The overall goal of this protocol is to isolate rat valve interstitial cells or VICs to study the mechanisms of calcific aortic valve disease. This method can help answer key questions in the valve calcification field, such as why do VICs calcify and what we can to prevent these cells from calcifying. The main advantages of this technique are the accessibility and potential to transform into cell lines for further studies.
Carry out all dissections in a ventilated hood previously disinfected with 70%ethanol to ensure sterility of samples and reagents. Sterilize dissection tools by autoclaving them, followed by immersing the tips of the tools in a beaker containing 70%ethanol prior to use. Soak the dissection tools in beakers of wash buffer before coming into contact with the animal or tissues.
After culling the rats as described in the text protocol, place the animal supine on a glass dissection board and disinfect the skin by spraying with 70%ethanol. To dissect out the heart, make a four centimeter incision in the midline of the rat with the aid of dissection scissors to expose the abdominal cavity, and carefully remove the ribcage and lungs to expose the heart. Remove the heart with a pair of sharp scissors.
Store the dissected heart in ice-cold wash buffer until all gross dissections of the rats are complete. To microdissect each heart, transfer the dissected heart into a Petri dish covered in wash buffer. Trim the cardiac muscle with a pair of spring straight scissors to be a left with a small area surrounding the ascending aorta and the aortic root.
Using the same spring straight scissors, carefully cut open the ascending aorta toward the left ventricle and expose the aortic valve leaflets. Next, transfer the opened aorta into a fresh sterile Petri dish filled with Hank's Balanced Salt Solution. Dissect out the aortic valve leaflets marked by their unique U-shape at the base of the aorta with a pair of Vannas scissors.
Store all leaflets in one milliliter of ice-cold wash buffer in a 1.5 milliliter microcentrifuge tube until all dissections are complete. Once all leaflets have been harvested, centrifuge them at 100g for one minute at four degrees Celsius to remove the wash buffer. To remove the valve endothelial cells or VECs, digest the leaflets in 100 microliters of 425 units per milliliter of Collagenase II for five minutes at 37 degrees Celsius.
Disrupt the digestion by gently pipetting up and down using a 200 microliter pipette tip. Centrifuge at 100g for 30 seconds to pellet the leaflets. After carefully discarding the supernatant, wash the leaflets with 500 microliters of wash buffer and re-pellet the cells by centrifugation at 100g for 30 seconds.
To harvest the VICs from the leaflets, digest with 100 microliters of the Collagenase II for 1.5 to two hours at 37 degrees Celsius. Then release the VICs by gently pipetting up and down using a 200 microliter pipette tip. Dilute the Collagenase II in 19 milliliters of culture medium and centrifuge at 670g for five minutes to pellet the VICs and remaining valve leaflet debris.
After discarding the supernatant, transfer the leaflets and the VICs to culture plates or flasks. Culture the VICs for five to seven days in culture medium until confluency is reached at 37 degrees Celsius in the presence of 5%CO2, changing the medium after 72 hours. To prepare primary rat VICs for in vitro calcification experiments, seed the cells at a density of 150, 000 cells per well in six-well plates.
Maintain the cells in culture medium until greater than 90%confluency. Treat the VICs with calcification versus control medium and incubate at 37 degrees Celsius in the presence of 5%CO2 for an additional 72 hours. To study the calcified primary rat VICs for subsequent downstream analyses, remove the calcification or control medium.
Wash the monolayers with wash buffer to remove non-bound calcium and phosphate ions before performing rat VIC characterization as detailed in the text protocol. For rat VIC calcification studies, stain the cell monolayers with 5%Alizarin Red S solution, gently rocking on a shaker for 20 minutes. Subsequently, wash three times with distilled water for five minutes each time.
To quantify calcium deposition, use a biochemical calcium assay kit. Leech calcium ions using 0.6 molar hydrochloric acid for 24 hours at four degrees Celsius with gentle agitation. Harvest the supernatant and measure the calcium concentration using a calcium assay kit following the manufacturer's guidelines.
Finally, calculate the calcium concentration as a fraction of total cellular protein as described in the text protocol. VIC phenotype of isolated cells was confirmed through immunofluorescence, probing for the VIC markers, vimentin and alpha smooth muscle actin. Additionally, the expression of the VIC growth regulator, tissue growth factor beta-1 and 2, was confirmed using PCR analysis.
Western blot analysis was also performed to verify that rat VICs were negative for the endothelial cell marker CD 31, using canine mitral VECs as a positive control. Rat VICs were exposed to elevated levels of calcium and phosphate, which mimic pathological hypercalcemia and hyperphosphatemia conditions. Treatment of VICs with calcium and phosphate induced calcification, as determined by Alizarin Red staining for calcium deposition and colorimetric determination of calcium levels following hydrochloric acid leaching.
Treatment of VICs with calcium and phosphate induced a significant increase in the mRNA expression of the osteogenic markers MSX2, ALPL, and PHOSPHO1. Once mastered, this technique can be done in one to two hours, excluding the digestion for 18 valve leaflets. While attempting this procedure, it's important to ensure sterility during dissection and digestion.
After watching this video, you should have a good understanding of how isolate primary rat VICs and to use them to investigate the mechanisms behind VIC calcification. Following this procedure, other methods like flow cytometry and microscopy analysis can be performed in order to study VIC physiology and behavior. After its development, this technique can pave the way for researchers in the field of cardiovascular research to explore the mechanisms of aortic valve classifications in humans using the rat model.
