Isolation and culture of human chorionic extrachorionic trophoblast cells

Summary

The normal exercise of trophoblast biological functions is a key component in maintaining a normal pregnancy. After embryo implantation, trophoblast cells divide, proliferate, and differentiate to form EVCTs and villous cytotrophoblasts (VCTs) with distinctly different biological properties. They are the only fetal cells at the maternal-fetal interface that are in direct contact with the maternal immune system, and play an important role in embryo implantation and maternal-fetal immune tolerance. As the terminal cell of trophoblast differentiation, EVCT can be adherent to the wall after in vitro culture, and gradually stretch from the initial ovoid shape to spindle, star or polygonal shape; EVCT can migrate and aggregate, but do not fuse, and have low proliferative capacity. Keratin 7 was expressed, but not waveform protein. HLA-G was only expressed in EVCT, which could be used as a screening marker to further improve the evaluation and identification of trophoblast cells.

Principle

The basic principle of human extrachorionic trophoblast isolation and culture is that EVCT has invasive ability, aggregates to form silos deep in the uterine meconium, has the closest contact with uterine meconium cells, and is an important part of the maternal-fetal immune microenvironment.

The chorionic tissue was selected from the meconium, milled and filtered through enzymatic digestion, and subjected to density gradient centrifugation, and the trophoblast cells were initially obtained by aspirating a 40% density layer according to the density of the cells, and the EVCT was further purified by short-term cultures of different durations.

Then, according to the morphology, phenotype and functional characteristics of the cells, immunohistochemistry or fluorescent antibody labeling cell surface antigens at the same time for flow cytometry sorting and other technical methods were used to identify, isolate and purify the cells, which laid the foundation for in-depth discussion of the biological functions of trophoblast cells.

Operation method

Isolation and culture of human chorionic extrachorionic trophoblast cells

Principle

The basic principle of human extrachorionic trophoblast isolation and culture is that EVCT has invasive ability, aggregates to form silos deep in the uterine meconium, has the closest contact with uterine meconium cells, and is an important part of the maternal-fetal immune microenvironment. The chorionic tissue was selected from the meconium, digested enzymatically, ground and filtered, and subjected to density gradient centrifugation, and the trophoblast cells were initially obtained by sucking up the 40% density layer according to the density of the cells, and the EVCT were further purified by short-term culturing at different times, and then, according to the morphology, phenotype, and function of the cells, they were identified, isolated, and separated from the cells using immunohistochemistry, or fluorescent antibody labeled with cell surface antigens and sorted by flow cytometry. Then, according to the morphological, phenotypic and functional characteristics of the cells, we used immunohistochemistry or fluorescent antibody labeled with cell surface antigen and flow cytometry sorting and other techniques to identify, isolate and purify the cells, which laid the foundation for in-depth discussion of the biological functions of trophoblast cells.

Materials and Instruments

Reagents:
PBS solution, D-Hanks solution, RBC lysate
DMEM/F12 culture solution, fetal bovine serum, trypsin
Collagenase Type IV, DNAzyme Type I, Fibronectin (FN)
Collagen type IV, Matrigel, Percoll stock solution, antibiotics
Instruments:
Ophthalmic scissors, forceps, sieves of different sizes (80 mesh, 300 mesh)
Assorted Petri dishes, syringe handles, plastic centrifuge tubes of different sizes
1.5 ml EP tubes, pipettes, bench-top freezing centrifuge
Magnetic poles and magnetic bead sorting columns, CO
2
Incubator, water bath shaker, flow cytometer

Move

The basic steps of human chorionic ectotrophic cell isolation and culture can be divided into the following steps:

1. Reagent preparation

(1) Type IV collagen working solution: use 0.25% glacial acetic acid to dissolve type IV collagen, overnight at 4 ℃ to make it fully dissolved, the final concentration of 2.0 mg / ml. 6 ~ 10 μg / c ㎡ when used to wrap the plate, overnight at 4 ℃. Cell culture plates covered with type IV collagen can be sterilized by UV irradiation overnight or by 75% ethanol rinsing.

(2) FN working solution: dissolve 1 mg of FN dry powder in 1 ml of sterile distilled water, leave it at room temperature for 30 minutes to dissolve naturally, and store at a concentration of 1 mg/ml. 20 μl of FN solution should be dispensed into 20 μl tubes and stored at -70 ℃.

Dissolve 20 μl of FN solution in 1 ml of FD medium (working concentration 20 μg/ml), pour it into a 35 mm diameter Pierce's Petri dish and incubate for 45 min at room temperature, then discard the medium.

The coated petri dishes can be used or stored at -20 ℃, but not more than 2 weeks.

2. Fresh meconium tissue was washed three times with 1xPBS to remove blood clots and fetal membranes, and the chorionic tissue was obtained and cut into 1 mm3 pieces with ophthalmic scissors.

3. 0.25% trypsin and 0.1% DNase I were added and digested in a 37 ℃ water bath with gentle shaking for 5 minutes, then the digested solution was aspirated and discarded.

4. Add 0.25% trypsin and 0.1% DNase I to the remaining tissue and continue digestion at 37 ℃ for 10 minutes, aspirate the digested liquid, and then add 10% calf serum to terminate digestion. Then add 10% calf serum to terminate the digestion. Repeat the digestion for 3~4 times and collect all the supernatant.

5. Take the digested supernatant rich in trophoblast cells, filter it through 80 mesh and 300 mesh sieve, collect the filtered liquid, centrifuge it at 300 g for 10 min, and discard the supernatant.

6. Resuspend the cell pellet on a discontinuous Percoll gradient (10% to 70%, one gradient per 10%) and centrifuge at 1000 g for 20 min.

7. Aspirate the cells in the 40% density layer (p=1.048-1.062 g/ml) as trophoblast cells, wash and add to DMEM high glucose culture medium containing 10% fetal bovine serum.

8. Adjust the cell density to 5x10/ml, and plant the cells in pre-coated type IV collagen (or FN or Matrigel) coated culture plates, and incubate them in adherent culture for 10 minutes, and remove the fibroblasts that are easy to be adhered to the wall. Incubate at 37 ℃ in 5% _CO2 incubator for 24 hours before the initial change of culture medium. 9.

9. Observe the growth status of the cells under an inverted phase contrast microscope after 24 h. EVCT could migrate and aggregate, but did not fuse and had a weak proliferative capacity.

10. Immunohistochemistry was performed to identify the obtained cells according to the characteristics of wave protein negative and CK7 positive.

11. HLA-G+ extrachorionic trophoblast cells can be sorted and purified by FACSAriaII after labeling with anti-HLA-G surface antibody, and the purity of sorting is >95%.

Caveat

1. According to the actual conditions in the laboratory, 1 mg/ml collagenase IV and 0.1 mg/ml DNase I can be used to digest the villous tissue.

2. EVCT has a weak proliferative ability, and some biological characteristics of the cells may be lost if too many passages are performed, so it is important to reduce the number of passages of cultured cells in the study.

3. EVCT and VCT both express CK7 and do not express waveform proteins, which can be differentiated and identified by c-erbB-2 immunohistochemistry for EVCT (c-erbB-2 positive) and VCT (c-erbB-2 negative).To obtain EVCT with high purity, labeled human anti-HLA-G antibody is required and flow cytometry is applied to sort EVCT.

For more product details, please visit Aladdin Scientific website.

https://www.aladdinsci.com/