Doryab Ali, Schmid Otmar
Institute of Lung Health and Immunity (LHI) and Comprehensive Pneumology Center (CPC), Helmholtz Munich, Member of the German Center for Lung Research (DZL), 85764 Munich, Germany.
Biomedicines. 2022 Jul 26;10(8):1791. doi: 10.3390/biomedicines10081791.
Chronic lung diseases are one of the leading causes of death worldwide. Lung transplantation is currently the only causal therapeutic for lung diseases, which is restricted to end-stage disease and limited by low access to donor lungs. Lung tissue engineering (LTE) is a promising approach to regenerating a replacement for at least a part of the damaged lung tissue. Currently, lung regeneration is limited to a simplified local level (e.g., alveolar−capillary barrier) due to the sophisticated and complex structure and physiology of the lung. Here, we introduce an extracellular matrix (ECM)-integrated scaffold using a cellularization−decellularization−recellularization technique. This ECM-integrated scaffold was developed on our artificial co-polymeric BETA (biphasic elastic thin for air−liquid interface cell culture conditions) scaffold, which were initially populated with human lung fibroblasts (IMR90 cell line), as the main generator of ECM proteins. Due to the interconnected porous structure of the thin (<5 µm) BETA scaffold, the cells can grow on and infiltrate into the scaffold and deposit their own ECM. After a mild decellularization procedure, the ECM proteins remained on the scaffold, which now closely mimicked the cellular microenvironment of pulmonary cells more realistically than the plain artificial scaffolds. We assessed several decellularization methods and found that 20 mM NH4OH and 0.1% Triton X100 with subsequent DNase treatment completely removed the fibroblasts (from the first cellularization) and maintains collagen I and IV as the key ECM proteins on the scaffold. We also showed the repopulation of the primary fibroblast from human (without chronic lung disease (non-CLD) donors) and human bronchial epithelial (16HBE14o−) cells on the ECM-integrated BETA scaffold. With this technique, we developed a biomimetic scaffold that can mimic both the physico-mechanical properties and the native microenvironment of the lung ECM. The results indicate the potential of the presented bioactive scaffold for LTE application.
慢性肺部疾病是全球主要死因之一。肺移植是目前治疗肺部疾病的唯一有效疗法，但仅限于终末期疾病，且受供体肺获取途径有限的限制。肺组织工程（LTE）是一种有前景的方法，可用于再生至少部分受损肺组织的替代物。目前，由于肺结构和生理的复杂精巧，肺再生仅限于简化的局部水平（如肺泡-毛细血管屏障）。在此，我们介绍一种使用细胞化-去细胞化-再细胞化技术的细胞外基质（ECM）整合支架。这种ECM整合支架是在我们的人工共聚物BETA（用于气液界面细胞培养条件的双相弹性薄片）支架上开发的，该支架最初接种了人肺成纤维细胞（IMR90细胞系），作为ECM蛋白的主要产生者。由于薄（<5 µm）BETA支架的相互连接的多孔结构，细胞能够在支架上生长并浸润到支架中，并沉积自身的ECM。经过温和的去细胞化程序后，ECM蛋白保留在支架上，现在该支架比普通人工支架更逼真地模拟了肺细胞的细胞微环境。我们评估了几种去细胞化方法，发现20 mM NH4OH和0.1% Triton X100并随后进行DNase处理可完全去除（首次细胞化后的）成纤维细胞，并使I型和IV型胶原蛋白作为关键ECM蛋白保留在支架上。我们还展示了来自人类（无慢性肺部疾病（非CLD）供体）的原代成纤维细胞和人支气管上皮（16HBE14o-）细胞在ECM整合的BETA支架上的重新接种。通过这种技术，我们开发了一种仿生支架，可以模拟肺ECM的物理机械特性和天然微环境。结果表明所提出的生物活性支架在LTE应用中的潜力。
