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Isolation and Characterization of Primary Rat Valve Interstitial Cells: A New Model to Study Aortic Valve Calcification
Published on: November 20, 2017
Aortic valve interstitial cells: an evaluation of cell viability and cell phenotype over time
Massimo Cimini1, Kem A Rogers, Derek R Boughner
1University of Western Ontario, Department of Anatomy and Cell Biology, London, Canada.
Insights
Porcine aortic valve interstitial cells remain viable and phenotypically stable for up to 96 hours post mortem. This finding supports the potential use of autopsy-derived human valve cells for in vitro research.
Area of Science:
- Cardiovascular Biology
- Tissue Engineering
- Cell Biology
Background:
- Understanding aortic valve interstitial cells is crucial for studying valve disease and developing tissue-engineered heart valves.
- Previous studies deemed autopsy samples unsuitable for immunocytochemical analysis due to post-mortem ischemic time.
- This study investigates cell viability and phenotype from porcine heart valves at extended post-mortem intervals.
Purpose of the Study:
- To determine the viability and phenotypic profile of cells explanted from porcine heart valves up to 120 hours post mortem.
- To assess the suitability of post-mortem heart valve tissue for cell culture and analysis.
- To inform potential strategies for utilizing human autopsy samples for valve research.
Main Methods:
- Porcine aortic valve leaflets were harvested at various post-mortem time points (0-120 hours).
- Cells were explanted and cultured, with viability assessed using trypan blue exclusion.
- Immunocytochemical and immunohistochemical analyses were performed to compare in vitro and in situ cell phenotypes.
Main Results:
- Cell viability in culture remained unaffected up to 96 hours post mortem.
- The number of explanted cells decreased significantly over time.
- Explanted cells consistently exhibited phenotypes similar to in situ cells from the same valves.
Conclusions:
- Porcine aortic valve interstitial cells can be successfully explanted up to 96 hours post mortem with preserved viability and phenotype.
- The findings suggest that human aortic valve interstitial cells harvested at autopsy could be viable for in vitro studies.
- This research provides a foundation for using post-mortem tissues in cardiovascular research and regenerative medicine.
Background And Aim Of The Study:
In investigating the mechanisms of aortic valve disease processes and to accurately construct tissue-engineered heart valve prostheses, a complete understanding of the native interstitial cell population is required. Previously, autopsy samples have been deemed unsuitable for immunocytochemical studies due to the ischemic time before harvesting. In this study, the viability and phenotypic profile of cells explanted from normal porcine heart valves up to 120 h post mortem was examined.
Methods:
The aortic valve leaflets of porcine hearts were excised at 0, 6, 24, 48, 72, 96 and 120 h post mortem; one half of the tissue was used to explant cells, and the other half was fixed in 3.7% formalin for sectioning. Samples taken at each time point were cultured and cell viability was determined using a trypan blue exclusion assay. Immunocytochemical and immunohistochemical analyses, using specific markers for fibroblasts, myofibroblasts and smooth muscle cells, were used to compare cell phenotypes both in vitro and in situ.
Results:
Absolute numbers of cells obtained from each leaflet decreased significantly over time; however, cell viability in culture was unaffected up to 96 h. At each time point, explanted cell populations expressed similar phenotypes when compared with histological samples prepared from the same valves.
Conclusion:
Porcine aortic valve interstitial cells may be explanted up to and including 96 h post mortem, with no statistically significant change in cell viability in vitro, and with a population that phenotypically resembles aortic valve interstitial cells in situ. These data suggest that human aortic valve interstitial cells may be successfully harvested at autopsy for in vitro studies.
