Umbilical cord mesenchymal stem cell separation method

A separation method and stem cell technology, which can be applied to animal cells, vertebrate cells, bone/connective tissue cells, etc., can solve the problems of mixed endothelial cells, low purity and high cost, improve separation efficiency, and have good application prospects. The effect of increasing the number

Inactive Publication Date: 2016-04-27
SICHUAN NEO LIFE STEM CELL BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the commonly used separation methods at home and abroad mainly include enzymatic digestion and tissue block adhesion. The advantages of simple operation, low cost and less cell damage make it more suitable for clinical application. However, the existing conventional method of tissue block adhesi

Method used

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  • Umbilical cord mesenchymal stem cell separation method
  • Umbilical cord mesenchymal stem cell separation method
  • Umbilical cord mesenchymal stem cell separation method

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Effect test

Embodiment 1

[0031] Embodiment 1 separation and purification method of the present invention

[0032] Petri dish pretreatment: 10cm cell culture dish was coated with 1ml 0.2% gelatin overnight to remove residual gelatin;

[0033] Isolation of umbilical cord mesenchymal stem cells by tissue block method: cut the fresh umbilical cord into 1 cm length with ophthalmic scissors, wash the blood stains of the umbilical cord with PBS; cut the umbilical cord to 2-3 mm per 1 cm length 3 Large and small tissue pieces, and evenly spread in the culture dish; place at 37°C 5% CO 2 Cultivate under the same conditions for 2 hours, then add 10ml of mesenchymal stem cell medium (STEMPROMSCSFM, purchased from Life Technologies) and continue to cultivate under the same conditions; half change the medium on the 5th day; fully change the medium on the 8th day and remove all tissue pieces ; Harvest the cells when the cell confluency is 30-40%.

Embodiment 2

[0034] Embodiment 2 separation and purification method of the present invention

[0035] Petri dish pretreatment: 10cm cell culture dish was coated with 1ml 2% Cellstart matrix overnight to remove residual Cellstart matrix;

[0036] Isolation of umbilical cord mesenchymal stem cells by tissue block method: cut the fresh umbilical cord into 1 cm length with ophthalmic scissors, wash the blood stains of the umbilical cord with PBS; cut the umbilical cord to 2-3 mm per 1 cm length 3 Large and small tissue pieces, and evenly spread in the culture dish; place at 37°C 5% CO 2 Cultivate under the same conditions for 2 hours, then add 10ml of mesenchymal stem cell medium (STEMPROMSCSFM) and continue to cultivate under the same conditions; half change the medium on the 5th day; fully change the medium on the 8th day and remove all tissue pieces; Cells were harvested when 30-40%.

Embodiment 3

[0037] Embodiment 3 separation and purification method of the present invention

[0038] Petri dish pretreatment: 10cm cell culture dish was coated with 1ml 0.1% gelatin overnight to remove residual gelatin;

[0039] Isolation of umbilical cord mesenchymal stem cells by tissue block method: cut the fresh umbilical cord into 1.5 cm length with ophthalmic scissors, wash the blood stains of the umbilical cord with PBS; cut the umbilical cord to 2-3 mm per 1.5 cm length 3 Large and small tissue pieces, and spread evenly in gelatin pretreated culture dishes; place at 37°C 5% CO 2 Cultivate under the same conditions for 2 hours, then add 10ml of mesenchymal stem cell medium (STEMPROMSCSFM) and continue to cultivate under the same conditions; half change the medium on the 5th day; fully change the medium on the 8th day and remove all tissue pieces; Cells were harvested when 30-40%.

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Abstract

The invention discloses an umbilical cord mesenchymal stem cell separation method. The umbilical cord mesenchymal stem cell separation method includes the steps of (1), taking fresh umbilical cords, cutting the fresh umbilical cords short and removing blood stains; (2) cutting the fresh umbilical cords into tissue blocks in the size of 2-3mm<3>, uniformly spreading the tissue blocks in a culture dish coated with an extracellular matrix, culturing at the temperature of 37 DEG C under 5% CO2 for 1-4 hours, adding a mesenchymal stem cell medium and continuing culturing at the temperature of 37 DEG C under 5% CO2; (3) renewing half of solution of the culture medium in the fifth day of culturing, removing all the tissue blocks and renewing all solution of the culture medium in the eighth day, and harvesting cells when cell confluence is 30-40%. The umbilical cord mesenchymal stem cell separation method is capable of separating the umbilical cord mesenchymal stem cells efficiently and is simple to operate and promising in application prospect.

Description

technical field [0001] The invention relates to a method for separating umbilical cord mesenchymal stem cells. Background technique [0002] Mesenchymal stem cells (MesenchymalStemCell, MSC) is a group of stem cells with strong proliferation ability and multi-lineage differentiation potential. It has remarkable effects in immune regulation and anti-inflammatory response, so it is widely used in clinical research and treatment, and its indications include transplantation Drug-versus-host reaction (GVHD), nervous system disease, diabetes, liver disease and other major diseases. [0003] Umbilical cord mesenchymal stem cells are isolated from the umbilical cord of newborns, and have the characteristics of more primitive, stronger proliferative ability and lower immunogenicity. At the same time, umbilical cord mesenchymal stem cells are rich in sources, convenient to obtain materials, and have no ethical restrictions. It is currently the most important source of mesenchymal ste...

Claims

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Application Information

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IPC IPC(8): C12N5/0775
Inventor 钟立武赖真阳范兆心伍明俊张博陈强
Owner SICHUAN NEO LIFE STEM CELL BIOTECH
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