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Method for separating and culturing endothelial clone formative cell

A technique of separating and culturing endothelium, applied in animal cells, vertebrate cells, artificial cell constructs, etc., can solve the problems of inconvenient bone marrow sampling, difficult cord blood sampling and operation, and low number of EPCs

Active Publication Date: 2013-07-24
AFFILIATED HOSPITAL CHINA ACADEMY OF MILITARY MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, EPCs are mainly derived from peripheral blood, bone marrow and umbilical cord blood. The main problems in obtaining them are: the number of EPCs in circulating blood is very low, it is inconvenient to obtain bone marrow, and it is difficult to obtain and operate umbilical cord blood.

Method used

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  • Method for separating and culturing endothelial clone formative cell
  • Method for separating and culturing endothelial clone formative cell
  • Method for separating and culturing endothelial clone formative cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 2

[0062] Example 2 Observation of the primary cell culture and passage process of umbilical cord-derived ECFCs

[0063] The primary cells of the umbilical cord-derived ECFCs isolated by the method in Example 1 adhered to the wall more than 24-36 hours later, and spindle-shaped and round adherent cells were seen. After being cultured for 3-7 days, scattered cell clones were observed under an inverted microscope. Fusiform, polygonal, round, with large cell bodies. After 7-10 days, the growth rate is faster, and many cell clones appear. About 10-14 days, 80% of the cells are fused, and they are arranged like paving stones. After 10 passages, the cell morphology did not change significantly. ( figure 1 )

[0064] figure 1 Cell morphology of umbilical cord-derived ECFCs. (A), primary cell culture for 36 hours; (B), primary cell culture day 5; (C), primary cell culture day 14; (D), P1 generation cells; (E), P3 generation cells; (F), P10 generation cells.

Embodiment 3

[0065] The determination of the growth curve of embodiment 3 umbilical cord source ECFCs

[0066] After the adherent cells obtained according to the method of Example 1 reached 80% confluence, they were digested with 0.25% trypsin, and then divided by 2×10 4 The cells / well were seeded in a 24-well plate, and 3 wells were digested every 24 hours, and the cells were collected, and the living cells were counted with 0.4% trypan blue, observed for 7 days, and the growth curve was drawn.

[0067] The growth curve of umbilical cord-derived ECFCs basically conformed to the law of cell growth curve, which was divided into incubation period, logarithmic period and plateau period, which were 1-2d, 3-7d and 7d later. It suggested that the proliferation of umbilical cord-derived ECFCs was relatively stable in vitro, and the growth status was normal. The cells proliferated rapidly, and in the logarithmic growth phase, the cell doubling time was about (43.53±5.38) h ( figure 2 ).

Embodiment 4

[0068] Example 4 Cell cycle assay of umbilical cord-derived ECFCs

[0069] In the logarithmic phase of the growth of the umbilical cord-derived ECFCs obtained in Example 1, digest the cells, freeze 75% ethanol and fix at 4°C for 1 hour, incubate with 10 μg / mL RNase A at 37°C for 30 minutes, add 50 μg / mL propidium iodide at 4°C and protect from light Incubate for 5 min, detect with flow cytometer (FACA Calibur, USA), and analyze the results with ModiFIT software.

[0070] Flow cytometry analysis of the cell cycle showed that G 0 / G 1 Period accounted for 75.58%, S+G 2 Cells in +M phase accounted for only 24.42% ( image 3 ).

[0071] The results showed that most of the cells in umbilical cord-derived ECFCs were in G0 / G1 phase, and a few were in S phase, which had typical characteristics of stem cell proliferation.

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Abstract

The invention relates to a method for separating and culturing endothelial clone formative cell, and concretely relates to the method for separating to obtain endothelial clone formative cell from umbilical cord. The method comprises the following steps: 1) digesting umbilical veins endangium by digestive enzyme; 2) flushing digested umbilical veins, collecting a flushing liquor; and 3) taking the flushing liquor for cell culture to obtain the endothelial clone formative cell. The cellular morphology, cell cycle, immunization phenotype, tube formation capability and phagocytosis capability of the separated umbilical cord source endothelial clone formative cell accord with the biological characteristics of endothelial progenitor cells, the umbilical cord can be taken as a new source of the endothelial progenitor cells, and the method provided by the invention provides the guarantee for experiment research and clinical application.

Description

technical field [0001] The invention relates to a method for isolating and culturing endothelial clone-forming cells, in particular to a method for isolating and culturing endothelial-cloning-forming cells from umbilical cords. Background technique [0002] Endothelial progenitor cells (EPCs) are the precursor cells of vascular endothelial cells, and also the progenitor cells with the ability to differentiate into mature vascular endothelial cells. Many studies have shown that EPCs participate in the process of embryonic blood vessels and postnatal angiogenesis, and have the functions of angiogenesis, repair of vascular damage, and secretion of various factors that promote neovascularization. It has important clinical application prospects in the process of vascular restenosis, vascular wound healing, and promotion of hematopoietic recovery. At present, EPCs mainly come from peripheral blood, bone marrow, and umbilical cord blood. The main problems in obtaining them are: th...

Claims

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Application Information

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IPC IPC(8): C12N5/071
Inventor 陈虎张颢张斌扈江伟汤永永
Owner AFFILIATED HOSPITAL CHINA ACADEMY OF MILITARY MEDICAL SCI
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