The use of collagenase III for the isolation of porcine aortic valvular interstitial cells: rationale and optimization.
OBJECTIVE
Substantial heart valve research relies on the isolation of valvular interstitial cells (VICs). While a wide variety of conditions have been reported for VIC isolation, the effectiveness of these methods has rarely been compared. It is also likely that valve donor age will influence these valvular tissue dissociation conditions. The study aim was to increase the efficiency and cost-effectiveness of VIC isolation, while taking into account possible differences due to valve donor age.


METHODS
Aortic valves were obtained from six-month-old (n = 24) and six-week-old (suckling) pigs (n = 45) within 24 h of death. After removal of endothelial cells, the tissues were minced and subjected to a variety of enzymatic digestions for variable lengths of time.


RESULTS
The optimal concentration of collagenase III was determined as 1 mg/ml for six-week-old pigs, and 2 mg/ml for six-month-old pigs. The optimal duration of digestion was 4 h for both ages. The addition of neutral protease (2 mg/ml) further increased yield, while additional DNAse and hyaluronidase had no effect. Yield was not influenced by the volume of enzyme solution, nor the use of previously frozen enzyme solution.


CONCLUSIONS
These findings provide age-specific conditions for improving the yield of VIC isolation, which should be of value in experimental studies of valvular cell biology and tissue engineering investigations.
Stephens
Elizabeth H
EH
Department of Bioengineering, Rice University, Houston, Texas 77251-1892, USA.
Carroll
Joshua L
JL
Grande-Allen
K Jane
KJ
D000818:Animals; D001021:Aortic Valve / Q000166:cytology* / Q000187:drug effects*; D002469:Cell Separation / Q000191:economics / Q000379:methods*; D003362:Cost-Benefit Analysis; D003851:Deoxyribonucleases / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D006821:Hyaluronoglucosaminidase / Q000494:pharmacology; D053509:Matrix Metalloproteinase 13 / Q000494:pharmacology*; D023421:Models, Animal; D010447:Peptide Hydrolases / Q000494:pharmacology; D012107:Research Design; D013552:Swine; D013997:Time Factors
D003851:Deoxyribonucleases; D006821:Hyaluronoglucosaminidase; D010447:Peptide Hydrolases; D053509:Matrix Metalloproteinase 13