3D Fibrotic Lung Extracellular Matrix Hydrogels Trigger Pro-Fibrotic Responses in Primary Lung Fibroblasts

Idiopathic Pulmonary Fibrosis (IPF) is characterized by the aberrant deposition and organization of extracellular matrix (ECM).
Increased stiffness of fibrotic lung tissue is a major contributor to fibrosis by perpetuating fibrotic responses, through modulation
of interactions between ECM-producing cells and fibrotic ECM. Human decellularized lung ECM-derived hydrogels can be used
as a model for mimicking the native three-dimensional (3D) microenvironment, including recapitulating the mechanical
environment in nonfibrotic (control) and fibrotic lung tissues. In this study, we aimed to characterize control and fibrotic human lung
ECM-derived 3D hydrogels seeded with primary lung fibroblasts to investigate the cellular remodeling responses dictated by the
origin of the microenvironment. IPF and control decellularized lung matrices were freeze-dried, ground to a fine powder, and
mixed (pool of 7 donors per diagnosis) before pepsin digestion. Primary lung fibroblasts were isolated from lung tissue of
patients with IPF (IPF) or macroscopically normal lung tissue derived from patients undergoing tumor resection (non-IPF). IPF or
non-IPF lung-derived fibroblasts (n=6 each) were resuspended in pH-neutralized ECM solutions and hydrogels were allowed to
form before being cultured at 37°C, 5% CO2. Cell-seeded ECM-derived hydrogels were harvested on day 14 and their stiffness
was measured using a Low-Load Compression Tester at 20% strain and compared with equivalent cell-free hydrogels. Cell-free
IPF hydrogels were stiffer than cell-free control hydrogels on day 14. Time in culture did not result in a difference in stiffness of cell-
free hydrogels. The stiffness of control hydrogels seeded with either IPF or non-IPF fibroblasts did not change over a 14-day
culture period (Figure 1A, 1B). In contrast, the stiffness of IPF hydrogels was increased in the presence of non-IPF lung
fibroblasts, compared to cell-free hydrogels over the 14 day period (Figure 1C) (p = 0.016). However, when IPF fibroblasts were
seeded in IPF hydrogels the stiffness of the hydrogel did not change compared to the cell-free counterpart (Figure 1D). These
results illustrate the importance of both the fibroblast-origin and the origin of the ECM in determining the response of the
fibroblasts to the 3D ECM microenvironment. Specifically, the fibrotic microenvironment evoked a profibrotic response in non-IPF
fibroblasts while IPF fibroblasts did not exhibit the same response in this microenvironment. Taken together, our data have
implications for cellular therapies that rely on adding non-diseased cells to tissues to promote disease resolution, as well as
revealing the complexity of the positive feedback between fibrotic ECM and fibroblasts.