Purification of HLA-G⁺ Extravillous Trophoblasts from Human Term Placental Tissues for Phenotyping and Functional Analysis

This protocol is designed to purify primary human trophoblasts from human placental tissues to study their functional properties and immunological properties in healthy pregnancy and under pathological conditions. So this protocol provides a reproducible platform to overcome some previous limitations with working with primary human extravillous trophoblasts, namely their viability. So this protocol is really optimized to enhance their viability and purity.

To begin, obtain a human placenta sample. Place the placenta with the fetal side facing upward so the cord insertion site is visible. Manually peel the amnion from the lateral membrane toward the umbilical cord insertion site and remove it.

Then use scissors to cut and discard the amnion. Cut the choriodecidual membrane into two inch strips starting from the base of the placenta upward while keeping each strip attached to the placenta. Place the choriodecidual membrane strip across two fingers, with the decidual side facing upward.

With a pair of sterile curved tip forceps, gently scrape the maternal decidua parietalis from the chorion. Collect the decidua parietalis into a 50 milliliter conical tube containing 20 milliliters of PBS for lymphocyte isolation. Then collect the chorionic membrane into a separate 50 milliliter conical tube containing 20 milliliters of wash medium A.Using scissors, cut the chorionic membrane inside the 50 milliliter tube into pieces smaller than two square millimeters.

Discard wash medium A from the tube. Wash the tissue repeatedly with PBS until the supernatant becomes clear. For placental dissection, place the placenta with the maternal side facing upward and remove blood clots.

Using sterile scissors, cut pieces of placenta basal plate while avoiding necrotic, calcified, or ischemic areas. Place the decidua basalis piece on the fingers with villi facing upwards. Then use scissors to carefully remove the villi to obtain a two to three millimeter membrane with only a thin villus layer remaining.

Collect the decidua basalis tissue into a 50 milliliter conical tube containing 20 milliliters PBS. Then chop the tissue into pieces smaller than two square millimeters. Next, discard the PBS from the tube.

Wash the decidua basalis repeatedly with PBS until the supernatant is clear without visible blood. Then split 15 milliliters of tissue suspension into two 50 milliliter conical tubes. Fill all tubes with PBS.

Centrifuge the tubes at 200G for one minute and pour off the supernatant. Add 150 milliliters of warm tissue digestion enzyme cocktail into a 300 milliliter glass bottle. Add 30 milliliters of chorionic membrane to the bottle.

Add 20 milliliters warm DMEM/F-20 and 80 milliliters warm tissue digestion enzyme cocktail into two separate 300 milliliter glass bottles. Then add 7.5 milliliters decidua basalis into each bottle. Incubate the bottles in a water bath.

Shake all bottles every three to four minutes during the 15 minute incubation. After digestion, filter the tissue through metal sieves with a mesh size of 40. Then add wash medium B to rinse tissues and dissolve gelatinous material.

Transfer the undigested tissue back into the glass bottles and repeat digestion. Then filter the cell suspensions through a 100 micrometer filter into labeled 50 milliliter conical tubes. Keep cells from the first and second digestions separate.

Then centrifuge the cell suspension at 650G for eight minutes. Discard the supernatant. Combine pellets of the same tissue type and digestion number into one tube.

Then make up the volume with wash medium B up to 25 milliliters. Add 12 milliliters ficoll to four 50 milliliter conical tubes labeled with the tissue type and digestion number. Carefully layer the cell suspension onto the ficoll.

Then centrifuge at 800G for 20 minutes at room temperature without breaking. Carefully aspirate the cell layer at the ficoll medium interface and transfer it into clean labeled tubes. Then fill tubes with wash medium B, centrifuge at 650G for eight minutes, and resuspend the pellets.

Place the resuspended pellets into sterile fluorescence-activated cell sorting tubes labeled with tissue type and digestion number. Add anti-CD45 BV785, anti-HLA GAPC, and anti-EGFR1 BV711 antibodies. Incubate for 20 minutes at room temperature in the dark.

After washing and resuspending the cells in wash medium B, immediately pass them through a 40 micrometer cell strainer. Sort CD45 negative HLAG positive EGFR positive extravillous trophoblast into tubes containing one milliliter EVT culture medium A.Flow cytometry analysis of lineage markers showed viable primary extravillous trophoblast culture until day four. Representative light microscopy images of primary chorionic and decidua basalis HLAG positive extravillous trophoblast were captured after 24 hours, 48 hours, and 72 hours of culture.

Extravillous trophoblast expressed polymorphic HLAC and immunosuppressive co-inhibitory molecules. Extravillous trophoblast increased the expression of HLAC, PD-L1, PD-L2, and HLA-E, but did not increase the expression of PVR and B7H3 when co-cultured with activated lymphocytes for 72 hours. No increase in extravillous trophoblast death was observed following co-culture with activated lymphocytes.

So this protocol allows people, scientists, to use primary extravillous trophoblasts for a wide variety of assays. The benefit is the high viability that allows for analysis using cell culture, cold culture, flow cytometry, proteomics, transcriptomics, using these really nicely viable high yield extravillous trophoblasts. So one of the most important aspects of our protocol is that you have to use fresh placental materials.

You cannot refrigerate them. Trophoblasts love dying, so please get the placental tissues as soon after delivery as you can, preferably within two hours after delivery, and keep them at room temperature at all time. This is crucial to obtain highly viable extravillous trophoblast.

So some of the more additional methods and more advanced innovative methods that you can use these primary trophoblasts for is long-term cell culture. So you can use them to establish primary four-day cell cultures. So cells will be viable for up to 96 hours, which is pretty long and it allows you to do cold cultures with immune cells, like T-cells and NK cells.

The other innovation is also that you can use them to establish trophoblast-like cell lines using the methods first described by Aoki, and that we also used to apply on these cultures to really establish long-term proliferating cell types that you can use for genome editing and other more long-term culture assays. So using these innovative methods, including the long-term cell culture, I think can really help you to design long-term mechanistic cultures where you can apply genome editing, knock-out, knock-in genes, proteins, over-express some of these trophoblasts to get really a lot more mechanistic detail on how these trophoblasts enforce tolerance, or allow immunity and more cytotoxicity when they are interacting with immune populations.