Weill Cornell Medical College, New York, New York, USA.
J Surg Res. 2012 May 1;174(1):39-47. doi: 10.1016/j.jss.2010.11.911. Epub 2010 Dec 23.
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.
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.
After 24 h of static seeding with 2.5 × 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).
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.
本研究使用从供体主动脉瓣叶分离的人瓣膜间质细胞（hVICs）对脱细胞人肺动脉瓣（hPVs）进行体外接种。建立了一种检测方法，使用整个 hPV 的预切割均匀大小的活检来评估接种效率、增殖和迁移的顺序。
使用渗透压、双去污剂、酶、多溶剂反复协议来创建脱细胞 hPV 的种子支架。接种 hVIC 的脱细胞瓣叶和窦壁支架在静态或循环压力生物反应器中孵育。在三个后续时间点，使用 8 个重复（n = 576 个活检，包括制造的支架对照）检测低、中、高初始细胞接种“剂量”密度。MTT 测定法检测代谢活性种子细胞。组织学定义细胞位置和形态。
在 24 小时的静态接种中，使用 2.5×10(5)个细胞（中剂量），2.5%的细胞附着在瓣叶上，100 ± 13 个细胞/mm(2)，而窦腔为 193 ± 21 个细胞/mm(2)（8%）。随后在静态培养 4 天，分别产生 894 ± 84 和 838 ± 50 个细胞/mm(2)，而在脉动培养中，分别产生 80 ± 12 和 79 ± 12 个细胞/mm(2)用于瓣叶和窦腔。然而，与静态培养的瓣叶相比，脉动生物反应器中的 76.0% ± 12.2%的细胞位于表面以下，而 21.4% ± 3.9%的细胞位于表面以下（P < 0.001）。
不同的接种模式表明，在静态接种中，表面增殖导致更高的绝对细胞数量与在循环压力生物反应器中细胞数量较少之间存在权衡，但有更多的细胞迁移到基质中。确定最佳的中等接种剂量实际上对于组织工程心脏瓣膜是可行的，并且可以通过相当传统的细胞扩增方法来实现。
