The overall goal of this technique is to culture endothelial and smooth muscle cells together in-vitro to create myoendothelial junctions that occur in small diameter resistance arteries. This method can help answer key questions in the cardiovascular field such as, protein localization and activation of signaling cascades in the arterial wall. The main advantage of this technique is that you can specifically isolate myoendothelial junction fractions from the endothelium and smooth muscle, which is impossible to do using an actual artery.
Visual demonstration of a paraffin embedding is particularly critical, as the embedding steps can be difficult to learn without seeing the process. Begin construction of the vascular cell co-culture or VCCC, by spraying 150 millimeter petri dishes and lids with disinfectant and then wiping with a paper towel or lint-free wipes. Then, spray the petri dishes with 70%ethanol and place them in the hood to air dry.
Open the plate of filter inserts under sterile conditions. Coat the bottom side of the filters with fiberactin solution by pipetting up to one milliliter of fiberactin solution through the side slits into the bottom of the plate. Make sure, that the bottom half of the filter is covered and leave the inserts in the hood, keeping the plate cover.
After 30 minutes of treatment with fiberaction solution, vacuum any excess solution without disturbing the filter inserts. Then, invert the plate, placing the filters into the bottom half of the clean petri dish. Trypsinize a 225 square centimeter flask of smooth muscle cells with three milliliters of pre-warmed trypsin EDTA solution.
Once the cells have lifted off, add nine milliliters of SMC medium to neutralize the trypsin. Transfer to a conical tube and mix well. Then, pipette 10 microliters onto a hemocytometer to determine cell number.
After performing a cell count, carefully plate 750 microliters of the cell suspension containing approximately 75, 000 smooth muscle cells onto the bottom side of each filter. Incubate the plate at 37 degree celsius overnight. On day two, fill each well of a clean six well plate with two milliliters of fresh pre-warmed SMC medium.
Remove the medium from an insert by suction and transfer it to the six well plate with SMC medium. Add one milliliter of a 0.5%bovine gelatin solution to the upper side of the inserts and place at 37 degree celsius, for at least 30 minutes. Trypsinize a 225 square centimeter flask of endothelial cells with three milliliters of pre-warmed trypsin EDTA solution.
With gentle tapping of the flask to lift the cells off the plate. After re-suspending the cells in EC medium and performing a cell count, remove the gelatin from the filter. Then, plate 360, 000 ECs in a one milliliter volume onto the top side of each filter insert.
Let the cells incubate undisturbed at 37 degree celsius for 24 hours. Begin fractionation, by suctioning off the medium from one six well plate of inserts in the cell culture hood and then, transport to a cold room on ice. In the cold room, pipette 10 microliters of PBS onto the SMC side of the inserts.
Use the cell lifter to scrape down the SMCs. Then, transfer the cells from the scrapper to a labeled petri dish containing lysis buffer. Once the cell scrapper has touched the lysis buffer, do not allow it to touch the insert filter until it has been completely wiped and dried off on paper towels.
Repeat the scrapping of the SMC filter once or twice more, to fully remove the remaining SMC cell debris. Next, pipette 10 microliters of PBS onto the endothelial cell side of the insert filter. Scrape the cells into an appropriately labeled petri dish of lysis buffer, as just demonstrated.
Collect the cell slurry from the EC side of the filter with the pipette and transfer to an appropriately labeled petri dish. Be very thorough in scrapping the cells off both sides of the filters to ensure minimal cell contamination in the myoendothelial junction fraction. Next, carefully use the scalpel to cut out the filters from the plastic insert.
Do this by cutting 70 to 80%of it away from the plastic and using forceps to pull the filter completely off the plastic insert structure. Point each filter into a labeled 50 milliliter conical tube containing lysis buffer. Ensure that the filters are immersed in buffer and remain wet.
After all the cells have been harvested from the filters, vortex the 50 milliliter MEJ tube on full strength for 15 seconds or until mixed well. Remove the filters from the MEJ conical with forceps, dragging them along the side walls of the tube as to leave the maximum amount of proteins and liquids in the tube. Once all the filters have been removed from the MEJ conical tube, do a quick spin in a centrifuge to pull all the liquid and proteins to the bottom of the tube.
After the spin, transfer the contents of the MEJ tube and the SMC and EC petri dishes into separate smaller centrifuge tubes. Then, centrifuge the tubes at close to 16, 000 x G for 15 minutes at four degree celsius. Remove the supernatants.
Then, assay cell lysates for protein content using a byzinconetic assay. On day five of VCCC incubation, add 4%PFA to each well containing a rinsed filter insert and incubate overnight with shaking at four degree celsius. After approximately 24 hours, transfer the inserts to 70%ethanol for at least 24 hours.
Process the filters on a long run on an automated tissue processor. After the run, remove a filter from its cassette using forceps to hold the filter at the edge and then, cut the filter in half with scissors. Lay each of the two halves on a cold plate and fill the embedding mold with liquid paraffin.
Very briefly touch the filled embedding mold to the cold plate, then embed each half of the filter into the embedding mold with the cut side down to ensure that the filter is sectioned from the center. Use forceps to hold the filter in place to prevent the halves from falling into each other until the paraffin is cool enough for them to stand alone. Then, move the embedding mold to a cold plate until completely solidify.
Embed and section the filtered insert in a vertical orientation. After sectioning, the VCCC can be immunostained for transverse immunofluorescence. This immunotransmission electron microscope image shows a mouse artery with expression of alpha globin at the MEJ labeled with gold beads, which appear as black dots.
This western blot demonstrates that plasminogen activator inhibitor one expression is enriched in the MEJ fraction versus the EC or SMC fractions. Plastic EC indicates ECs grown on a plastic dish which do not form MEJ. Immunostaining reveals the tight compartmentalization of the VCCC model.
The alpha-1 adrenergic receptor one is seen only in the smooth muscle cell layer, the bradykinin receptor is seen only in the endothelial cell layer. F actin staining is throughout both cell types at the in-vitro MEJ as indicated by the white arrow. While attempting this procedure, it is important to remember to keep everything sterile until the time of harvest, carefully plate the cells and thoroughly scrape the filters.
Following this procedure, other methods like western blotting or immunofluorescence can be performed in order to answer additional questions lik protein localization, expression levels or activation.
