Method for ex vivo expansion of human vascular endothelial progenitor cells in low oxygen condition

A technology for the expansion of endothelial progenitor cells in vitro, which is applied in the field of expanding human vascular endothelial progenitor cells in vitro, and can solve the problems that the stemness of progenitor cells cannot be fully maintained and large-scale production cannot be achieved.

Inactive Publication Date: 2018-11-06
广州赛琅生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Moreover, although the culture technology of endothelial progenitor cells can obtain a certain amount for scientific research n

Method used

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  • Method for ex vivo expansion of human vascular endothelial progenitor cells in low oxygen condition
  • Method for ex vivo expansion of human vascular endothelial progenitor cells in low oxygen condition
  • Method for ex vivo expansion of human vascular endothelial progenitor cells in low oxygen condition

Examples

Experimental program
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Effect test

Embodiment 1

[0022] Example 1 In vitro expansion of human vascular endothelial progenitor cells under hypoxic conditions

[0023] 1. EPCs were isolated from human bone marrow and cultured under different oxygen concentrations.

[0024] After obtaining informed consent, bone marrow was obtained from 3 volunteers. Bone marrow mononuclear cells (MNC) were isolated from bone marrow using human peripheral blood lymphocyte separation medium (TBD, catalog number: LTS1077), and 4×10 5 cells / cm 2 Inoculate on fibronectin (BD, product number: 354008) coated 25cm 2 Easy-to-use culture bottles (NUNC, catalog number: 156367). The medium for culturing vascular endothelial progenitor cells was EGM-2 medium, and then the culture flasks were placed at 37°C, 5% CO2, 1% O 2 (low oxygen) or 20% O 2 cultured under (normal oxygen) conditions.

[0025] EGM-2 medium is a set, including 500mL EBM-2 basal medium and adding 0.5mLhEGF (human recombinant epidermal growth factor), 0.2ml Hydrocortisone (hydrocorti...

Embodiment 2

[0035] Example 2 Effects of Hypoxic Conditions on EPCs Differentiation and Stemness

[0036] In order to detect the effect of cultured under hypoxic conditions on the function of EPCs, we conducted angiogenesis experiments and Transwell migration experiments on EPCs cultured under the two conditions to analyze the angiogenesis function and homing ability of EPCs in vitro.

[0037] 1. Migration experiment

[0038] Transwell migration experiments were performed in 24-well plate Transwell chambers (pore size 8.0 μm; Corning). EPCs (200 μL, 1×10 5 cells, obtained in Example 1) were inoculated in the upper chamber of Transwell, and 500 μL of EBM-2 medium supplemented with 10% FBS was added to the lower chamber of Transwell. respectively at 37°C / 1%O 2 with 20%O 2 After 24 hours of incubation, the cells on the surface of the filter paper were carefully removed with a cotton swab, fixed with 75% alcohol and stained with 0.1% crystal violet, and five random fields of view were take...

Embodiment 3

[0043] The influence of embodiment 3 hypoxic conditions on the gene expression of EPCs

[0044] 1. RNA sequencing and preprocessing

[0045] The cells cultured in Example 1 were preserved with Trizol, and then total RNA was extracted using the RNAsimple Total RNA Extraction Kit (TIANGEN, product number: DP419), and stored in DEPC aqueous solution containing RNase inhibitors (Abcam, product number: ab146240) middle. After the RNA sample is detected by the Agilent 2100 bioanalyzer, the total amount is not less than 2ug, the concentration is not lower than 50ng / ul, and the RIN value is not lower than 7. Samples that meet this standard are constructed using NEB's transcriptional library building kit (NEB, catalog number: E7530). After passing the library inspection, the RNA was sequenced through the sequencing platform of Illumina Company, and each sample generated no less than 8Gb of data, the sequencing mode was 150PE, and the insert size was 280bp.

[0046] After the data is...

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Abstract

The invention provides a method for ex vivo expansion of human vascular endothelial progenitor cells in a low oxygen condition. The method comprises the following steps: separating EPCs from human bone marrow, culturing the EPCs in an incubator in the low oxygen condition, and ensuring that the low oxygen condition adopts a 1 percent O2 low oxygen condition. Gene expression to the EPCs by the lowoxygen condition comprises various gene differential expression, and at least relates to the following biological processes: reaction of cells to fatty acid, and adjustment of positive regulation of the activity of ubiquitin-protein ligase with transformed caryomitosis cell cycles. Compared with normoxia culture, the culture method has the advantages that low oxygen environment is more similar tobone marrow microenvironment, and is more beneficial to keeping the primary characteristics of the EPCs; the method performs molecular level evaluation on change of gene expression in EPCs caused by different culture methods, gives differential expression genes in which the low oxygen condition or anoxic condition affects cell dryness and molecular pathways of the differential expression genes, and is applied to improvement of the current stem cells.

Description

technical field [0001] The invention belongs to the field of medicine and biology, and in particular relates to a method for expanding human vascular endothelial progenitor cells in vitro under hypoxic conditions. Background technique [0002] Bone marrow contains progenitor cells that can migrate to peripheral blood and differentiate into mature endothelial cells, which are called vascular endothelial progenitor cells (EPCs), which are the precursor cells of vascular endothelial cells, mainly found in umbilical cord blood, adult peripheral blood, bone marrow. Endothelial progenitor cells and hematopoietic stem cells come from the common mesoderm precursor blood angioblasts. Under the stimulation of physiological or pathological factors, they can mobilize from bone marrow to peripheral blood to participate in the repair of damaged blood vessels, while endothelial progenitor cells in cord blood originate from in the fetal liver. Studies in recent years have shown that vascu...

Claims

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Application Information

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IPC IPC(8): C12N5/071
CPCC12N5/0692C12N2500/02
Inventor 陈镇洲林依秋刘兵钟健冯卓威
Owner 广州赛琅生物技术有限公司
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