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Separation culture method for endothelial progenitor cells and kit of method

An endothelial progenitor cell, separation and culture technology, which is applied in the field of the separation and culture method of umbilical cord blood endothelial progenitor cells and a kit thereof, can solve the problems of limited endothelial progenitor cells, low amplification efficiency, slow cell proliferation and the like, and achieves cell surface antigen The effect of high expression rate, large cell harvest, and high cell confluency

Inactive Publication Date: 2015-08-19
GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, each cord blood contains limited endothelial progenitor cells, in order to meet the requirements of therapeutic transplantation, EPCs need to be further expanded in vitro
The existing method of separating and culturing endothelial progenitor cells in blood generally adopts the method of coating the culture dish, and then isolates mononuclear cells from the blood and then adheres to the wall for culture. This method is inefficient and can only isolate a small amount of endothelial progenitor cells from blood. The subsequent amplification efficiency is also low, and the cell proliferation is slow

Method used

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  • Separation culture method for endothelial progenitor cells and kit of method
  • Separation culture method for endothelial progenitor cells and kit of method
  • Separation culture method for endothelial progenitor cells and kit of method

Examples

Experimental program
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Effect test

Embodiment 1

[0040] Example 1 Isolation and culture of umbilical cord blood EPCs

[0041] Umbilical cord blood was obtained from the umbilical cords of full-term healthy newborns, anticoagulated with compound sodium citrate (CPD-A), and separated within 24 hours after collection.

[0042] Dilute the umbilical cord blood with PBS at a ratio of 1:1, centrifuge at 400g at 20°C for 10 minutes, discard the supernatant and fat layer, resuspend the cell pellet with an appropriate amount of PBS, and carefully add an equal volume of Ficoll-paque lymphocyte separation medium (density 1.077× 10 3 g / L), centrifuge at 600g for 30min at 20°C, and collect the mononuclear cell layer.

[0043] After washing twice with PBS, the mononuclear cells were resuspended in DMEM+10% FBS complete culture solution, and the cell concentration was adjusted to 10 7 / mL, planted in a 35mm Petri dish covered with gelatin, the culture condition is DMEM medium containing 10% FBS, add VEGF 20ng / mL, FGF 20ng / mL, IGF 1ng / mL, ...

Embodiment 2

[0046] Example 2 Isolation and culture of umbilical cord blood EPCs

[0047] Umbilical cord blood was obtained from the umbilical cords of full-term healthy newborns, anticoagulated with compound sodium citrate (CPD-A), and separated within 24 hours after collection.

[0048] Dilute the umbilical cord blood with PBS at a ratio of 1:1, centrifuge at 400g at 20°C for 10 minutes, discard the supernatant and fat layer, resuspend the cell pellet with an appropriate amount of PBS, and carefully add an equal volume of Ficoll-paque lymphocyte separation medium (density 1.077× 10 3 g / L), centrifuge at 600g for 30min at 20°C, and collect the mononuclear cell layer.

[0049] After washing twice with PBS, the mononuclear cells were resuspended in DMEM+10% FBS complete culture solution, and the cell concentration was adjusted to 10 7 / mL, planted in a 35mm Petri dish covered with gelatin, the culture condition is DMEM medium containing 10% FBS, add VEGF 20ng / mL, FGF 20ng / mL, IGF 1ng / mL, ...

Embodiment 3

[0052] Example 3 Isolation and culture of umbilical cord blood EPCs

[0053] Umbilical cord blood was obtained from the umbilical cords of full-term healthy newborns, anticoagulated with compound sodium citrate (CPD-A), and separated within 24 hours after collection.

[0054] Dilute the umbilical cord blood with PBS at a ratio of 1:1, centrifuge at 400g at 20°C for 10 minutes, discard the supernatant and fat layer, resuspend the cell pellet with an appropriate amount of PBS, and carefully add an equal volume of Ficoll-paque lymphocyte separation medium (density 1.077× 10 3 g / L), centrifuge at 600g for 30min at 20°C, and collect the mononuclear cell layer.

[0055] After washing twice with PBS, the mononuclear cells were resuspended in DMEM+10% FBS complete culture solution, and the cell concentration was adjusted to 10 7 / mL, planted in a 35mm Petri dish covered with gelatin, the culture condition is DMEM medium containing 10% FBS, add VEGF 20ng / mL, FGF 20ng / mL, IGF 1ng / mL, ...

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Abstract

The invention relates to the technical field of separation culture of endothelial progenitor cells of umbilical blood, particularly a separation culture method for endothelial progenitor cells of umbilical blood and a kit of the method. The method comprises the following steps: carrying out adherent culture on a mononuclear cell of umbilical blood in advance; and carrying out cell culture on non-adherent cells, wherein a culture medium for cell culture is a full culture medium containing fetuin. Compared to cells obtained by conventional methods, the endothelial progenitor cells of umbilical blood cultured by the method provided by the invention are relatively uniform in shape, relatively high in convergence degree, relatively great in cell harvest yield, relatively fast in cell proliferation and relatively high in antigen presentation rate on cell surface.

Description

technical field [0001] The invention relates to the technical field of separation and culture of endothelial progenitor cells, in particular to a method for separation and culture of umbilical cord blood endothelial progenitor cells and a kit thereof. Background technique [0002] Cardiovascular and cerebrovascular diseases are collectively referred to as cardiovascular and cerebrovascular diseases, and generally refer to ischemic or hemorrhagic diseases in the heart, brain, and systemic tissues caused by hyperlipidemia, blood viscosity, atherosclerosis, and high blood pressure. . It is a common disease that seriously threatens the health of human beings, especially middle-aged and elderly people over 50 years old. Even with the most advanced and perfect treatment methods, more than 50% of the survivors of cerebrovascular accidents still cannot take care of themselves completely. The number of people dying from cardiovascular and cerebrovascular diseases is as high as 15 mi...

Claims

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Application Information

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IPC IPC(8): C12N5/0789
Inventor 陈海佳王一飞葛啸虎黎娟妹王小燕李平何炜均
Owner GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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