This article details a method for culturing placental villous and decidual tissues to study their interactions. The process involves microdissection and careful handling of tissues to maintain their viability and functionality.
Begin with placental villous and decidual parietalis specimens.
Dissect the villous — a well-vascularized tissue containing extravillous trophoblast, or EVT, cell columns.
Transfer the villous over an extracellular matrix, ECM-coated transwell placed in a tissue culture well.
Take the next specimen; microdissect the decidua — a uterine tissue surrounding the developing fetus.
Place the decidua over another ECM-coated transwell.
Add a suitable medium to the tissue culture wells to maintain humidity, while keeping the transwells devoid of medium.
Without a medium, the EVT cell columns secrete proteinases that cleave ECM proteins.
This initiates ECM remodeling, creating space for the EVT cells to invade and firmly adhere to the matrix.
Meanwhile, the other cells of the villous and decidua attach to the ECM by activating integrin receptors, which cluster to bind cooperatively to the matrix.
These processes strengthen the cell-to-ECM adhesion, securely embedding the tissues.
Finally, add tissue-specific media to the transwells for culture growth.
Using sterile technique in a tissue culture hood, thaw extracellular matrix files on ice, then, dilute the warmed extracellular matrix at a 1 to 1 ratio with ice cold collection medium. Mix the suspension with pipetting, taking care not to induce bubbles.
Next, place transwell inserts with 0.4-micron pores into individual wells of a 6-well tissue culture plate and coat each insert with 100 microliters of the extracellular matrix suspension. Place the plate on ice, and rinse the specimens 2 times in collection medium, transferring the tissue to a sterile petri dish under a dissecting microscope after the second wash.
The specimens contain 2 distinct types of decidual tissues; decidua parietalis and decidua capsularis. Using sterile springer dissecting scissors and forceps, microdissect the decidua parietalis into 3-cubic-millimeter pieces, using the forceps to tease away any clotted maternal blood and use forceps to transfer 3 or 4 pieces of decidua into each transwell.
Identify the well-vascularized villi with prominent extravillous trophoblast cell columns and use forceps to transfer 3 or 4 villous trees into each transwell. Adjust the branches, so that the trees are positioned flat atop the extracellular matrix and separate any clumped branches. Then, add 1 milliliter of collection medium to the bottom of each well and incubate the cultures overnight at 37 degrees Celsius in 5% carbon dioxide. 12 to 16 hours later, add 1 milliliter of the appropriate culture medium to the top of each transwell.
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