Efficient amplification culture system for human vascular endothelial progenitor cells

A technology for endothelial progenitor cells and endothelial cells, which is applied in the field of high-efficiency expansion and culture system of human vascular endothelial progenitor cells, and can solve the problems such as the inability to fully maintain the stemness of progenitor cells and the inability to achieve large-scale production.

Active Publication Date: 2016-02-17
BIOPHARMAGEN CORP FANGZHOU SUZHOU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, the culture technology of endothelial progenitor cells can obtain a certain amount for scientific resear

Method used

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  • Efficient amplification culture system for human vascular endothelial progenitor cells
  • Efficient amplification culture system for human vascular endothelial progenitor cells
  • Efficient amplification culture system for human vascular endothelial progenitor cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0124] Embodiment 1, the preparation of culture medium

[0125] The culture system of the present invention comprises CD34 + Stem cell expansion, endothelial progenitor cell adherent expansion and differentiation in two parts:

[0126] 1. CD34 + Stem cell expansion phase: stem cell expansion medium

[0127] Adopt ModifiedIMDM medium (serum-free) as basal medium, add the cytokine selected from any formula in Table 3 wherein:

[0128] Table 3 (unit: ng / mL)

[0129]

Formula A

Formula B

Formula C

SCF

200

100

400

FLT-3L

200

100

400

TPO

20

10

150

IL-3

10

5

200

GM-CSF

12.5

8

20

Sall4B

3

2

8

[0130] 2. Adherence expansion period: endothelial progenitor cell expansion and differentiation medium

[0131] Adopt EGM-2 medium (20% fetal calf serum) as basal medium, add the cytokine and other components selected from any formula in Table 4 th...

Embodiment 2

[0134] Embodiment 2, on human umbilical cord blood CD34 + Expansion and differentiation of endothelial progenitor cells derived from stem cells (I)

[0135] Day 0:

[0136] (1) Preparation of CD34 + Stem Cell Expansion Medium

[0137] Add various cytokines to ModifiedIMDM medium (serum-free), so that the final concentration of cytokines is SCF200ng / mL, Flt-3L200ng / mL, IL-310ng / mL, TPO20ng / mL, Sall4B3ng / mL, GM-CSF12.5ng / mL (i.e. formula A in Example 1) as the expansion medium.

[0138] (2) Separation and purification of cord blood CD34 + stem cell

[0139] Take fresh umbilical cord blood from full-term healthy newborns, and use Miltenyi's MACS magnetic bead sorting system to separate CD34 + Mononuclear cells, the number of cells obtained was 1.5 x 10 6 cells. CD34 + Stem cell expansion medium to resuspend the cells and adjust the cell concentration to 15×10 5 cells / mL, aspirate 100 μL of cell suspension and add 1 mL of CD34 per well in a 24-well plate + Stem cell ex...

Embodiment 3

[0159] Embodiment 3, after mobilizing human peripheral blood CD34 + Expansion and differentiation of endothelial progenitor cells derived from stem cells (I)

[0160] Peripheral blood collection after human bone marrow mobilization: 5 days before collection, healthy adult volunteers were given G-CSF at 5 μg / kg, mobilized continuously for 3 days, and 200-300 mL of peripheral blood was collected each time.

[0161] Day 0:

[0162] (1) Preparation of CD34 + Stem Cell Expansion Medium

[0163] Add various cytokines to ModifiedIMDM medium (serum-free), so that the final concentration of cytokines is SCF200ng / mL, Flt-3L200ng / mL, IL-310ng / mL, TPO20ng / mL, Sall4B3ng / mL, GM-CSF12.5ng / mL (i.e. formula A in Example 1) as the expansion medium.

[0164] (2) Separation and purification of peripheral blood CD34 + stem cell

[0165] Use 10 mL of fresh mobilized peripheral blood, dilute it 20 times with PBS, and use Miltenyi’s MACS magnetic bead sorting system to separate CD34 + Mononuc...

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Abstract

The invention relates to an efficient amplification culture system for human vascular endothelial progenitor cells and provides an efficient amplification culture method for endothelial progenitor cells, giving effective and mass amplification to CD34+ mononuclear cells at the premise of keeping their original dryness. The inventor also optimizes an endothelial growth factor combination attachment culture technique to amplify and differentiate endothelial progenitor cells. The technical scheme of the invention may provide mass endothelial progenitor cells and solve the source and quantity problems of endothelial progenitor cells or endothelial cells of a patient with ischemic disease and hemophilia A during transplantation therapy.

Description

technical field [0001] The invention relates to the field of cell biology, more specifically, the invention relates to a high-efficiency expansion culture system of human vascular endothelial progenitor cells. Background technique [0002] Endothelial progenitor cells (EPCs) are the precursor cells of vascular endothelial cells, which mainly exist in umbilical cord blood, adult peripheral blood and bone marrow. Endothelial progenitor cells and hematopoietic stem cells come from the common mesoderm precursor blood angioblasts (Hemangioblast). 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 cells in cord blood Progenitor cells originate in the fetal liver. Recent studies have shown that endothelial progenitor cells play an important role in cardiovascular and cerebrovascular diseases, peripheral vascular diseases, tumor angiogenesis, ...

Claims

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Application Information

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IPC IPC(8): C12N5/071
Inventor 秦蒙蒋永平戴卫
Owner BIOPHARMAGEN CORP FANGZHOU SUZHOU
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