Poster O17 - Three Dimensional Pulmonary Fibrosis Modeling with Decellularized ECM Cryogels

Introduction: Idiopathic pulmonary fibrosis (IPF) is a condition found in many interstitial lung diseases and affects approximately 250,000 Americans (PFF 2024). This disease features inflammation and scarring of the lung tissue around the alveolar sacs due to increased collagen deposition, thereby limiting gas exchange and decreasing lung elasticity (Sauleda 2018). Current in vitro models of IPF are composed of hydrogels fabricated with common ECM components such as collagen and elastin; however, these models are limited in representing the cellular signaling and remodeling between resident fibroblasts and the extracellular matrix. Hydrogels derived from single source components are homogeneous in composition and mechanical properties (ie stiffness, elastic modulus, etc), while the ECM in healthy and diseased states is more dynamic due to protein diversity and constant remodelling.While these models have been successful in myofibroblast differentiation, this study aims to mimic IPF in a bio active model through the use of decellularized based gel modeling through the use of cryo and hydrogels. Cryogels are formed through the freezing and subsequent lyophilization of precursor gel solution which provides controlled pore dimensions and increased elastic modulus. We hypothesize that cryogels composed of lung decellularized extracellular matrix will provide increased surface area for cellular attachment and aid in myofibroblast differentiation in presence of TGF-b1 . The objective of this work is to develop an in vitro IPF model that is able to both stimulate myofibroblasts differentiation and be modified by increased collagen production.

Materials and

Methods: Both the cryogels and hybrid gels were fabricated using acid based digestion of cryomilled decellularized human lung tissue (Pouliot et al 2016). Normal human lung fibroblasts (NHLF) were used for experimentation due to their prominence within the lung interstitium (White 2015). While cryogels were formed by freezing the gel precursor solution, lyophilizing, and seeding with NHLF, hybrid gels combine the completed cryogel with additional gel precursor solution containing NHLF in suspension. In order to stimulate fibrosis in the gel models, human latent transforming growth factor beta one TGFb-1(1 ng/ml)was suspended within the treatment media and was used in the cell culturing process of normal human lung fibroblasts. Live/Dead staining was conducted in order to confirm whether cells had properly adhered to the cryogel structure, while RNA was collected from cellular lysate for qPCR. In order to optimize the cryogel model, the initial cell seeding concentration was decreased to 70,000 cells/gel and adherence time increased to 24 hours to promote cellular adherence.

Results, Conclusions, and Discussions: The cryogels were found to have a pore size of 98.8 um +/- 15.6 and fiber diameter of 44.4 um +/- 7.8 which mirrors the structure of the distal lung. When examining the mechanical properties of the cryogel using nanoindentation analysis there was no significant difference between cryogels and native lung tissue with regards to hardness and elasticity. Cellular viability and adherence within the gel structure was confirmed via Live/Dead staining and fluorescence microscopy. where the most cells were found within the first 48 hours and fell dramatically upon reaching 72 hours. To quantify myofibroblast differentiation, qPCR was used in order to determine whether alpha smooth muscle actin (aSMA) and transcription factor EGR1 had been upregulated. Within the cryogel model, aSMA displayed over 75 fold change when compared to controls while EGR-1 showed 10 fold change. Both gene expression for both proteins taper off significantly after 24 hours where aSMA showed little over 6 fold change and EGR1 approximately 3 fold change.
Conclusions and Discussions
We have engineered a lung ECM cryogel model that is able to both sustain cellular culture and allow for the differentiation of normal human lung fibroblasts into the more fibrotic phenotype of myofibroblasts. Optimization criteria from the fibrotic cryogels will then be extrapolated in the hybrid gel models. Cellular adherence would be easier to promote due to cellular encapsulation by the softer hydrogel component and could increase the lifespan of the model.

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