Method for separating and purifying mesenchymal stem cells originated from formation tissue

A mesenchymal stem cell and tissue technology, applied in the field of separation and purification of mesenchymal stem cells, can solve the problems of cumbersome separation and purification methods, high cost, and inapplicability of mesenchymal stem cell separation and purification, achieve good sorting effect and improve purification efficiency Effect

Active Publication Date: 2009-09-16
海南迪森生物科技有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this separation and purification method is cumbersome and costly, and is not suitable

Method used

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  • Method for separating and purifying mesenchymal stem cells originated from formation tissue
  • Method for separating and purifying mesenchymal stem cells originated from formation tissue
  • Method for separating and purifying mesenchymal stem cells originated from formation tissue

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Take the decidua tissue on the maternal side of the placenta, cut it into small pieces, take 5g of the fragments and add it to 10mL containing 0.1% (w / v, ie 1g / L, 1g of enzyme per 1L of digestive juice) type IV collagenase (Sigma company, 125U / mg) in the digestive solution (distilled solution solvent is double-distilled water) and digested for 30 minutes to collect the cells, with cell culture medium (adding 10% fetal bovine serum (Hangzhou Sijiqing Bioengineering Materials Co., Ltd.) and 2mM L-glucose Aminoamide (Sigma Company) was suspended in DMEM-LG (Hyclone Company) culture medium (prepared by adding 100 mL fetal bovine serum and 2 mmol L-glutamine to 900 mL of basic DMEM-LG culture liquid, the same below), and the suspended cells were collected for different Time period of adherent culture.

[0038]1. After the collected suspension cells are cultured in a 6-well culture plate for 8 hours, collect the unattached suspension cells, keep the cells that have been attac...

Embodiment 2

[0044] Example 2: Screening of clones of cells:

[0045] According to the results of Example 1, the cells sorted by the optimal adherence time period (72 hours) were screened for cell clone culture. Attached cells were digested with Tryspin-EDTA and harvested for a period of 72 hours. The harvested cells were washed once with PBS and then suspended in the cell culture medium (preparation: 100mL fetal bovine serum, 2mmol L-glutamine, 900mL basic DMEM-LG medium mixed), and carried out cell density gradient release by limiting dilution method Afterwards, cell clones were cultured in 96-well culture plates. After the cell clones are formed, the clones of single cells are collected, and then transferred to 24-well culture plates for subculture and expansion culture. See Figure 1(A) for the cell clone formed by a single cell; Figure 1(B) for the cell morphology after the cloned cells are expanded and layered in a 24-well culture plate, which is long fiber-like cells.

[0046] The...

Embodiment 3

[0047] Embodiment 3: comparative analysis of amplification ability

[0048] Collect the cells attached to the best adherent time period (72 hours), and placenta tissue cells digested with 0.1% type IV collagenase directly adhere to the adherent cells in a 6-well culture plate and culture them for 72 hours (not carried out). Adhered cells sorted by time-period adherence) were used as control, cultured in a 24-well culture plate with cell culture medium (preparation: 100mL fetal bovine serum, 2mmol L-glutamine, 900mL basal DMEM-LG medium mixed) , and assayed for cell expansion capacity. Experimental results such as figure 2 , where —◇—indicates adherent cells that have not been sorted by time-period adherence, and —◆—indicates cells that adhere to the best adherence time period (72 hours).

[0049] From figure 2 According to the analysis of the comparison results, the cells attached in the best time period (72 hours) have a significant growth advantage: the cells grow rapidly...

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Abstract

The invention provides a method for separating and purifying mesenchymal stem cells originated from formation tissues, the invention implements adherence screening of different time periods in accordance with the adherence ability of different types of the cells in the formation tissues, screens adherent cells having the optimal adherence time period after a specific surface antigen assay of the mesenchymal stem cells of the adherent cell in different time periods, and performs clone culture formed by cell colony subsequent to collecting and suspending the cells so as to obtain the purified mesenchymal stem cells of the formation tissues. The invention has the beneficial effects that: (1) the inventive method can reach relatively good screening effect; (2) using a single-cell clone method for the cells screened by the optimal adherence time period further improves the purification rate of the mesenchymal stem cells of the formation tissues; (3) the mesenchymal stem cells of the formation tissues screened by the optimal adherence time period have high proliferation potential; (4) the mesenchymal stem cells of the formation tissues screened by the optimal adherence time period has outstanding multi-differentiation potential.

Description

(1) Technical field [0001] The invention relates to a method for separating and purifying mesenchymal stem cells derived from formed tissues. (2) Background technology [0002] Stem cells with certain differentiation potential may exist in various tissues of the human body. Relevant studies have shown that, in addition to bone marrow and umbilical cord blood, there are also cells that can differentiate into osteoblasts, chondrocytes, adipocytes, and nerve cells in other formed tissues, such as fat, neonatal umbilical cord, and placenta. These stem cells are collectively referred to as mesenchymal stem cells (MSCs). These mesenchymal stem cells may be of great value in cell transplantation and tissue engineering in medicine. [0003] Since no specific marker molecule for mesenchymal stem cells has been found so far, the isolation and purification of the stem cells is still an indirect method: that is, screening and purification are carried out by the method of adhesion to t...

Claims

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Application Information

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IPC IPC(8): C12N5/08
Inventor 王金福袁文佶
Owner 海南迪森生物科技有限责任公司
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