Modeling fibrosis: Studying growth and barrier integrity in lymphatic endothelial cells on temporally stiffened methacrylated collagen.

Introduction:: Lymphangiogenesis—new lymphatic capillary growth—is triggered by fibrosis-related tissue stiffening and soluble factor signaling. Despite the consistency of lymphangiogenesis during fibrosis and subsequent tissue stiffening, in vitro models of fibrotic diseases and lymphatics rarely include pathophysiological stiffness levels (e.g., 10-40 kPa for breast cancer tissue). Specifically, the temporal stiffness changes in the extracellular matrix (ECM) are a component of fibrosis yet to be included within in vitro models of lymphatic vasculature. Fibrotic diseases like tumors, kidney disease, and lymphedema have significant lymphatic involvement but have conflicting therapeutic approaches (anti vs. pro lymphangiogenic). These differences likely arise from a conflation of lymphatic vessel growth and function (vessel barrier integrity), but current preclinical in vitro models do not adequately separate and address the two processes, which may be why in vitro outcomes do not match what is observed in vivo. The objective of this work is to establish in vitro model conditions to investigate the impact of temporal ECM stiffening on lymphatic growth and barrier integrity using methacrylated type I collagen (ColMA) and photo-crosslinking within a microfluidic device. Overall, the proposed work contributes to the long-term goal of developing dynamic, predictive in vitro models of disease with significant fibrotic elements and lymphatic involvement. These models can then be used to develop, test, and improve therapies that target mechanisms of disease progression, rather than the symptoms of disease to lead to better therapeutic outcomes.

Materials and Methods:: Rheological time sweeps (oscillatory shear) were used to determine the temporal change in shear storage modulus (G’) of ColMA (8 mg/mL; Advanced BioMatrix) during self-assembly and photo-crosslinking. The photoinitiators Irgacure5929 (365nm), lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP; 405nm), and Ruthenium/Sodium Persulfate (RUT; 400-450nm) were compared for their dynamic stiffness ranges and temporal control over ColMA stiffness. Lymphatic endothelial cell (LEC) viability was tested under photo-crosslinking conditions (90s light exposure) using Calcien AM/Ethidium homodimer. Human dermal LECs (PromoCell) were seeded at 20,000 cells/cm2 on top of 2D ColMA gels that were un-crosslinked (soft, 0.5kPa), crosslinked (stiff 4kPa) and photo-crosslinked midway through culture (temporal) were assessed for differences in their morphology (cell shape) and expression of common LEC markers (VE-Cadherin – cell-cell junction; VEGFR3 – growth factor receptor; Lyve1 – LEC specific) using immunofluorescence. 3D printing and soft lithography were used to fabricate polydimethylsiloxane microfluidic devices with two fluid channels (300 µm diameter) completely surrounded by a 3D ColMA gel. LECs were seeded 500,000 cells/mL within the device channels to assess attachment to the ColMA surface and cell morphology. Additional work is ongoing to assess LEC proliferation, LEC migration, and barrier integrity of sprouting LECs using immunofluorescence, FITC-dextran perfusion through LEC sprouts, and permeability measurements across the capillary wall, respectively, within the device.

Results, Conclusions, and Discussions:: Results

LAP and RUT were shown to have the greatest stiffness ranges (3-5 kPa), with RUT with the highest consistency. LEC viability was not significantly affected by the presence of photoinitiator or light exposure (not shown). Morphological assessment, via immunostaining, allow for observations of LEC barrier integrity, evidenced by the cell spreading and cell-cell junctions (VE-Cadherin) . Microfluidic devices were successfully fabricated and seeded with LECs that remained attached to the ECM channels over 7 days.

Discussions

ColMA, paired with RUT, provides a hydrogel with tunable stiffness in close to pathophysiological ranges (Figure 1). The use of a stiffening hydrogel in an in vitro model of lymphatic growth during fibrosis is novel not only in the high stiffness values but also the temporal stiffening capabilities. This material will allow for recapitulation of the progressive nature of fibrosis. Cell seeding on soft and stiff matrices, and immunofluorescent staining, suggest LEC mechanosensing. The LEC morphological differences between soft and stiff matrices, with cell spreading and junctions suggesting an increase in barrier integrity on stiff ColMA (Figure 2). Microfluidics provide a platform to study both LEC growth and barrier integrity in soft and stiff substrates. Specifically, this platform will allow for testing of temporal stiffness changes on LEC growth and barrier integrity.

Conclusions

In vitro models have yet to study the effects of progressive ECM stiffening on lymphangiogenesis in the context of fibrosis. Our results demonstrate that ColMA, in combination with LAP or RUT, provides a natural polymeric material with temporally adjustable stiffness that can be used in models of fibrosis with dynamic biophysical cues at pathological stiffness levels. Moreover, results suggest that ColMA can be used to separately assess changes in lymphatic capillary growth and barrier integrity during fibrotic progression, which is important when considering how to resolve lymphatic dysfunction. Overall, this in vitro model has potential to provide a clearer picture of how physical interactions during fibrosis affect lymphatic vessel formation and function. Study outcomes will eventually provide insights to better direct lymphatic treatments and support the development of new therapies that do more than just attenuate symptoms.

Acknowledgements (Optional): : This work is funded by the National Science Foundation ERI, Award #2138841

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