A 3D Human Lymphatic Vessel-on-Chip Reveals the Roles of Interstitial Flow and VEGF-A/C for Lymphatic Sprouting and Discontinuous Junction Formation.

Introduction: Lymphatic vessels (LVs) maintain fluid homeostasis by draining excess interstitial fluid, which is accomplished by two distinct LVs: initial LVs and collecting LVs. The interstitial fluid is first drained into the initial LVs through permeable "button-like" lymphatic endothelial cell (LEC) junctions. Next, the drained fluid ("lymph") transports to lymph nodes through the collecting LVs with less permeable "zipper-like" junctions that minimize loss of lymph. Despite the significance of LEC junctions in lymphatic drainage and transport, it remains unclear how luminal or interstitial flow affects LEC junctions in vascular endothelial growth factors A and C (VEGF-A and VEGF-C) conditions. Moreover, it remains unclear how these flow and growth factor conditions impact lymphatic sprouting. Methods: We developed a 3D human lymphatic vessel-on-chip that can generate four different flow conditions (no flow, luminal flow, interstitial flow, both luminal and interstitial flow) to allow an engineered, rudimentary LV to experience those flows and respond to them in VEGF-A/C. Results: We examined LEC junction discontinuities, lymphatic sprouting, LEC junction thicknesses, and cell contractility-dependent vessel diameters in the four different flow conditions in VEGF-A/C. We discovered that interstitial flow in VEGF-C generates discontinuous LEC junctions that may be similar to the button-like junctions with no lymphatic sprouting. However, interstitial flow or both luminal and interstitial flow stimulated lymphatic sprouting in VEGF-A, maintaining zipper-like LEC junctions. LEC junction thickness and cell contractility-dependent vessel diameters were not changed by those conditions. Conclusions: In this study, we provide an engineered lymphatic vessel platform that can generate four different flow regimes and reveal the roles of interstitial flow and VEGF-A/C for lymphatic sprouting and discontinuous junction formation. Supplementary Information: The online version contains supplementary material available at 10.1007/s12195-023-00780-0.