Method for obtaining human endometrial mesenchymal stem cells from curettage samples

A quality stem cell and sample technology, applied in the field of stem cell isolation, culture and preservation, can solve problems such as ethical restrictions and difficulties in the source of stem cells, and achieve the effects of quality assurance, convenient collection, and rapid proliferation

Inactive Publication Date: 2011-12-28
HANGZHOU S EVANS BIOSCI LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The purpose of the present invention is to find a more efficient method for isolating and culturing endometrial stem cells that has a wide r

Method used

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  • Method for obtaining human endometrial mesenchymal stem cells from curettage samples
  • Method for obtaining human endometrial mesenchymal stem cells from curettage samples
  • Method for obtaining human endometrial mesenchymal stem cells from curettage samples

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1: Separation, cultivation, expansion, cryopreservation, and recovery of human endometrial stem cells obtained through curettage culture:

[0051] 1), Prepare the collection kit and prepare the culture medium:

[0052] Negative pressure collection bottle (sterile).

[0053] Collection solution: Add DMEM basal medium (ie, DMEM medium) to 2000 mg of ciprofloxacin, 5000 mg of kanamycin and 400 units of heparin until the volume reaches 500 ml to obtain a collection solution.

[0054] That is, the 500 ml collection fluid contains 400 units of heparin, ciprofloxacin at a concentration of 4 mg / mL, and kanamycin at a concentration of 10 mg / mL.

[0055] Before use, the collection solution was stored at 2-4°C.

[0056] Prepare Chang’s complete medium: under sterile conditions, add 65mL of MEM-alpha medium (MEM alpha, Invitrogen Company), 18mL of Chang B base solution (Irvine Scientific Company), 2mL of Chang C base solution (Irvine Scientific Company) into a sterile con...

Embodiment 2

[0096] Embodiment 2, cell recovery culture

[0097] The frozen samples obtained in the above-mentioned Example 1 were taken out from the liquid nitrogen storage tank, and immediately put into a 37° C. water bath to thaw. When it is observed that the ice nuclei in the freezing tube are only the size of soybeans, take out the freezing tube and put it into the ice-water mixture immediately. Under sterile conditions, gently aspirate the sample in the cryopreservation tube, add it to a 50mL sterile centrifuge tube, then immediately add 10mL of Chang's complete medium, mix well and wash, and centrifuge at 4°C and 1000rpm for 10 minutes, remove the supernatant from the tube. Repeat the operation once. Cell counts and viability analysis were performed with disc blue. According to 10000 pieces / cm 2 Cells were seeded at a density of 75 cm (using Chang's complete medium) 2 In a culture bottle, put in 37°C, saturated humidity, 5% CO 2 Incubator cultivation. According to the growth ...

Embodiment 3

[0098] Example 3. Flow Cytometry

[0099] The endometrial mesenchymal stem cells cultured at passage P5 obtained in Example 1 were selected for flow cytometric detection according to the following steps.

[0100] Each test tube contains a concentration of 5*10 6 / ml of P5 generation cells 2ml.

[0101] 1) Cell suspension preparation:

[0102] The cells were digested with 1 ml of 0.25% (mass concentration) trypsin, centrifuged at 500 rpm for 5 min, and the supernatant was discarded. Add PBS to wash 2 times, then resuspend the cells in 2ml PBS, so that the cell concentration is 5*10 6 / ml.

[0103] 2) Fix with 4% paraformaldehyde (prepared in PBS) for 15 minutes, then wash with PBS twice (1500 rpm, centrifuge for 5 minutes).

[0104] 3) Fix with 5% BSA for 40 minutes.

[0105] 4) According to the dilution requirement of flow cytometry antibody (direct standard) every 10 6 20ul of antibodies (CD29, CD45, CD105, CD117, CD90, CD34, CD73, CD44, HLA-DR) were added to each cell...

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Abstract

The invention discloses a method for obtaining human endometrial mesenchymal stem cells from curettage samples, which comprises the following steps: 1) mixing artificial abortion products and collection liquid according to the volume ratio of 1:0.9-1.1; obtaining curettage samples; 2 ) Separation and culture of endometrial stem cells: after the curettage sample is incubated on a shaker, it is filtered; the filtrate is obtained; then the fragmented tissue and monocytes in the blood are collected by density gradient centrifugation; then the cells are cultured, expanded and Purification; 3) After the P1 generation cells obtained in the above steps covered the bottom of the bottle, digest the cells with trypsin to collect the cells, resuspend the cells with pre-cooled freezing solution to obtain a cell suspension, and freeze the above cell suspension, The resulting frozen samples were stored in liquid nitrogen storage tanks. The method of the invention can obtain target cells with high purity and large quantity, that is, endometrial stem cells.

Description

technical field [0001] The invention belongs to the technology related to the separation, culture and preservation of stem cells. Background technique [0002] Stem cell technology is one of the hottest technologies in life science today. Its research content involves almost all biomedical fields of life science. In addition to playing an important role in cell therapy, tissue / organ transplantation and gene therapy, it is also discovering new genes , Gene function analysis, developmental biology models and new drug development have an important impact. In recent years, major breakthroughs have been made in the research of stem cells. In 1999 and 2000, the world's most authoritative American magazine "Science" listed stem cells and the Human Genome Project as the top 10 scientific breakthroughs of the year for two consecutive years. Stem cells are likely to lead to revolutionary progress in the medical field, so they have immeasurable medical value. Stem cells and the Human ...

Claims

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Application Information

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IPC IPC(8): C12N5/0775
Inventor 项春生
Owner HANGZHOU S EVANS BIOSCI LTD
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