Department of Surgery, Connecticut Children's Medical Center, 282 Washington Street, Hartford, CT 06106, USA; Department of Vascular Biology, University of Connecticut Health Center, 263 Farmington Avenue, MC3501, Farmington, CT 06030, USA.
Biomaterials. 2013 Dec;34(38):10043-55. doi: 10.1016/j.biomaterials.2013.09.055. Epub 2013 Oct 1.
The optimal method for creating a de-cellularized lung scaffold that is devoid of cells and cell debris, immunologically inert, and retains necessary extracellular matrix (ECM) has yet to be identified. Herein, we compare automated detergent-based de-cellularization approaches utilizing either constant pressure (CP) or constant flow (CF), to previously published protocols utilizing manual pressure (MP) to instill and rinse out the de-cellularization agents. De-cellularized lungs resulting from each method were evaluated for presence of remaining ECM proteins and immunostimulatory material such as nucleic acids and intracellular material. Our results demonstrate that the CP and MP approaches more effectively remove cellular materials but differentially retain ECM proteins. The CP method has the added benefit of being a faster, reproducible de-cellularization process. To assess the functional ability of the de-cellularized scaffolds to maintain epithelial cells, intra-tracheal inoculation with GFP expressing C10 alveolar epithelial cells (AEC) was performed. Notably, the CP de-cellularized lungs were able to support growth and spontaneous differentiation of C10-GFP cells from a type II-like phenotype to a type I-like phenotype.
尚未确定一种能够制备出无细胞和细胞碎片、免疫惰性且保留必要细胞外基质 (ECM) 的脱细胞肺支架的最佳方法。在此，我们比较了利用恒压 (CP) 或恒流 (CF) 的自动化去污剂脱细胞方法与之前利用手动压力 (MP) 灌输和冲洗去污剂的公布方案。对每种方法得到的脱细胞肺进行评估，以确定是否存在剩余的 ECM 蛋白和免疫刺激性物质，如核酸和细胞内物质。我们的结果表明，CP 和 MP 方法更有效地去除细胞物质，但 ECM 蛋白的保留情况不同。CP 方法的额外好处是更快、更可重复的脱细胞过程。为了评估脱细胞支架保持上皮细胞的功能能力，通过气管内接种 GFP 表达的 C10 肺泡上皮细胞 (AEC) 进行了评估。值得注意的是，CP 脱细胞肺能够支持 GFP 表达的 C10 细胞从 II 型样表型自发分化为 I 型样表型的生长。
