Isolation And Culture Of First Trimester Human Trophoblast Cell

Objective:Trophoblast cell derives from extraembryonic trophoblast, and growsvery quickly. There are many microvillus called “villi”, appears on theembryonary sac. Trophoblast cells are firstly formed by flat cuboidal cells,then gradually differentiates into two layers when villi forms. The cellsbetween the inner layer and interstitial is called “cytotrophoblast”; cellsbetween outer layer and uterine decidua, called “syncytiotrophoblast”.Trophoblast cell invades endometrium、 myometrial and helicine artery,therefore uteroplacental circulation is established. The proliferation andinvasion of trophoblast cell is closely related to successful nidation、placentalformation and embryo development. Embryo implantation requirestrophoblast invasion to the uterine stroma. Characteristic of extravilloustrophoblast in embryonic trophoblast is similar to tumor. Through theproliferation and invasion of the uterus, embryo adhere to the uterine wall, andthen gradually finishes embryo development. But Unlike tumor cell,proliferation and invasion of trophoblast cell is a highly controlled processwhich needs the coordination of various paracrine factors derived frommaternal deciduas. The adjustment disorder of proliferation and invasivenesscan lead to a series of pregnancy related diseases. invasiveness inadequate willlead to spontaneous abortion, intrauterine growth retardation, preeclampsia,pregnancy-induced hypertension syndrome etc. Whereas excessive invasionof trophoblast cell will lead to gestational trophoblastic disease (GTD), suchas hydratidiform mole (HM), invasive mole (IM), choriocarcinoma (CC) andrare placental site trophoblastic tumor, etc. The studys of a vitro model oftrophoblast cell is necessary for studies on trophoblastic diseases.At present, there are two models applied on the the studys of trophoblastic diseases or the research of placental function: parentaltrophoblast cells and some transfected trophoblastic cell lines. There aresome human transfected trophoblastic cell lines such as SGHPL, TCL-1,JAR, JEG-3, HTR-8/SVneo, BeWo, SM10, TEV-1, Rcho-1, HPT-8etc. Buttransfected trophoblast cells cannot replace the parental trophoblast cell. Thecultrual method of parental trophoblast cells is Low yield, low purity and belimited by clinical. So it’s important to seek a simple and efficient method ofcultrual parental trophoblast cells in vitro.The human first trimester villous tissue includes syncytiotrophoblastcell, cytotrophoblast cell, red cell, macrophages, fibroblasts, endothelial cells,uterus matrix cell, decidual cell etc. Cytotrophoblast cell is the storeroom ofvarious types of the human trophoblast cell. There are some studies onestablishing human tyophoblast cells in vitro, but quantities and purity aredifferent with different methods. The study on parental first trimester humantyophoblast cells is not perfect in vitro. So it’s important to seek a simplemethod that can obtain enough purity of human tyophoblast cells. It providesan important cytological basis for the study on trophoblastic diseases.Method:1.MaterialsHuman first trimester villous tissues were obtained under sterileconditions.2. IsolationTrimester villous tissues were washed three times with PBS to removethe blood. Whole placenta was put into a sterile15-mL bottle, and then thedigestion buffer was added and incubated for15–20min at37°C. Supernatantwas collected in a beaker; FBS was added to stop enzyme digestion. Repeatabove step several times until all the trophoblastic cells were digested from theplacenta under microscope, and the remnant is white fibriform tissue. All thecolation supernatant were collected into centrifuge tubes. Then supernatantswere removed and discarded, taking care not to remove the pellet at bottom，latter, add3mL DMEM cell suspension medium into the tubes. 3. PurityAll cell suspensions were laid very carefully onto Percoll gradient,centrifuged without using the brake. White band was collected between60%percoll and35%percoll, cleaned two times, then supernatant andresuspend pellet were removed with the cell culture medium. Count cellsolution with hemocytometer and culture cells at1×105finally.4. PassageTrypsin digestion5. Observation of cell with inverted microscopeThe cells were observed with inverted microscope and shooted every day.6. Identification the purity of tyophoblast cellsWe observed the positive expression of cytokeratin-7and negativeexpression of vimentin by immunohistochemical method andimmunocytochemical methodResults：1. Activity of cellsThe percentage of living cell is more than95%.2. Cell morphology and characterThe cell isolated from tissue is big and round,1hour later, some adherentcells could be seen. More than80%cells extend in24hour. The cells shapebecomes to triangle or polygon, we can see a round and big nucleus, full ofendochylema. We can see many evection of endochylema at high power lens.It is the character of trophoblast cell. The cells reached approximately80%after4-5days, trophoblast cells subcultured after5days, later one passageevery3days.3. The results of tissues by HE staining and immunohistochemicalmethodWe observed the location and morphous by HE staining.The expression of cytokeratin-7is positive and Vimentin is negative introphoblast cell. Vimentin is positive and cytokeratin-7is negative in theinterior cells such as fibroblasts. All the negative control groups are negative. 4. The results by immunocytochemical methodThe positive expression of cytokeratin-7is more than90%, theexpression of Vimentin is negative or poor positive occasionally.Conclusion：1. According to the special position of trophoblast cells in chorionic villi,we utilize the method of digesting the whole first trimester placenta andfinished digestion of cells Timely, we digest tissue by several times and keepthe cell vitality better.2. If we cut tissue into pieces and then digestion, the mixed cells Shouldbe fully purified by classic Percoll gradient. we can obtain High puritytrophoblast cells by digest the whole tissue, so we purified cells by simplePercoll gradient. This operation is more convenient.3. The cell suspension contain trophoblast cells mainly, reduces theinterference with mesenchymal cells when purifing trophoblast cells by simplePercoll gradient. We can obtain tyophoblast cells much higher purity andhigher yield.4. High purity and high yield of trophoblast cells are obtained by usingonly one or two cases of human first trimester villi through this method, whichcan also reduce individual variation in different placenta and Short the time ofclinical operation. Also we can obtain the first trimester placent easier andeduces the clinical restrictions.