A high-efficiency expansion culture system of non-human primate vascular endothelial progenitor cells

A primate, endothelial progenitor cell technology, applied in the field of high-efficiency expansion culture system of non-human primate vascular endothelial progenitor cells

Active Publication Date: 2019-06-21
BIOPHARMAGEN CORP FANGZHOU SUZHOU
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, there is currently no expansion method for non-human primate-derived vascular endothelial progenitor cells in this field. In order to better conduct preclinical in vivo functional studies at the level of non-human primates, an optimized non-human The culture technology of human primate vascular endothelial progenitor cells needs to be established urgently

Method used

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  • A high-efficiency expansion culture system of non-human primate vascular endothelial progenitor cells
  • A high-efficiency expansion culture system of non-human primate vascular endothelial progenitor cells
  • A high-efficiency expansion culture system of non-human primate vascular endothelial progenitor cells

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Experimental program
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Embodiment 1

[0106] Embodiment 1, the preparation of culture medium

[0107] The culture system of the present invention comprises two parts: CD34+ stem cell expansion and endothelial progenitor cell adherent expansion and differentiation:

[0108] 1. CD34+ stem cell expansion stage: stem cell expansion medium

[0109] Modified IMDM medium (serum-free) is adopted as the basal medium, and cytokines selected from any formula in Table 3 are added thereto:

[0110] Table 3 (unit: ng / mL)

[0111] Formula A Formula B Formula C SCF 200 100 400 FLT-3L 200 100 400 TPO 20 10 150 IL-3 10 5 20 GM-CSF 12.5 8 20 G-CSF 12.5 8 20 Sall4B 3 2 8 VEGF 50 25 75

[0112] 2. Adherence expansion period: endothelial progenitor cell expansion and differentiation medium

[0113] Adopt EBM-2 medium (10% or 20% fetal bovine serum) as basal medium, add the cytokine and other components selected from any formula in Table 4 wherein:

[...

Embodiment 2

[0116] Example 2. Expansion and differentiation of endothelial progenitor cells derived from monkey bone marrow CD34+ stem cells (I)

[0117] Day 0:

[0118] (1) Preparation of CD34 + Stem Cell Expansion Medium

[0119] Various cytokines were added to Modified IMDM medium (serum-free), so that the final concentration of cytokines was SCF200ng / mL, Flt-3L 200ng / mL, IL-310ng / mL, TPO 20ng / mL, Sall4B3ng / mL, GM- CSF 12.5 ng / mL, G-CSF 12.5 ng / mL, and VEGF 50 ng / mL (formula A in Example 1) were used as the expansion medium.

[0120] (2) Separation and purification of CD34 in bone marrow + stem cell

[0121] Take 8-10 mL of femoral bone marrow from the anterior superior iliac spine of healthy young cynomolgus monkeys, dilute it 10 times with PBS, obtain mononuclear cells by density gradient centrifugation, and use BD’s CD34 antibody (APC-mouse anti-human CD34) and US Tianyi company MACS magnetic beads (Rat anti-mouse IgG) two-step sorting method to separate CD34 + Mononuclear cel...

Embodiment 3

[0137] Example 3. Expansion and differentiation of endothelial progenitor cells derived from monkey bone marrow CD34+ stem cells (II)

[0138] Day 0:

[0139] (1) Preparation of CD34 + Stem Cell Expansion Medium

[0140] Various cytokines were added to Modified IMDM medium (serum-free), so that the final concentration of cytokines was SCF100ng / mL, Flt-3L 100ng / mL, IL-35ng / mL, TPO 10ng / mL, Sall4B2ng / mL, GM- CSF 8 ng / mL, G-CSF 8 ng / mL, and VEGF 25 ng / mL (ie formula B in Example 1) were used as the expansion medium.

[0141] (2) Separation and purification of CD34 in bone marrow + stem cell

[0142] Take 6-8 mL of femoral bone marrow from the anterior superior iliac spine of healthy young cynomolgus monkeys, dilute it 10 times with PBS, and use the corresponding method in Example 2 to sort CD34 + cells, the number of cells obtained was 1.2×10 6 cells. CD34 + Stem cell expansion medium to resuspend the cells and adjust the cell concentration to 5×10 5 cells / mL, added to a...

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Abstract

The invention relates to an efficient amplification culture system of non-human primate animal blood vessel endothelial progenitor cells, and provides a novel efficient amplification culture method of non-human primate animal blood vessel endothelial progenitor cells. The method has the advantages that endothelial progenitor cells / endothelial cells with the endothelial active function can be efficiently obtained, so that the cell number required in the preclinical study of primate animal self body transfusion transplantation can be met; an effective path is provided for applying an endothelial progenitor cell amplification culture technology to clinic treatment.

Description

technical field [0001] The invention belongs to the field of cell biology, and more specifically, the invention relates to a high-efficiency expansion culture system of non-human primate vascular endothelial progenitor cells. Background technique [0002] Cell therapy is a new disease treatment technology that has emerged in recent years. It uses the characteristics of certain cells with specific functions to achieve the purpose of treating diseases through in vitro expansion and special culture. Endothelial progenitor cells (EPCs) are the precursor cells of vascular endothelial cells, mainly present in umbilical cord blood, adult peripheral blood, and bone marrow. Recent studies have shown that endothelial progenitor cells play an important role in cardiovascular and cerebrovascular diseases, peripheral vascular diseases, tumor angiogenesis, and wound healing. It can be used as a cell therapy approach for FVIII-deficient hemophilia A. It has been reported that normal endot...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/071C12N5/0783
Inventor 蒋永平秦蒙关欣
Owner BIOPHARMAGEN CORP FANGZHOU SUZHOU
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