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

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
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Example Embodiment

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

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

[0025] The amniotic fluid sample was diluted 1:1 with PBS supplemented with EDTA; centrifuged at 1200 r / min for 5 min; discarded the supernatant, added the special medium for amniotic fluid and repeatedly pipetted and mixed; inoculated in a gelatin-coated petri dish; 2 The growth of amniotic fluid stem cells can be observed after 4-5 days of culture under the environmental 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 The digestion was terminated with complete medium containing 10% fetal bovine serum when it started to fall off the culture dish; the mixture w...

Example Embodiment

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

[0030] The amniotic fluid stem cells of the 3rd and 6th passages were obtained by subculture, counted, and the growth curve was drawn according to the obtained data, with 1×10 4 Inoculated into a 24-well plate, three wells were taken every 24 hours, and the average number was recorded. as figure 1 Amniotic fluid stem cell growth graph shown.

Example Embodiment

[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, inoculated on a 24-well plate coated with gelatin, 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 (each wash is 10 minutes);

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

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

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

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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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