The overall goal of this trophoblast cell culture model is to measure the effect of obesity-related inflammation on the regulation of critical pathways in trophoblasts. The main advantage of this control system is it establish a relationship between TNF alpha mediated inflammation and the gene regulation in placenta in the context of maternal obesity. The main advantage of this experimental system is it establishes relationships between TNF-alpha mediated inflammation and gene regulation in placenta in the context of maternal obesity.
Demonstrating the procedure will be Bailey Simon and Matt Bucher, technicians from my lab. Villous tissue should be sampled from the placenta as soon as possible following delivery, preferably in 30 minutes or less. With the chorionic plate facing upwards, use forceps and scissors to excise, at random, two to three full-thickness sections, two to three centimeters in from the periphery of the placenta.
Avoid parts of the placenta that appear abnormal. Trim away the chorionic and basal plates and any large blood vessels. Place the resulting villous tissue in warm, complete medium.
Within 30 minutes of villous tissue sampling, begin trophoblast isolation. Place each piece of villous tissue in a 50 milliliter conical tube filled with room temperature PBS and gently agitate to release excess blood in the tissue. Decant the PBS and replace with fresh PBS.
Repeat the PBS rinse until most of the excess blood is removed. The PBS rinse will be light red or pink when the tissue is thoroughly rinsed. Place the villous tissue in a sterile petri dish and remove as many blood vessels as possible by using a microscope slide to gently scrape off the soft villous tissue from the vessels.
Using scissors, finely mince the resulting villous tissue. Transfer the minced villous tissue to a sterile bottle with digestion solution according to the calculated volumes based on the specific activity of trypsin and DNase. Incubate the digestion bottle in a 37 degree Celsius water bath with shaking at 70 rpm for 35 minutes.
After 35 minutes, tilt the digestion bottle on its side and allow the undigested pieces of tissue to settle at the bottom of the bottle. Carefully draw up the supernatant with a serological pipette, avoiding the settled tissue. Dispense the supernatant through a 100 micron cell strainer equally between 50 milliliter conical tubes.
Start the second digestion by adding digestion solution to the remaining settled tissue and incubating the bottle in the 37 degrees Celsius water bath. Gently layer three to five milliliters of newborn calf serum, or NCS, underneath the strained supernatant from the first digestion by slowly dispensing from a serological pipette at the bottom of the tube. A meniscus between the strained supernatant containing trophoblasts and NCS should be visible.
Centrifuge the supernatants over NCS at 1, 250 times g at 20 degrees Celsius for 15 minutes. The resulting pellet will include red blood cells in the lowermost layer, followed by a white layer containing trophoblast cells. In the same way, strain the supernatant from the second and third digestion.
Layer NCS underneath the strained supernatant and centrifuge at 1, 250 times g at 20 degrees Celsius for 15 minutes. Once all supernatants have been centrifuged and the NCS and supernatant layers removed, re-suspend each pellet in five milliliters of warm, complete medium and then pool the suspensions. Divide the cell suspension equally between two 50 milliliter conical tubes and centrifuge at 1, 250 times g at 20 degrees Celsius for 15 minutes.
Gently remove the supernatant and re-suspend each of the cell pellets in six milliliters of warm, complete medium. To begin this procedure, divide the cell suspension equally between four density gradients by using a transfer pipette to slowly and carefully layer the cell suspension on top of the density gradients. Centrifuge the density gradients at 1, 250 times g at 20 degrees Celsius for 20 minutes with minimum acceleration and deceleration.
This should produce distinguishable bands of sedimented cells. Slowly and carefully remove the top layer of density gradient media until the opaque band containing trophoblast cells is reached. Transfer the trophoblast bands into two 50 milliliter conical tubes and fill with warm, complete medium.
Gently invert the tubes three to six times to mix and centrifuge at 1, 250 times g at 20 degrees Celsius for 15 minutes. Remove the supernatant and re-suspend each cell pellet in five milliliters of warm, complete medium. Combine the cell suspensions and count viable cells using a hemocytometer and trypan blue.
The next step after cell counting is to plate the cells. Plate three million cells per well in each six well plate. Gently rock the plate back and forth and side to side to evenly distribute the cells.
Leave the plated cells in the laminar flow vent for approximately 30 minutes to allow the cells to evenly distribute, settle, and begin to adhere to the bottom of the wells. After 30 minutes, place the plates in a 37 degree Celsius incubator with 5%carbon dioxide and 95%humidity. Examine the trophoblasts under a microscope after 24 hours of culture to confirm attachment and proper trophoblast morphology.
The cells can be treated after 24 hours of culture. Aspirate the culture media from the cells and replace with two milliliters of TNF alpha supplemented medium per well. Return the plates to the 37 degree Celsius incubator.
After 24 hours of TNF alpha exposure, replace the TNF alpha supplemented medium with complete medium. Incubate for another 24 hours. 24 hours after removal of TNF alpha, aspirate media and gently rinse the cells with room temperature PBS.
Add directly to each well 80 microliters of ice cold radioimmunoprecipitation assay buffer containing freshly added protease and phosphatase inhibitors. Remove the cells from the plate with a cell scraper. Transfer the cells to a 1.5 milliliter microcentrifuge tube, pooling wells within treatment groups.
Ice the cells by vortexing the tubes on high for at least three intervals of 15 seconds. Incubate the cells at four degrees Celsius with rocking for 15 minutes. Vortex the tubes again on high.
Centrifuge at 10, 000 times g for five minutes at four degrees Celsius to pellet cellular debris. Transfer the supernatant to a new 1.5 milliliter microcentrifuge tube. Store the cellular protein samples at 80 degrees Celsius until analysis by Western blotting for the protein of interest.
After 72 hours of culture, immunocytochemical analysis of cytokeratin 7, shown in red, in primary trophoblasts confirm that the cells underwent syncytialization evidenced by the presence of multi-nucleated cell masses. Furthermore, immunocytochemical analysis of vimentin, also in red, revealed very few contaminating fibroblasts. Nuclear counter-staining is shown in blue in both images.
Treatment with TNF alpha up to 250 picograms per milliliter up-regulated rubicon expression. At TNF alpha concentrations greater than 250 picograms per milliliter, rubicon expression levels decreased back toward baseline and even below. Likewise, rubicon is significantly up-regulated in flash frozen villous tissue biopsies from placentas from obese pregnancies with female fetuses compared to lean controls.
Once mastered, isolation of trophoblasts from villous tissue can be done in six to seven hours if it is performed properly. While attempting this procedure, it's important to prepare materials and reagents ahead of time so you can work deliberately and efficiently. Following this procedure, other assays, such as those that measure cellular bioenergetics, can be performed to answer additional questions, like does inflammation in the obese intrauterine environment affect the metabolic capacity of trophoblasts?
After this development method paved the way for placental researcher to establish the effect of maternal environment on trophoblast function. After watching this video you will have a good understanding of how to isolate trophoblasts from villous tissue for your cell culture experiment and how to determine the effect of TNF alpha mediated inflammation in regulation of critical pathways in the trophoblasts. Don't forget that working with human tissue, cultured cells, DMSO, acrylamide and reducing reagents can be extremely hazardous and precautions, such as wearing proper PPE and using aseptic technique should always be taken while performing this procedure.
