Isolation of Human Primary Valve Cells for In vitro Disease Modeling

This protocol describes the collection of human aortic valves extracted during surgical aortic valve replacement procedures or from cadaveric tissue, and the subsequent isolation, expansion, and characterization of patient specific primary valve endothelial and interstitial cells. Included are important details regarding the processes needed to ensure cell viability and phenotype specificity.

Transcript

The isolation of human primary valve endothelial and interstitial cell is critical for understand the underlying mechanism of the pathogenesis of calcific aortic valve disease. When the valve is excised from the native tissue, the cell viability drop. So we have identified a method that prolong cell viability.

After receiving the extracted valve tissue samples, extract the aortic root and submerge the tissue in a 50 milliliter conical tube containing sterile rinsing solution. After a 10 minute incubation in an ice bucket on a rocker, spray down the tubes with 70%ethanol. In a sterile tissue culture hood, transfer the tissue to a Petri dish and excise two valve leaflets.

Place one leaflet in a cryogenic vial and snap freeze the tissue in liquid nitrogen for minus 80 degrees Celsius storage. To fix the tissue for calcium content staining, cut the second leaflet in half from nodule to hinge and place both tissue pieces into a cassette that is submerged in 4%paraformaldehyde. Then place the entire setup on the rocker at room temperature for a minimum of two but not more than four hours.

To isolate valve interstitial cells, transfer the leaflet into a new 50 milliliter conical tube containing ice cold PBS, and place the tube on a rocker for two minutes at room temperature. After mixing, transfer the tissue to a 60 millimeter dish containing five to seven milliliters of cold collagenase solution. Use forceps to dip both sides of the leaflet three to four times into the solution before incubating the tissue in the cell culture incubator for five to 10 minutes at 37 degrees Celsius with gentle rocking of the tissue three to four times every two minutes.

At the end of the incubation, transfer two milliliters of solution from the dish to a sterile 15 milliliter conical tube. Placing forceps at the nodule of the leaflet, use a sterile cotton swab to swipe the tissue from the forceps to the hinge while twirling the swab along the leaflet. After swiping, rinse the swab in the tube of collagenase solution and swab the other side of the tissue as just demonstrated.

After rinsing, repeat the swab on both sides of the tissue and use a one milliliter pipette and collagenase solution to rinse any dislodge cells from both sides of the leaflet surface into the dish. After rinsing, transfer all of the solution from the dish into the tube containing the cells from the swab and place the remaining valve tissue into a new 15 milliliter conical tube containing seven milliliters of sterile collagenase solution. Pellet the isolated valve endothelial cells by centrifugation and resuspend the cell pellet in three milliliters of vascular endothelial cell growth medium.

After a second centrifugation, resuspend the cells in one milliliter of fresh growth medium for counting and seed the cells at a 5 x 10 to the fifth cells per square centimeter concentration in collagen coated six-well plates, refreshing the medium every three to four days. When the valve endothelial cell patches cover more than 80%of the plate, split the cells at a 1.3 x 10 to the fourth cells per square centimeter concentration, depending upon their growth rate. Once the cells have been expanded, confirm their valve endothelial cell phenotype by immunofluorescent staining for valve endothelial cell markers of interest.

To isolate the valve interstitial cells, place the tube containing the swab to leaflet tissue into the cell culture incubator for 12 to 18 hours with the cap slightly open. At the end of the incubation, use a serological pipette to gently dissociate the tissue to ensure release of the valve interstitial cells and filter the cell suspension through a 70 micron strainer into a 50 milliliter conical tube. Add seven milliliters of valve interstitial cell medium to the tube and collect the cells by centrifugation.

Resuspend the pellet in one milliliter of fresh valve interstitial cell growth medium for counting and seed the cells at a 1.3 x 10 of the fourth cells per square centimeter concentration in a 60 millimeter diameter tissue culture treated dish. After one to two days, wash the cultures two times with PBS and add fresh medium to the cells. When the cells reach a 90%confluency, wash the cultures two times with DPBS and treat the cells with two to three milliliters of pre-warmed dissociation reagent for two to three minutes at 37 degree Celsius.

When the cells have detached, transfer the cell suspension to a conical tube for centrifugation and gently resuspend the pellet in three to four milliliters of pre-warmed valve interstitial cell growth medium for counting. Then seed the cells at a one to two ratio to the original culture density and assess the valve interstitial cell growth phenotype by immunofluorescent staining as demonstrated. Calcific aortic valve disease tissue samples exhibit an altered morphology with heavy nodules of calcification compared to control uncalcified tissue samples.

When the calcification is assessed by Von Kossa staining, dark brown or black precipitation is observed in the diseased leaflet tissue. Cold storage solution greatly stabilizes the excised valve tissue cells with an approximately 40%viability observed for both types of recovered cells up to 61 hours post valve extraction. Morphological analysis of the valve endothelial cells reveals packed cobblestone-like growth contact inhibited cells while the valve interstitial cells demonstrate a spindle shaped morphology similar to that observed for myofibroblasts.

Immunostaining confirms that the majority of expanded valve endothelial and valve interstitial cells express the expected tissue specific markers. Remember that gentle but firm swabbing of the leaflet is critical for the release of the endothelial cell layer, and that the overnight incubation with collagenase is required for the release of the interstitial cell population from inside the dense tissue. Once the cell lines has been established, they can be used for mechanical study regarding specific aortic valve disease pathogenesis, including disease onset, progression, and treatment.

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Journal (Biology)

Journal (Biology)

Isolation of Human Primary Valve Cells for In vitro Disease Modeling

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