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Application of mesenchymal stem cell culture from human umbilical cord or supernatant of mesenchymal stem cell culture

A technology of mesenchymal stem cells and cultures, applied in the field of mesenchymal stem cells, can solve the problem of high treatment costs

Inactive Publication Date: 2018-10-30
ACADEMY OF MILITARY MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are still certain potential risks in MSC transplantation, and the treatment cost is high, so it is particularly important to explore new strategies for MSC treatment of skin injuries.

Method used

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  • Application of mesenchymal stem cell culture from human umbilical cord or supernatant of mesenchymal stem cell culture
  • Application of mesenchymal stem cell culture from human umbilical cord or supernatant of mesenchymal stem cell culture
  • Application of mesenchymal stem cell culture from human umbilical cord or supernatant of mesenchymal stem cell culture

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1 MSC Separation and Identification of Umbilical Cord Origin

[0050] Take the umbilical cord from normal delivery, cut the umbilical cord into 1cm pieces in a sterile environment, put the cut umbilical cord tissue into α-MEM complete medium containing 10% FBS and culture it for 72 hours, and replace the culture medium every 2-3 days , when the crawled cells reached 80% confluency, add 0.25% trypsin to digest and passage. The primary human umbilical cord-derived MSCs were cultured for 5 days, and observed under a microscope, fibrous and adherent MSCs crawled out around the umbilical cord tissue. With the subculture MSC grows faster and the purity becomes higher, and all the cells grow in the shape of a long spindle and vortex adherent, which is consistent with the characteristics of MSC culture in vitro, such as figure 1 (A) shown. Collection of P3 generation MSCs showed high expression of CD105, CD73, CD166, etc., low expression or no expression of CD34, CD45...

Embodiment 2

[0051] Example 2 Growth Factors Highly Expressed by MSCs

[0052] The growth and metabolism of MSC cells cultured in vitro will secrete some growth-promoting factors, so the expression of regenerative factors in MSC was detected. 0.25% trypsin digested and cultured MSCs of the P3 generation, extracted the total RNA of MSCs, and reverse-transcribed them into cDNA. The q-PCR results showed that MSCs could highly express TGF-β, EGF, FGF, VEGF and other related growth factors, and the expression level of VEGF highest, such as figure 2 shown.

Embodiment 3

[0053] The preparation of embodiment 3 culture or culture supernatant lyophilized powder

[0054] P3 generation MSCs were collected, cultured in serum-free medium for 48 hours, the serum-free MSC culture supernatant was aspirated, dead cells and debris were discarded by centrifugation, and the collected culture or culture supernatant was collected.

[0055] The collected collected culture or culture supernatant is desalted and decolorized by gel filtration, put into the freeze-drying chamber of the vacuum freeze-dryer, and carried out according to the procedures set by the biological characteristics of the protein, such as pre-cooling and freeze-drying. Preparation of dry powder, collecting the powder after the program runs. The freeze-drying procedure is as follows:

[0056] 1. Pre-freezing stage: -50℃, 2h;

[0057] 2. Initial evaporation stage: -28°C, 8h;

[0058] 3. Drying: 8°C, 6h;

[0059] 4. Freeze-drying: 27°C, 6h.

[0060] Weigh 30mg of powder and dissolve it in 3...

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PUM

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Abstract

The invention discloses application of a mesenchymal stem cell culture from a human umbilical cord or a supernatant of the mesenchymal stem cell culture. The invention provides the application of themesenchymal stem cell culture from the human umbilical cord or the supernatant of the mesenchymal stem cell culture in preparing drugs for promoting skin injury repair, promoting vascular growth, diminishing inflammation and promoting collagen fiber accumulation. The mesenchymal stem cells are derived from umbilical cord tissues and do not need to be cultured under hypoxia and other conditions, and only normal cell culture and regulation are needed; the passage number of cells is relatively-low and the cells are P3 and P4 generation cells which do not affect the dryness of the mesenchymal stemcells. Therefore, mesenchymal stem cell culture supernatant components disclosed by the application are closer to natural growth metabolic components of MSC, purer and better in treatment effect.

Description

technical field [0001] The invention belongs to the technical field of application of mesenchymal stem cells, and in particular relates to the use of human umbilical cord-derived mesenchymal stem cell cultures or culture supernatants thereof. Background technique [0002] Mesenchymal stem cells (mesenchymal stem cells, MSCs) are a type of adult stem cells with self-renewal, multilineage differentiation and immune regulation. They can be isolated from bone marrow, fat, and perinatal umbilical cord and placenta. It is not suitable for tumorigenicity and does not involve ethical controversy, so it is widely used in clinical trials and basic research. [0003] Wound healing has always been an important problem. As a special organ of the barrier between the body and the outside world, the skin plays a very important role in wound healing. Previous studies have confirmed that MSCs are recruited to inflammatory sites by small molecules such as chemokines, and the damaged microenvi...

Claims

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Application Information

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IPC IPC(8): A61K35/51A61K9/19A61P9/14A61P17/00A61P17/02A61P29/00C12N5/0775
CPCA61K9/19A61K35/51A61P9/14A61P17/00A61P17/02A61P29/00C12N5/0665
Inventor 张毅刘伟江刘元林王鹏张伟李雪王洋樊月滕天怡周娜孟祥宇
Owner ACADEMY OF MILITARY MEDICAL SCI
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