人間の成人および胎児心標本由来分離とプライマリの心外膜細胞の培養

The overall goal of this procedure is to isolate and culture human epicardial-derived cells in order to study epicardial cell behavior. This method can help answer key questions in the field of cardiac research, such as how is the epicardium activated during cardiac development and disease. The main advantage of this technique is that it can be applied to human cardiac tissue samples, which may help translation of results to a clinical setting.

Our protocol describes the investigation of epithelial to mesenchymal transition of epicardial-derived cells. However, it can also be used to study proliferation or secretion of factors. To begin, fill a 100-millimeter cell culture dish with PBS inside the laminar flow cabinet.

Place approximately 200 microliters of PBS into the lid, then add 5 milliliters of pre-warmed EPDC medium to a 15-milliliter tube. Place the cardiac tissue, isolated from an adult human heart, in the dish. Under a stereo microscope, use forceps to carefully remove the epicardial layer.

Next, collect the pieces of epicardial tissue in the lid and use a scalpel to cut them into 0.5 cubed millimeter pieces. Add one milliliter of trypsin to the tissue pieces. Using a P1000 pipette tip, transfer them into a 1.5-milliliter conical centrifuge tube.

Then, incubate the tube for 10 minutes at 37 degrees Celsius inside a water bath with constant shaking at 60 RPM. After incubation, clean the tube with 70%ethanol and allow the tissue to settle at the bottom. Next, carefully transfer the supernatant which contains epicardial cells into a 15-milliliter tube with 5 milliliters of EPDC medium to inactivate the trypsin.

Add one milliliter of trypsin to the remaining tissue in the centrifuge tube and mix by gentle pipetting. Then, repeat the trypsin incubation step two more times. After the final collection, transfer the remaining epicardial tissue into the tube containing the cell suspension.

Thoroughly homogenize the cell suspension by gentle pipetting, then use a 10-milliliter syringe with a 19 gauge needle to mechanically dissociate the cells in the suspension and transfer the suspension into a fresh 15-milliliter tube. To further dissociate the cells, pass the cell suspension through a 10-milliliter syringe with a 21 gauge needle. Next, use a 10-milliliter pipette to transfer the cell suspension onto a 100-micrometer cell strainer on top of a 50-milliliter tube.

Allow the suspension to pass through the strainer to remove all remaining clumps. Add 5 milliliters of EPDC medium onto the strainer to collect residual cells. After a brief gentle mixing, transfer the cell suspension into a 15-milliliter tube and centrifuge at 200 g for five minutes at room temperature.

Next, remove the supernatant and resuspend the cell pellet in EPDC medium. Remove the coating media from the gelatin-coated plate and add an appropriate amount of cell suspension. Incubate the cells for at least 48 hours at 37 degrees Celsius in 5%CO2.

To culture epicardial cells, replenish the medium every three days. Use EPDC medium supplemented with 10 micromolar ALK 5-kinase inhibitor SB431542. Use a microscope to inspect the cells during culture period.

Upon reaching confluency, passage the cells in a 1:2 surface ratio. To passage the cells, use a pipette or aspirator to remove the medium and carefully add PBS to the cells. Gently rotate the plate and aspirate PBS.

Add trypsin and gently rotate the plate to spread it over the cells. Incubate the plate for one minute at 37 degrees Celsius. Next, tap the plate to mechanically detach the cells and use a microscope to confirm cell detachment.

Add the required volume of EPDC medium supplemented with 10-micromolar SB431542 to the plate. Mix by gentle pipetting and transfer the suspension to a new gelatin-coated plate. To induce EMT, passage the cells using the previously described protocol and incubate for at least 24 hours at 37 degrees Celsius in 5%CO2.

After reaching 50-70%confluency, aspirate the medium and wash the cells with PBS. Next, stimulate the cells with one nanogram per milliliter of TGF beta-3 supplemented EPDC medium and incubate for five days at 37 degrees Celsius in 5%CO2 and monitor cells daily. After five days, cells that underwent EMT should acquire a spindle-shaped morphology.

Culture these cells using the previously described protocol using EPDC medium without any supplementation. To validate proper dissection of the epicardial layer, human adult heart tissue is immunostained with WT1 and cTnI antibodies before and after the dissection. Absence of epicardial marker WT1 after dissection indicates complete removal of the epicardial layer.

Presence of myocardial tissue marker cTnI indicates that subepicardial extracellular matrix and myocardial tissue are intact after the dissection. Adult EPDCs cultured in the presence of ALK 5-kinase inhibitor should maintain cobblestone morphology. Transition from cobblestone morphology to a spindle-shaped morphology of EPDCs after TGF beta-3 treatment indicates epithelial to mesenchymal transition.

To further confirm EMT, EPDCs are immunostained with a mesenchymal marker alpha Smooth Muscle Actin before and after TGF beta-3 treatment. Presence of mesenchymal marker in TGF beta-3 treated cells insures the induction of EMT. The mRNA levels of EMT-related genes in adult EPDCs are represented here.

TGF beta-3-induced downregulation of epicardial marker WT1 and upregulation of mesenchymal markers MMP3, N-Cadherin, periostin, alpha-SMA, and collagen 1A1 confirm the induction of EMT. While attempting this technique, it is important to remember to perform it as efficiently as possible. A shorter isolation time will improve cell survival.

Once mastered, this technique can be done in about 60 minutes. Because this method relies on primary cells, each isolation can behave differently. Therefore, it is important to monitor cell morphology and density before starting an experiment.

After watching this video, you should have a good understanding of how to handle human primary epicardial-derived cells.