Endometrial metamorphosis is a critical step for embryo implantation and successful pregnancy. The cellular composition of the metaphase tissue is quite complex, with the metaphase stromal cells accounting for approximately 75% of the total metaphase cell number and being the main component cells of the maternal-fetal interface. These cells originate from fibroblasts in the mesenchyme, have a large, polygonal shape, and secrete a variety of hormones at high levels, including prolactin (PRL), which is involved in the nutritional supply of the meconium and the formation of the endocrine microenvironment. The cytoplasm is rich in waveform proteins and free of cytokeratin 7 (CK7), which can be used for the identification of metaphase stromal cells.
Principle
The basic principle of isolation and purification of human meconium stromal cells is that mesenchymal-derived DSC can participate in the maintenance of maternal-fetal immune tolerance by regulating the function of meconium immune cells, and the thicker meconium is removed from aborted tissues.
After enzymatic digestion, grinding and filtration, and density gradient centrifugation, depending on the density of the cells, 20% to 40% density layer can be aspirated to obtain DSC initially, and the suspended cells are removed by short-term culture to further purify DSC.
Then, according to the morphology, phenotype and functional characteristics of the cell population, the cells were identified by immunohistochemistry; and a large number of DSC with high purity were obtained by in vitro passaging culture, which provided the basis for more in-depth study of the role of cells in maternal-fetal tolerance.
Operation method
Isolation and purification of human metaphase stromal cells
Principle
The basic principle of isolation and purification of human metaphase stromal cells is that mesenchymal-derived DSC can participate in the maintenance of maternal-fetal immune tolerance by regulating the function of metaphase immune cells. thicker metaphase is removed from aborted tissue, digested enzymatically and then ground and filtered, and centrifuged in density gradient. 20% to 40% density layer can be aspirated to obtain DSC initially according to the density of cells. According to the density of the cells, DSC can be initially obtained by absorbing the 20%~40% density layer, and further purified by removing the suspended cells through short-term culture, and then identified by immunohistochemistry according to the morphology, phenotype and functional characteristics of the cell populations; and a large number of DSC with high purity can be obtained through in vitro passaging culture, which can provide the basis for more in-depth study of the role of the cells in maternal-fetal tolerance. Provide the basis for more in-depth study of the role of cells in maternal-fetal tolerance.
Materials and Instruments
Reagents: Move The basic steps for isolation and purification of human meconium stromal cells can be divided into the following steps: 1. Pick the thick ecdysis tissue, rinse it repeatedly in pre-cooled D-Hanks solution, blow to remove blood stains, then place the clean tissue in a sterile petri dish and cut it into 1 mm3 pieces with ophthalmic scissors. 2. Add 0.1% Collagenase IV (or combine with 50 μg/ml DNase I to digest the tissues) and digest the tissues at 37 ℃ for 1 hour, then terminate the digestion with DMEM/F12 cell culture medium containing 10% FBS. The cell suspension was centrifuged at 300 g for 10 min after grinding and filtering through a 100-mesh sieve, and the supernatant was discarded. 3. Resuspend the cell pellet in 3 ml of D-Hanks solution, carefully spread on the discontinuous Percoll density gradient interface (60%, 40%, 20% gradient), and centrifuge at 1000 g for 20 min. 4. Aspirate the cells between the 20% and 40% gradient layers (p=1.031-1.056), i.e. the preliminary isolated DSC, and resuspend them in DMEM/F12 cell culture medium containing 10% FBS after washing. 5. The obtained cells were incubated in 37 ℃, 5% CO2 incubator for 30 minutes, and then the liquid was changed to remove the suspended cells, and the adherent cells were the purified DSC. 6. Place the cells in a 37 ℃, 5% CO2 incubator and continue to cultivate for 5-7 days, when the cells in the culture flasks spread all over the wall of the flasks, and the degree of fusion reaches 80% can be passed on. First discard the culture medium, D-Hanks solution washed gently 2 times, add 2 ml of digestive enzyme solution to digest the cells for 2 minutes, inverted microscope to observe 90% of the cells rounded, add 2 ml of FD complete culture medium to terminate the digestion. Then add 2 ml of FD complete culture solution to terminate the digestion, and make single cell suspension by repeated blowing with a pipette, inoculate into new culture flasks respectively, and continue to cultivate and pass on the cells in the incubator at 37 ℃, 5% CO2 and saturated humidity. Caveat 1. According to the actual situation of each laboratory, the digestive enzyme solution prepared by 0.25% trypsin and 0.02% ethylenediaminetetraacetic acid (EDTA) at the ratio of 1:1 can be used for the digestion of the tissue of the ecdysis, and the digestive enzyme solution can be shocked in the 37 ℃ thermostatic water bath for 20 minutes.(2) The feature of high secretion of PRL by DSC at the beginning of pregnancy can be utilized to carry out immunohistochemistry of PRL for cell identification. For more product details, please visit Aladdin Scientific website.
PBS solution, D-Hanks solution, RBC lysate
RPMI 1640 cell culture solution, DMEM/F12 culture solution
Fetal bovine serum, trypsin, collagenase type IV, DNAase type I
Percoll stock solution, antibiotics
Instruments:
Ophthalmic scissors, forceps, sieves of different sizes (100 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.