Trophoblast Cell Recovery from Angiogenesis-Tube Formation Assay for Differentiation Marker Expression Analysis

We studied the process of extravillous trophoblast, EVT cell invasion into the uterus to form the placenta for correct maternal blood supply. The invasion and differentiation of EVT are identified as the region of preeclampsia. We employ an easy and accessible in vitro model to study the differentiation process of EVT.

Experimental challenges include mimicking the dynamic microenvironment of the placenta in vitro and replicating trophoblast maternal interactions accurately. Additionally, understanding the precise timing and triggers of preeclampsia onset remains elusive, hampering preventive strategies, obtaining longitudinal data for comprehensive analysis process, logistical and ethical hurdles.

The protocol addresses the challenge of separating trophoblast cells from the basement membrane matrix in vitro to obtain pure cell extract for biochemical analysis. By overcoming this obstacle, the protocol enable a deeper investigation into trophoblast differentiation and marker expression, filling a gap in understanding the molecular mechanism of placenta development.

The protocol offers a simple, fast, and cost-effective method for isolating tube-like structures for the basement member matrix, allowing for destruction of high-quality RNA and protein for biochemical analysis. This approach provides a more efficient way to study trophoblast behavior and marker expression compared to traditional techniques.

We intend to explore RECK's involvement in early pregnancy development and investigate the impact of extracellular vesicles on preeclampsia symptoms. We want to understand not signaling trophoblast mesenchymal to endothelial transition and give better roles in trophoblast fate. Additionally, examine dopamine stimulation of maternal immune response and platelets' role in placental development will be pursued.

[Narrator] To begin, obtain the reagents to prepare a complete growth medium under laminar airflow. Combine 190 milliliters of RPMI 1640 with 10 milliliters of FBS to create a 5% FBS solution. Warm this mixture in a water bath at 37 degrees Celsius. Thaw the basement membrane matrix at four degrees Celsius for three to four hours. Then, keep the matrix on ice and dilute it to 70% with ice cold RPMI 1640 without FBS. Dissolve EDTA and PBS to prepare a 50-millimolar EDTA solution and store it at four degrees Celsius. Then, obtain a multi-well six-dish pre-cooled at minus 20 degrees Celsius for one hour. Add 60 microliters per square centimeter of the diluted basement membrane matrix to the dishes and incubate to promote matrix polymerization. Carefully seed 80,000 cells per square centimeter of HTR-8/SVneo cells over the polymerized matrix and the uncoated dishes as a control. Incubate the plates for 12, 24, and 48 hours at 37 degrees Celsius with 5% carbon dioxide. HTR-8/SVneo cells generated tube-like structures at 12, 24, and 48 hours after incubation over the basement membrane matrix, whereas no tube-like structures were observed in the control dishes. To begin, obtain HTR-8/SVneo cell culture on the basement membrane matrix. Gently wash the cells three times with ice cold PBS to eliminate the cell debris. After adding one milliliter of cold PBS, acquire microphotographs within 10 minutes. Then, remove PBS and add 700 microliters of PBS-EDTA to each well. Incubate the plate on ice for three to five minutes. Using a micropipette, gently recover the suspension of the tubular network in a 1.5-milliliter tube and centrifuge to obtain a white pellet of cells. Invert the tube to eliminate the supernatant and dry the pellet. To remove the dissolved basement membrane matrix and EDTA, wash the cells with one milliliter of PBS at four degrees Celsius. After at least three washes, resuspend the pellet in one milliliter of PBS and aliquot into different tubes depending on the requirements. Finally, centrifuge the tubes and obtain the cell pellet.