Determining Cell Seeding Dosages for Tissue Engineering Human Pulmonary Valves

Background. This study examines in vitro seeding of decellularized human pulmonary valves (hPVs) with human valve interstitial cells (hVICs) isolated from unrelated donor aortic valve leaflets. An assay was developed to assess seeding using precut uniform sized biopsies from whole hPVs for sequential evaluation of seeding efficiency, proliferation, and migration. Materials and Methods. Scaffolds for seeding were created from decellularized hPVs using a reciprocating osmolality, double detergent, enzyme, multiple solvent protocol. hVICs seeded decellularized leaflet and sinus wall scaffolds were incubated in either static or cyclic pressure bioreactors. Low, medium, and high initial cell seeding "dosing" densities were assayed at subsequent three time points, using eight replicates each (n=576 biopsies including manufactured scaffold controls). Metabolically viable seeded cells were quantified by MTT assay. Histology defined cell locations and morphology. Results. After 24 h of static seeding with 2.5 x 10(5) cells (medium dose), 100 +/- 13 cells/mm(2) (2.5%) attached to leaflets, compared with 193 +/- 21 cells/mm(2) (8%) for sinuses. Subsequent 4 d in static culture yielded 894 +/- 84 and 838 +/- 50 cells/mm(2) versus pulsatile culture yielding 80 +/- 12 and 79 +/- 12 cells/mm(2) for leaflet and sinus, respectively. However, 76.0% +/- 12.2% of cells in leaflets in the pulsatile bioreactor were subsurface as compared to 21.4% +/- 3.9% in statically cultured leaflets (P < 0.001). Conclusion. Different seeding modes suggest a tradeoff between surface proliferation resulting in higher absolute cell numbers for static seeding versus fewer cells in a cyclic pressure bioreactor but with a greater percentage having migrated into the matrix. The medium seeding dose determined to be optimal is actually feasible for tissue engineering heart valves, and can be achieved by fairly traditional cell amplification methods. (C) 2012 Elsevier Inc. All rights reserved.