Method for storing endometrial stem cells

A technology of endometrial stem cells and cells, which is applied in the corresponding culture and storage technology field, and can solve problems such as ethical restrictions and difficulties in the source of stem cells

Inactive Publication Date: 2011-08-17
HANGZHOU S EVANS BIOSCI LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Aiming at the problems of existing stem cell source difficulties or ethical restrictions, the present invention seeks a more efficient

Method used

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  • Method for storing endometrial stem cells
  • Method for storing endometrial stem cells
  • Method for storing endometrial stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1: A method for storing endometrial stem cells, that is, separation, culture, expansion, cryopreservation, and recovery of human endometrial stem cells; the following steps are performed in sequence:

[0042] 1). Prepare collection kit and preparation medium:

[0043] Collection tube 1: It contains 20ml Hank's Balanced Salt Solution (HBSS), and add the following ingredients to the following concentrations: Vancomycin (Vancomycin) 70μg / mL, Cephalexin (Claforan) 200μg / mL, Kanamycin (Amikacin) ) 100μg / mL, Gentamycin (Gentamycin) 140μg / mL, Amphotericin B (Amphotericin B) 2.5μg / mL and 400 units of heparin added.

[0044] Collection tube 2: filled with 10ml HBSS (Hank's balanced salt solution), and add 180 units of heparin.

[0045] Before starting the collection of menstrual blood in step 2), the collection tube 1 and collection tube 2 should be stored at 4°C.

[0046] Prepare Chang complete medium: under aseptic conditions, add 65mL MEM-alpha medium (MEM alpha, Invitrogen co...

Embodiment 2

[0082] Example 2. Cell resuscitation culture

[0083] The frozen sample obtained in Example 1 above was taken out of the liquid nitrogen storage tank and immediately put into a 37°C water bath to thaw. When it is observed that the ice core in the cryopreservation tube is only the size of a soybean, remove the cryopreservation tube and immediately put it into the ice-water mixture. Under aseptic conditions, gently aspirate the sample from the cryopreservation tube, add it to a 50mL sterile centrifuge tube, and immediately add 10mL of Chang complete medium, mix well, wash, and centrifuge at 4℃ and 1000rpm for 10 Minutes, remove the supernatant from the tube. Repeat the operation once. Use plate blue for cell counting and viability analysis. According to 10000 / cm 2 The density (using Chang complete medium) to inoculate the cells to 75 cm 2 In the culture flask, put CO at 37℃, saturated humidity, and 5% volume fraction 2 Culture in an incubator. According to the growth of the cel...

Embodiment 3

[0084] Example 3. Flow Cytometry

[0085] The endometrial mesenchymal stem cells cultured at generation P5 obtained in Example 1 were selected for flow cytometry as follows.

[0086] Each test tube contains a concentration of 5*10 6 / ml 2ml of P5 generation cells.

[0087] 1) Preparation of cell suspension:

[0088] Digest the cells with 1 ml of 0.25% (mass concentration) trypsin, centrifuge at 500 rpm for 5 min, and discard the supernatant. Wash twice with PBS, then resuspend the cells in 2ml PBS to make the cell concentration 5*10 6 / ml.

[0089] 2) Fixed with 4% paraformaldehyde (prepared in PBS) for 15 minutes, and then washed twice with PBS (1500 rpm, centrifugation for 5 minutes).

[0090] 3) 5% BSA is fixed for 40 minutes.

[0091] 4) According to the dilution requirement of flow cytometry antibody (direct standard) every 10 6 Add 20ul antibodies (CD39, CD44, CD90, CD34, HLA (ABC), HLA (DR-DP-DQ)) to each cell / 200ul, protect from light, and incubate at 37°C for 30 min.

[0092] 5) ...

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Abstract

The invention discloses a method for storing endometrial stem cells, which comprises the following steps: 1) preparing a collection tool set and preparing a culture medium; 2) collecting menses; 3) performing a sterility test; and 4) subjecting endometrial stem cell to separation culture, namely, filtering mixed liquid, which is obtained by the step 2), in a collection tube 1, collecting single karyoplasts by using a density gradient centrifugation method, inoculating the obtained single karyoplasts in a Chang complete culture medium at an inoculation density of single karyoplasts of 1*10<5> to 1*10<6>/ml, and culturing in a culture tank which is at 37 DEG C and saturated humidity and contains CO2 at a volume percentage concentration of 5 percent; 5) amplifying and purifying cells; and 6) storing cells by freezing. When the method of the invention is used, a large number of high-purity target cells, namely endometrial stem cells, can be obtained.

Description

Technical field [0001] The present invention belongs to the related technology of cell separation, culture and preservation, especially separation of endometrial mesenchymal stem cells from menstrual blood, establishment of endometrial stem cell strains, and corresponding culture and preservation techniques. Background technique [0002] Stem cell technology is one of the most popular technologies in life sciences today. Its research content covers almost all biomedical fields of life sciences. 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. 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 the stem cell and human genome project as the top 10 scientific breakthroughs of the year, especially in 199...

Claims

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Application Information

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IPC IPC(8): C12N5/0775A01N1/02
Inventor 项春生
Owner HANGZHOU S EVANS BIOSCI LTD
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