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Efficient separation and culture method of fat EPCs (endothelial progenitor cells) without enveloping

A technology for endothelial progenitor cells and cells, applied in the field of obtaining endothelial progenitor cells, can solve the problems of FN and serum, complicated operation procedures, etc., and achieve the effects of clear components, low operation requirements, and reduced material costs.

Active Publication Date: 2019-06-18
BEIJING JING MENG STEM CELL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method is not only cumbersome to operate, but also has heterogeneous components such as FN and serum.

Method used

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  • Efficient separation and culture method of fat EPCs (endothelial progenitor cells) without enveloping
  • Efficient separation and culture method of fat EPCs (endothelial progenitor cells) without enveloping
  • Efficient separation and culture method of fat EPCs (endothelial progenitor cells) without enveloping

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 Separation, extraction and enrichment of fat-derived EPC

[0040] 1. Configuration of complete medium: complete medium is to add respectively in IMDM serum-free medium: vascular endothelial growth factor VEGF, epidermal growth factor EGF, basic fibroblast growth factor bFGF each 10ng / mL and 5‰ ( V / V) platelet lysate.

[0041] 2. Collection of human adipocytes: collect 40ml of adult fat under aseptic conditions to a biological safety cabinet, transfer it into two 50ml centrifuge tubes on average, add 20ml of PBS respectively, mix thoroughly and centrifuge at 1000r / min for 10min, and collect the upper layer of fat Tissues were washed twice as above, and rinsed to remove interstitial fluid and blood cells in the tissues.

[0042] 3. Add 20ml of collagenase I (0.1%) to a 50ml centrifuge tube containing 20ml of fat, and place it on a shaking shaker at 37°C for 1 hour to digest until there are no obvious tissue pieces. During the digestion process, shake and mix ev...

Embodiment 2

[0048] Example 2 Differential digestion and enrichment of EPC

[0049] According to the aforementioned research results, the primary EPCs obtained from the 8h group were thus enriched to obtain high-purity primary EPCs for cell differential attachment time.

[0050] For the EPC cells after secondary attachment in each group in Example 1, when the cobblestone-like cell colony confluence reached ≥80%, after slowly washing the cells with PBS, use 0.1% (m / v) trypsin at 37°C Digested for 1 minute, found scattered non-clonal elongated spindle-shaped mesenchymal cells curled off, while EPC clones showed no signs of floating, rinsed with normal saline to remove, then added the same amount of trypsin to continue digestion for 2 minutes, until the cobblestone-like cells shrank After being rounded, the cells were collected by centrifugation to obtain enriched primary EPCs.

[0051] Here, the differential digestion separation method is used to enrich the EPCs of cultured adipose tissue. ...

Embodiment 3

[0052] Example 3 Continuous expansion capability and cell performance verification of EPC

[0053] The primary cells obtained in Example 2 were continuously expanded in vitro using complete medium, including the following specific operations:

[0054] Cell density according to 1*10 4 / cm 2 Inoculate into the culture plate T25 (or T75), and the cell fusion rate reaches 80%-90% about every three days. After washing with PBS, directly digest with 0.1% trypsin for 2 minutes to amplify. The amplification ratio is 1:3-1 : Between 4.

[0055] In order to further identify the purity of the cells obtained in the different attachment time groups, flow cytometric detection was performed on the first-generation EPCs isolated and obtained in different attachment time groups (results are shown in Table 1). It was found that the endothelial cell surface marker CD31 was highly expressed in the EPCs isolated from the 4h, 8h and 12h groups, and the EPC-specific marker VEGFR-2 was also expres...

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Abstract

The invention discloses a method for acquiring EPCs (endothelial progenitor cells) from autologous fat tissue of human beings or animals. The method comprises steps of efficient separation and enrichment of the fat EPCs, a culture dish is not required to be enveloped in advance in the separation process, multiplication culture is performed in vitro, the purity of the obtained EPCs is high, the dryness and the proliferation capability of the cells are high, and the EPCs have the typical EPC form and cell phenotype as well as low-density lipoprotein uptake and blood vessel formation capability.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the separation and purification of endothelial progenitor cells, in particular to a method for obtaining endothelial progenitor cells from fat samples. Background technique [0002] Endothelial progenitor cells (EPCs) are primitive cells that can proliferate, migrate, adhere and differentiate into vascular endothelial cells, and play an important role in repairing vascular endothelium and promoting angiogenesis. Studies have shown that EPC plays an important role in the research of cardiovascular and cerebrovascular diseases, diabetic vascular diseases, hypertension, liver cirrhosis and tumor therapeutic drugs targeting angiogenesis. [0003] EPCs originate from bone marrow and exist in bone marrow, umbilical cord blood, and peripheral blood. The latest research shows that EPCs are also found in umbilical cord, placenta, fat, and the adventitia of organs. Currently, the main sources...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071
Inventor 赵亚李志刚杜颖王煜骏丁炜侯小强宫晓艳索维克·蒙代尔任春林
Owner BEIJING JING MENG STEM CELL TECH
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