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Method for preparing CD34 positive cells from umbilical cord mesenchymal stem cells

A technology of mesenchymal stem cells and umbilical cords, applied in the field of preparation of CD34+ cells, which can solve the problems of limited supply of human umbilical cord blood and limited number of CD34+ cells

Active Publication Date: 2017-02-22
BOYALIFE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Isolation and purification of primary CD34 directly from human umbilical cord blood by the above method + cells, in view of the limited supply of human cord blood, the CD34 + The number of cells is also extremely limited

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0123] Embodiment 1, the pretreatment of human umbilical cord sample

[0124] Under sterile conditions, take the near-fetal umbilical cord of normal full-term production, disinfect the surface of the umbilical cord tissue with alcohol, and cut the umbilical cord from the middle;

[0125] Wash thoroughly with PBS containing 100U / mL penicillin and 100U / mL streptomycin to remove residual blood stains;

[0126] Divide the umbilical cord into 0.5cm umbilical cord segments, carefully remove the arteries and veins, take 50mL centrifuge tubes to hold the umbilical cord segments, 1 segment / centrifuge tube;

[0127] In a centrifuge tube, cut the umbilical cord segment into pieces with a size of about 1mm×1mm×1mm, and add LG-DMEM culture solution dropwise during the cutting process to keep the tissue moist;

[0128] Wash twice with PBS buffer solution containing 100 U / mL penicillin and 100 U / mL streptomycin (pH7.2, sodium phosphate salt, phosphate root concentration 0.025M, if not spec...

Embodiment 2

[0129] Embodiment 2, enzymolysis

[0130] The umbilical cord fragments obtained in Example 1 were mixed with enzyme solution (0.1% type I collagenase, 0.1% trypsin, 0.1% hyaluronidase, 0.1% DNase, 0.02% EDTA; % represents the percentage of mass concentration), Digest at 37°C for 2h;

[0131] After digestion, centrifuge for 15 minutes with a centrifugal force of 400g and a temperature of 4±2°C;

[0132] Discard the mixed enzyme solution to obtain single cells derived from umbilical cord.

Embodiment 3

[0133] Embodiment 3, primary culture of cells

[0134] The cells obtained from the treatment in Example 2 were divided into 1×10 6 The density of cells / ml was inoculated into T-25 culture flasks, placed in 20ml of RPMI1640 culture medium supplemented with 10% (v / v) fetal bovine serum, placed at 37°C, 5% CO 2 (v / v), incubating and cultivating for 3 days in an incubator with 95% humidity;

[0135] Change the medium for the first time after 3 days to remove unattached cells;

[0136] The culture medium was replaced every 48 h thereafter.

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Abstract

The invention relates to a method for preparing CD34 positive cells from umbilical cord mesenchymal stem cells. The method specifically comprises the following steps: (a) providing mesenchymal stem cells from an umbilical cord; (b) performing primary culture on the mesenchymal stem cells; (c) performing subculture on the mesenchymal stem cells; (d) in a subculture process, adding an inductive agent in a culture medium for inducing the mesenchymal stem cells; (e) obtaining the CD34 positive cells. The invention also relates to cell products of the CD34 positive cells, prepared through the method and application of the cell products. The method disclosed by the invention has excellent technical effects described in the description.

Description

technical field [0001] The present invention belongs to the field of treating clinical diseases with cell biology techniques. Specifically, the present invention relates to a method for preparing CD34 from umbilical cord-derived mesenchymal stem cells + cell method. Background technique [0002] Stem cells mediate regeneration and the transmission of genetic information to subsequent cell generations. They can self-renew and produce differentiated progeny. In recent years, advances have been made in our understanding of the molecular mechanisms underlying the interactions between stem cells and their tissue microecological environment. This has led to a better understanding of the molecular regulatory mechanisms at work in stem cells. [0003] While gene therapy is still experimental, the technology holds the promise of having a major impact on human health. The scope and definition of gene therapy has changed and expanded over the past few years. In addition to correc...

Claims

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Application Information

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IPC IPC(8): C12N5/0783C12N5/0775A61K35/17A61P9/00
CPCA61K35/17C12N5/0636C12N5/0665C12N2501/599C12N2506/1369
Inventor 许晓椿朱业峰肖海蓉
Owner BOYALIFE
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