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Method for separating endothelial colony-forming cells

A separation method and cell technology, applied in the direction of animal cells, vertebrate cells, artificial cell constructs, etc., can solve the problems of inability to separate cells and collagen tissues, insufficient separation by enzymatic digestion, and low separation efficiency, reaching clinical Excellent application prospects, high separation efficiency, and large number of cells

Active Publication Date: 2014-01-22
SICHUAN NEO LIFE STEM CELL BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, with the mechanical scraping method, the endothelial colony-forming cells and the collagen tissue cannot be successfully separated, and the separation efficiency is low, and the separation by the enzymatic digestion method is often not thorough enough, and the separation efficiency is low

Method used

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  • Method for separating endothelial colony-forming cells
  • Method for separating endothelial colony-forming cells
  • Method for separating endothelial colony-forming cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1 Isolation method of endothelial colony-forming cells of the present invention

[0025] The umbilical cords were collected from healthy puerperas with full-term vaginal delivery or cesarean section. After collection, they were stored in PBS solution containing double antibodies at 4°C and processed within 48 hours. Wash repeatedly, and flush the umbilical cord vein with phosphate-buffered solution (PBS) until the outflow fluid is transparent.

[0026] Clamp one end of the umbilical cord with a hemostat, add type II collagenase (concentration 0.2%) with a pipette gun and fill the umbilical cord vein, and clamp the other end of the umbilical cord with a hemostat; digest on a shaker at 37°C for 30 minutes; after digestion, take Lower the hemostat, collect the digestive juice into a 50ml centrifuge tube; cut the umbilical cord along the umbilical vein to expose the inner surface of the umbilical vein, and gently scrape the umbilical vein with a cell scraper for 5 m...

Embodiment 2

[0028] Example 2 Isolation method of endothelial colony-forming cells of the present invention

[0029] The umbilical cords were collected from healthy puerperas with full-term vaginal delivery or cesarean section. After collection, they were stored in DPBS buffer containing double antibodies at 4°C and processed within 48 hours. Wash repeatedly, and flush the umbilical cord vein with phosphate-buffered solution (PBS) until the outflow fluid is transparent.

[0030] Clamp one end of the umbilical cord with a hemostat, add type II collagenase (concentration 0.2%) with a pipette gun and fill the umbilical cord vein, and clamp the other end of the umbilical cord with a hemostat; sterilize on a shaker at 37°C for 15 minutes; after digestion is complete, take Lower the hemostat, collect the digestive juice into a 50ml centrifuge tube; cut the umbilical cord along the umbilical vein to expose the inner surface of the umbilical vein, and gently scrape the umbilical vein with a cell s...

Embodiment 3

[0032] Example 3 Isolation method of endothelial colony-forming cells of the present invention

[0033] The umbilical cords were collected from healthy puerperas with full-term vaginal delivery or cesarean section. After collection, they were stored in Hanks buffer containing double antibodies at 4°C and processed within 48 hours. Wash repeatedly, and flush the umbilical cord vein with phosphate-buffered solution (PBS) until the outflow fluid is transparent.

[0034] Clamp one end of the umbilical cord with a hemostatic clamp, use a pipette gun to add a mixed enzyme (dual enzyme) composed of type II collagenase (concentration: 0.2%) and trypsin (concentration: 0.5%) at a ratio of 1:1 (v / v) And fill the umbilical cord vein, clamp the other end of the umbilical cord with a hemostatic forceps; 37 ℃ shaker for 60 minutes; after the digestion is completed, remove the hemostatic forceps, collect the digestive juice into a 50ml centrifuge tube; cut the umbilical cord along the umbili...

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Abstract

The invention discloses a method for separating endothelial colony-forming cells. The method comprises the following steps: (1) enzymatic digestion: taking an umbilical cord, washing an umbilical cord vein with an alkaline or neutral buffer solution till effluent liquid is transparent, filling collagenase or a mixed enzyme of collagenase and trypsin into the umbilical cord vein, digesting for 15-60min, and collecting a digestion solution; (2) mechanical scraping: dissecting the umbilical cord vein, exposing the inner surface of the umbilical cord vein, scraping the inner surface of the umbilical cord vein with a cell scraper for 3-10min, washing with the alkaline or neutral buffer solution, and collecting a washing solution; (3) merging the digestion solution in the step (1) and the washing solution in the step (2), centrifuging, and removing supernatant liquid for preparation. By adopting the method for separating the endothelial colony-forming cells, disclosed by the invention, the endothelial colony-forming cells can be effectively separated from the umbilical cord vein, and clinical application prospects are excellent.

Description

technical field [0001] The present invention relates to methods for isolating endothelial colony-forming cells. Background technique [0002] Endothelial progenitor cells are the precursor cells of endothelial cells and play an important role in maintaining the stability of the vascular endothelial environment. According to the clonal morphology, appearance time and cell proliferation potential of endothelial progenitor cells (EPCs), peripheral blood MNCs cultured in vitro can differentiate into early and late EPCs. Endothelial colony-forming cells are a late-stage EPCs subpopulation discovered in recent years. They are ideal seed cells for transplantation because of their stronger proliferation and angiogenesis capabilities. Endothelial colony-forming cells can be isolated from bone marrow, umbilical cord blood, peripheral blood, umbilical cord and other tissues. [0003] The umbilical cord vein contains a large number of endothelial colony-forming cells, which is easy to...

Claims

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Application Information

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IPC IPC(8): C12N5/071
Inventor 赖真阳陈强李雪莲钟立武
Owner SICHUAN NEO LIFE STEM CELL BIOTECH
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