3D culture method of chicken embryo primordial germ cells
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A technology of primordial germ cells and culture methods, applied in germ cells, 3D culture, cell culture support/coating, etc., can solve the problem that it is difficult to obtain chicken embryo PGCs in large quantities
Active Publication Date: 2018-11-06
FOSHAN UNIVERSITY
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[0004] At present, there are two in vitro culture systems for chicken embryo primordial germ cells, namely feeder layer culture and feeder layer-free serum-free culture. It is difficult to obtain a large number of chicken embryo PGCs in these two culture systems
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Embodiment 1
[0019] Example 1: 3D culture of chicken embryo primordial germ cells
[0020] A method for cultivating chicken embryo primordial germ cells in 3D, comprising the following steps:
[0021] 1) Select 20 fresh breeding eggs, wipe the surface with 75% alcohol, equilibrate at room temperature for 2 hours, place in an incubator at 38°C and a relative humidity of 60% for incubation, and hatch until 14HH of the chicken embryo development period, and take the embryonic blood;
[0022] 2) Inject embryonic blood into a 48-well plate containing cPGCs feeder-free culture system at 37°C, 5% CO 2 incubator cultivation;
[0023] 3) After culturing until the 15th day, take the cells in the culture plate into a 500 μL centrifuge tube, centrifuge at 3000 rpm for 5 minutes, discard the supernatant to remove blood cells, resuspend the cells in the complete culture medium into a 48-well culture plate, and store at 37°C for 5 %CO 2 incubator cultivation;
[0024] 4) Take the cells of the second ...
Embodiment 2
[0026] Example 2: Comparison of cell viability of chicken embryo PGCs between 3D culture method and 2D culture method
[0027] 1) The experiment was divided into two groups, namely the 3D culture group and the 2D group, i.e. the traditional culture method, from the second generation chicken embryo PGCs obtained in Example 1, 1 × 10 per hole 5 cells were seeded in a 24-well plate, and each group was set up with 3 replicate wells;
[0028] 2) DAPI staining:
[0029] 2-1) In the 3D group cells, add 0.25% trypsin to hydrolyze the sodium alginate hydrogel, digest for 2 minutes, stop the digestion with complete medium containing 10% fetal bovine serum, centrifuge at 3000rpm for 5 minutes, and discard the supernatant;
[0030] 2-2) Centrifuge the 2D group at 3000rpm for 5min, discard the supernatant;
[0031] 2-3) Add 4% paraformaldehyde solution to fix for 30 minutes, centrifuge at 3000rpm for 5min, resuspend the cells in PBS containing 5% FBS, centrifuge at 3000rpm for 5min, disc...
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Abstract
The invention discloses a 3D culture method of chicken embryo primordial germ cells. The 3D culture method comprises the following steps: 1), placing collected 14HH chicken embryo blood in a cPGCs complete medium for culturing, and purifying cells; 2), placing the purified chicken embryo primordial germ cells into the cPGCs complete medium again for in-vitro culture amplification, taking subcultured second-generation cells, and centrifuging to obtain a chicken embryo primordial germ cell precipitate; 3), adding a sodium alginate solution to the precipitate; 4), sucking a cell suspension by using a short-needle insulin syringe, dropwise adding into a culture dish containing a CaCl2 solution to form sodium alginate hydrogel spheres, standing for 15min and then culturing. Through introductionof sodium alginate into a culture condition, the in-vivo growth environment of the chicken embryo primordial germ cells can be simulated very well in a 3D cell culture mode, so that obtainment of a large number of chicken embryo primordial germ cells is facilitated, and thus application of the chicken embryo primordial germ cells to research and production is facilitated.
Description
technical field [0001] The invention relates to the technical field of bioengineering, in particular to a method for culturing chicken embryo primordial germ cells. Background technique [0002] Primordial germ cells (PGCs) are the primordial progenitor cells of germ cells. In female and male animals, PGCs can form oogonia and spermatogonia, respectively, and have developmental totipotency. Avian embryos are powerful models for studying development and stem cell biology, and they have certain advantages, such as their easy availability, small size, convenient in vitro manipulation, and short reproductive cycle. As reproductive stem cells, PGCs can be used to study the development and differentiation of germ cells, and can also be used as carriers of exogenous genes. They have broad applications in the preparation of gonad chimeras, the production of transgenic animals, and the protection of germplasm resources of rare species. prospect. [0003] Sodium alginate is a highly...
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