Decellularized Precision Cut Lung Slices as a model to study in vitro lung regeneration

Abstract

Objective: Idiopathic lung fibrosis is a disease characterized by continuous injury and consequent repair of the distal epithelial lung compartment, which is facilitated by cells with regenerative capacity from the conducting airways and the alveolar compartment. The extent of this regenerative capacity, as well as possible stimuli inducing regeneration of tissue have yet to be extensively characterized. We therefore sought to establish a method for assessing the regenerative capability of diverse lung epithelial cell populations by using decellularized precision cut lung slices (PCLS) to offer a cell-free scaffold with similar mechanical properties, three-dimensional-structure and protein composition as the native lung. Materials and methods: In this work we developed a method to generate extracellular lung matrix scaffolds for extended cell culture of unfractionated lung cells. To that end, we took a step-wise approach involving several detergents (SDS, TritonX, SDC and CHAPS) and decellularization regimens, to provide a reliable, optimized, and scalable method for efficiently generating high quality extracellular matrix scaffolds. Additionally, we developed a reseeding protocol to facilitate repopulation of the resultant ECM-scaffolds with unfractionated lung cells and study changes in the cellular composition as well as regenerative potential in decellularized PCLS. Results: Detergent based protocols showed differences in both the efficacy of cell removal as well as the preservation of the histological appearance and protein composition of the scaffold. The final decellularization protocol was based on CHAPS and yielded completely decellularized extracellular matrix scaffolds with largely intact morphology and protein structure. These scaffolds were successfully used for extended culture of unfractionated lung cells for up to 3 weeks and supported the growth of different subpopulations including fibroblasts, macrophages, AEC I and AEC II. Both cell adhesion to the matrix as well as proliferative activity could be observed. Conclusions: We conclude that our method of culture of unfractionated lung cells on decellularized extracellular matrix lung scaffolds described herein provides a feasible approach to enable and enhance translational research in regenerative medicine. The provided model is suited for extended research on the regenerative capability of various cell populations in the lung.