living cells through a series of biological mechanisms such as endocytosis-fusion-efflux and is excreted out of a cell membrane in an active secretion mode, and is essentially a lipid bilayer, the diameter of the vesicle is 30-100nm, and the density of the vesicle is 1.15-1.19 g/mL. Cells that have been shown to secrete exosomes are: mast cells, lymphocytes, dendritic cells, tumor cells, mesenchymal stem cells, and the like; exosomes are currently considered to be specifically secreted membrane vesicles, involved in intercellular communication, and increasingly interested in exosome research, whether for studying their function or for understanding how they are used in the development of minimally invasive diagnostics. The separation and purification of exosomes are always the concerns of researchers, the batch preparation of exosomes is crucial to the subsequent research, and people mostly adopt ultracentrifugation, immunomagnetic beads or kits and other methods to realize the extraction and separation of exosomes at present; the prior art has complex process, the extracted cell exosome stock solution has high content of foreign protein and high purity, and the subsequent application and research of the cell exosome are greatly influenced.

Research published by the professor's schwann of Jiangsu university college of medicine in Theranostics proves that DIM up-regulates Wnt11 expression in hucMSCs-derived exosomes, and Wnt11 knockdown inhibits beta-catenin activation and stem cell induction in DIM-hUCMSCs, and eliminates the therapeutic effect thereof in vivo, so the research result shows that DIM promotes the dryness of hucMSCs by increasing the autocrine signal of the exosome Wnt11, and provides a new strategy for improving the therapeutic effect of hucMSCs on wound healing. Human umbilical cord mesenchymal stem cells (hucMSCs) are considered as promising therapeutic means in regenerative medicine, but their therapeutic effects need to be further improved.

Disclosure of Invention

The invention discloses a stem cell extract and a preparation method and application thereof, wherein the stem cell extract is safer without exogenous animal components, and the obtained extract presents abundant cell factors besides exosomes, especially the extract has an effect on inflammation and can be directly used as an anti-inflammatory preparation or mixed with auxiliary materials to prepare an anti-inflammatory drug after being frozen; particularly, the method of the invention does not need ultracentrifugation treatment, greatly simplifies the operation steps, retains the mixed components of the exosomes and overcomes the step that the cell supernatant needs to be removed in the prior art.

The invention adopts the following technical scheme:

the preparation method of the stem cell extracting solution comprises the following steps: inoculating the primary stem cells into a culture medium, carrying out primary culture, changing the culture medium every other day, removing the culture medium when the primary stem cells grow to 80-90%, rinsing the rest stem cells, mixing with normal saline, carrying out induction culture, and carrying out centrifugal separation to obtain a supernatant; then filtering the supernatant to obtain the stem cell extracting solution.

The invention discloses the application of the stem cell extracting solution in medicaments; furthermore, the medicine can be used as an anti-inflammatory medicine, an anti-tissue fibrosis medicine, an anti-tumor medicine and an anti-aging medicine.

The stem cell extracting solution prepared by the invention is liquid, in particular to physiological saline solution containing various components, can be directly used as an anti-inflammatory active substance, and can also be used as an active ingredient to be prepared into medicines with various states with conventional pharmaceutical excipients for anti-inflammation. During the culture process of the cells, a plurality of cytokines and exosomes are generated and secreted into culture supernatant, the components act in a direct or indirect way to perform biological functions, and the cytokines are mutually influenced and interacted; the actions of various cytokines are not isolated, and a cytokine network (cytokine) is formed by complex interactions such as mutual regulation of synthesis and secretion, mutual regulation of receptor expression, mutual influence of biological effects and the like, so that compared with a single cytokine, a mixed cytokine naturally produced by a cell can obtain an irreplaceable technical effect.

In the invention, the primary stem cells are stem cells which are not cultured, are derived from human tissues such as human umbilical cord mesenchymal stem cells, and avoid the problems of limited source and safety ethics compared with embryonic stem cells and the problem of invasive acquisition compared with bone marrow stem cells.

In the present invention, the primary stem cells are inoculated into the culture medium at a density of 10⁵～10⁷a/T150 bottle, preferably 10⁶Per T150 bottles; the culture medium is serum-free MSC culture medium (ScienCell corporation); every other day liquid change is the current term; primary cell culture at 37 ℃ with 5% CO₂Is carried out in an incubator; when the growth density of the stem cells is close to 80-90%, removing the culture medium, and continuously rinsing for 3 times by using normal saline; during induction culture, the dosage of the physiological saline is 20-30 mL/T150 bottle, preferably 25mL/T150 bottle; inducing culture at 37 deg.C with 5% CO₂The time in the incubator of (1) is 48 to 96 hours, preferably 72 hours.

Furthermore, vitamin E is added during induction culture, preferably, the dosage of the vitamin E is 5-10 muL/L of normal saline, and the best dosage is 8 muL/L of normal saline.

In the invention, the centrifugal separation is 1500rpm centrifugation for 10 minutes, cells are removed, and supernatant is collected; filtering the supernatant with filter membrane diameter of 0.1 micrometer to obtain stem cell extractive solution.

The stem cell extracting solution disclosed by the invention is safe in components, is rich in stem cell exosomes, various cell factors and nutrient substances, and has obvious effects of improving inflammation and tissue fibrosis.

Drawings

FIG. 1 shows the VEGF content in exosomes obtained by various methods;

FIG. 2 shows the bFGF content of exosomes obtained by various methods;

FIG. 3 is a Western-blot method for identifying exosomes in the obtained stem cell extract;

FIG. 4 shows the results of the experimental pulmonary fibrosis;

FIG. 5 shows the experimental results of pulmonary fibrosis in the control group;

fig. 6 shows the results of the blank pulmonary fibrosis experiment.

Detailed Description