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The Mechanical Environment Modulates Lymphatic Tube Formation In Vitro
Gene K. Lee, MD, MPH, David P. Perrault, BS, Yi-Chen Wu, MS, Josephine Y. Fang, PhD, Sun Young Park, MS, Bo Han, PhD, Young-Kwon Hong, PhD, Alex K. Wong, MD, FACS.
University of Southern California, Los Angeles, CA, USA.

Purpose: Clinical lymphedema can be tempered by exogenous administration of pro-lymphangiogenic molecules such as VEGF-C or retinoic acids at the time of lymphatic injury. In this study, we sought to explore the effect of three-dimensional mechanical environment on VEGF induced lymphangiogenesis. Improved understanding of growth factor induced lymphangiogenesis will aid clinical translation of novel therapies against lymphedema.
Methods: Human dermal lymphatic endothelial cells (LEC) were isolated from human foreskin. The cells were expanded in traditional 2D culture in EBM in LEC media with 15% fetal bovine serum supplement and 1% Ampicillin/Gentamycin. Transglutaminase-crosslinked collagen hydrogels (Col-Tgel) were prepared with a 12% gelatin stock gel washed with PBS, and further diluted for various gel concentrations. Mechanical tests were carried out with an indentation test, and the deformation distance of the gel construct was used as an indicator of relative gel stiffness. A total of 5 different concentrations were used with relative stiffness including: <1, 5, 9, 18 and 25 kPa. The 3D gels were cultured as a single droplet on a 48-well suspension cell culture plate with exchange of fresh media and 100ng/mL of VEGF-A/C every 2-3 days. The gels were directly observed daily under the light microscope and recorded.
Results:
Figure 1. Lymphatic tubes in 3D gel cultured with VEGF-A/C as seen 4 days after plating under 20x magnification. Red arrows indicate lymphatic tubes.
Conclusions: The 3D hydrogel served as the interstitial substrate to support LEC tube formation under different mechanical properties. Our experimental results showed a high density of tube formation in LECs cultured in ~18 kPa Col-Tgels beginning as early as 48 hours after plating. Shorter and early stages of tubule structure formation was also visible in the ~9 kPa gel. Col-Tgels with stiffness lesser than 9 kPA or greater than 18 kPa were not conducive to lymphatic tube formation. Lymphatic tube formation was similarly seen in the 18 kPa gels cultured with LEC media alone without soluble cytokines, but only after a 72-96 hour lag compared to the gels with cytokine treatment (images not shown). Our results show that the 3D environment plays an integral role in both cell-cell and cell-extracellular matrix (ECM) interactions. LECs respond not only to soluble factors associated with the ECM, but also the biomechanical cues for tubulogenesis.


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