Primary isolation and culture method of amniotic fluid mesenchymal stem cells

A technology for isolating and cultivating high-quality stem cells, applied in the field of stem cell culture, can solve the problems of tediousness, cell damage, and high cost, and achieve the effect of simple operation and avoiding damage and pollution

Active Publication Date: 2018-04-13
GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, immunomagnetic bead sorting is mostly used for the sorting of amniotic fluid stem cells, but this method has a certain impact on cell viability, and the cost is high
There are also some methods that use mechanical separation, but the method of artificially scraping endothelial cells is very cumbersome and easily causes pollution, and it is very easy to cause cell damage, resulting in a decrease in cell activity

Method used

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  • Primary isolation and culture method of amniotic fluid mesenchymal stem cells
  • Primary isolation and culture method of amniotic fluid mesenchymal stem cells
  • Primary isolation and culture method of amniotic fluid mesenchymal stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] The purification process of the amniotic fluid mesenchymal stem cells provided by the present invention is specifically:

[0046] 1. Primary culture:

[0047] 1. Take the amniotic fluid of the caesarean section, put it in a sterile container, and put it into the ultra-clean table.

[0048] 2. Fill the amniotic fluid into a 50mL centrifuge tube, centrifuge at 2000rpm for 5min, discard the supernatant.

[0049] 3. Add 40mL PBS to each 50mL centrifuge tube for resuspension, centrifuge at 2000rpm for 5min, and discard the supernatant.

[0050] 4. Add 0.1% gelatin to the six-well plate, 1 mL / well, incubate at room temperature for 30 minutes, and discard the gelatin solution after completion.

[0051] 5. Resuspend the pellet with lonza complete medium and adjust the cell density to 5×10 5cell / mL, inoculated into a gelatin-incubated six-well plate, 2 mL / well; transferred to a cell culture incubator for cultivation.

[0052] 6. After culturing for 12 hours, transfer the cul...

Embodiment 2

[0061] The purification process of the amniotic fluid mesenchymal stem cells provided by the present invention is specifically:

[0062] 1. Primary culture:

[0063] 1. Take the amniotic fluid of the caesarean section, put it in a sterile container, and put it into the ultra-clean table.

[0064] 2. Fill the amniotic fluid into a 50mL centrifuge tube, centrifuge at 2000rpm for 5min, discard the supernatant.

[0065] 3. Add 40mL PBS to each 50mL centrifuge tube for resuspension, centrifuge at 2000rpm for 5min, and discard the supernatant.

[0066] 4. Add 0.1% gelatin to the six-well plate, 1 mL / well, incubate at room temperature for 30 minutes, and discard the gelatin solution after completion.

[0067] 5. Resuspend the pellet with lonza complete medium and adjust the cell density to 1×10 5 cell / mL, inoculated into a gelatin-incubated six-well plate, 2 mL / well; transferred to a cell culture incubator for cultivation.

[0068] 6. After culturing for 13 hours, transfer the cu...

Embodiment 3

[0077] The purification process of the amniotic fluid mesenchymal stem cells provided by the present invention is specifically:

[0078] 1. Primary culture:

[0079] 1. Take the amniotic fluid of the caesarean section, put it in a sterile container, and put it into the ultra-clean table.

[0080] 2. Fill the amniotic fluid into a 50mL centrifuge tube, centrifuge at 2000rpm for 5min, discard the supernatant.

[0081] 3. Add 40mL PBS to each 50mL centrifuge tube for resuspension, centrifuge at 2000rpm for 5min, and discard the supernatant.

[0082] 4. Add 0.1% gelatin to the six-well plate, 1 mL / well, incubate at room temperature for 30 minutes, and discard the gelatin solution after completion.

[0083] 5. Resuspend the pellet with lonza complete medium and adjust the cell density to 1×10 6 cell / mL, inoculated into a gelatin-incubated six-well plate, 2 mL / well; transferred to a cell culture incubator for cultivation.

[0084] 6. After culturing for 11 hours, transfer the cu...

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Abstract

The invention relates to the technical field of stem cell culture, in particular to a primary isolated culture method for amniotic fluid mesenchymal stem cells. The method includes that according to a differential attachment principle, endothelial cells are enabled to grow along a wall under proper conditions while the mesenchymal stem cells are not adhered to the wall yet. By accurate control of culture time, culture solution containing the amniotic fluid mesenchymal stem cells is transferred after wall adherence of the endothelial cells is finished, and the culture solution is subjected to subculturing to obtain the amniotic fluid mesenchymal stem cells. The method is simple in operation, less devices are required, manual scraping operations are avoided, and accordingly cell damages and contamination are avoided. According to detection results, viability of the amniotic fluid mesenchymal stem cells contained in the culture solution acquired at the step 1 is not lower than 89.28%; after three generations, flow cytometry detection results show that surface antibodies of the cells still accord with characteristics of the stem cells, and differentiating tendency is avoided.

Description

technical field [0001] The invention relates to the technical field of stem cell culture, in particular to a method for primary isolation and culture of amniotic fluid mesenchymal stem cells. Background technique [0002] Stem cells are a kind of primitive undifferentiated cells with self-replication ability and multi-directional differentiation potential, which are in an undifferentiated state and have the ability to proliferate. Under specific conditions, stem cells can differentiate into different types of mature cells with characteristic morphology, specific molecular markers and special functions. According to different sources of stem cells, they can be divided into embryonic stem cells (embryonicstem cells, ESCs) and tissue stem cells or adult stem cells (adultstem cells, ASCs) derived from postnatal organs or adult individual tissues. From the perspective of sources, it is difficult to obtain a large number of stem cells. Therefore, finding new sources of stem cell...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0775
Inventor 葛啸虎陈海佳王一飞卢瑞珊王小燕李平马岩岩
Owner GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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