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Simple and quick culture method for amniotic fluid-derived stem cells (AFSCs)

A stem cell culture and stem cell technology, applied in the field of bioengineering, can solve the problems of infection risk of unknown pathogens, cumbersome and inconvenient culture steps, high operating costs, etc., achieve good growth activity, reduce experimental costs, and simplify experimental operation steps.

Inactive Publication Date: 2017-12-19
FOSHAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In order to overcome the cumbersome and inconvenient operation of the existing amniotic fluid stem cell culture steps, the high cost, or the possible risk of infection by unknown pathogens, the present invention ensures that amniotic fluid stem cells with good growth activity can be obtained under simpler and cost-saving operations. Some biological characteristics, broad application prospects

Method used

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  • Simple and quick culture method for amniotic fluid-derived stem cells (AFSCs)
  • Simple and quick culture method for amniotic fluid-derived stem cells (AFSCs)
  • Simple and quick culture method for amniotic fluid-derived stem cells (AFSCs)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1: Isolation and culture of amniotic fluid stem cells

[0024] (1) Primary culture of amniotic fluid stem cells:

[0025] Dilute the amniotic fluid sample with EDTA-added PBS 1:1; centrifuge at 1200r / min for 5min; discard the supernatant, add amniotic fluid-specific medium and repeatedly blow and mix; inoculate on a gelatin-coated Petri dish; 2 The growth of amniotic fluid stem cells can be observed after 4-5 days of culture under the environment conditions.

[0026] (2) Subculture of amniotic fluid stem cells:

[0027] When the primary cultured amniotic fluid stem cells reach 80-90% confluence, discard the cell culture medium; wash three times with PBS; cover the cells with 0.25% trypsin containing 0.02% EDTA to dissociate the cells; wait for the cells to shrink and become round And stop digestion with complete medium containing 10% fetal bovine serum when it starts to fall off from the culture dish; blow the mixture repeatedly until the adherent cells fall o...

Embodiment 2

[0029] Embodiment 2: Drawing of growth curve of amniotic fluid stem cells

[0030] The amniotic fluid stem cells of passage 3 and passage 6 were obtained by subculture, counted, and the growth curve was drawn according to the obtained data, with a scale of 1×10 4 Inoculate into a 24-well plate, take three wells every 24 hours, and record the average number. like figure 1 The growth curve of amniotic fluid stem cells is shown.

Embodiment 3

[0031] Example 3: Immunofluorescence identification of amniotic fluid stem cells

[0032] (1) The amniotic fluid stem cells of the third generation were obtained by subculture, seeded on a gelatin-coated 24-well plate, and when 70% confluence was reached, the culture medium was discarded and washed twice with PBS;

[0033] (2) Add 3.7% paraformaldehyde solution to the 24-well plate to fix for 30 minutes, and wash the cells three times with PBS containing 5% FBS (10 minutes each time);

[0034] (3) Add permeabilization agent (0.1% TritonX-100, PBS) to the 24-well plate, discard after incubating at room temperature for 15 minutes, wash with PBS three times, 5 minutes each time;

[0035] (4) Add blocking solution (10% FBS:PBS) to the 24-well plate, block at 37°C for 1 hour, discard it, wash with PBS three times, 5 minutes each time;

[0036] (5) Incubate overnight at 4°C with primary antibodies against CD34, CD44, and CD90 (1:200, Santa Cruz, CA). After removing the primary anti...

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Abstract

The invention provides a simple and quick culture method for AFSCs. The culture method comprises the following steps: diluting amniotic fluid samples and EDTA-added PBS in a ratio of 1: 1, carrying out centrifugation for 5min at 1200r / min; discarding a supernatant, and adding an amniotic fluid special medium to be repeatedly blown and uniformly mixed; inoculating in a gelatin-coated petri dish; culturing for 4 to 5 days under the environmental conditions that the temperature is 37 DEG C and the concentration of CO2 is 5%, thereby being capable of observing growth of AFSCs. The method breaks through the traditional method, simplifies experimental operation steps, saves the time and reduces the experiment cost, and lowers risks of cell contamination due to cumbersome experimental steps; cultured cells still possess due biological characteristics.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a simple and quick method for culturing amniotic fluid stem cells. Background technique [0002] Recent advances in cell therapy, regenerative medicine, and stem cell research have significantly increased the need for various types of readily available cell suspensions, especially for multipotent stromal cells (mesenchymal stem cells, MSCs). The main reason for the popularity of MSCs in current medical practice is their unique immunomodulatory properties. However, the growth ability and differentiation ability of MSCs decrease with age, and the cell activity and differentiation ability of cells obtained from adult tissues such as fat, dermis, bone, synovium and bone marrow are far inferior to those of fetal origin, and in these Adult stem cells have certain difficulties in isolation, purification and maintenance, and it is difficult to reach the quantity required for tran...

Claims

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Application Information

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IPC IPC(8): C12N5/0775
CPCC12N5/0668C12N2509/00
Inventor 赵姝灿陈志胜郑桂纯
Owner FOSHAN UNIVERSITY
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