(20e) “Lymphatics-on-a-Chip” to Reconstitute Lymphatic Drainage Function and Lymphedema
AIChE Annual Meeting
2017
2017 Annual Meeting
Food, Pharmaceutical & Bioengineering Division
Cells, Organs, and Labs on a Chip
Sunday, October 29, 2017 - 4:42pm to 5:00pm
Among many lymphatic diseases, âLymphedema (LE)â, featured by abnormal tissue swelling, is the most common lymphatic disorder, influencing 150 million individuals worldwide. In physiological condition, normal LVs properly drain excess interstitial fluid that is leaked from blood capillaries; and the drained lymph fluid travels through the lymphatic system to go back to the blood circulation through the subclavian veins. These recirculating processes maintain interstitial fluid homeostasis in normal tissues. However, any failures in the LV drainage trigger abnormal fluid accumulation in the tissues, causing âswellingâ. Currently, mechanisms of LV drainage in normal or LE condition are poorly understood, thus therapeutic options for LE treatment are limited. There is no clinically available drug for LE, and widely practiced conservative therapies, such as massage and compression garments are palliative.
One of the major obstacles to better understanding and curing LE is a lack of appropriate experimental tools for evaluating lymphatic drainage function. Lymphatic drainage is the most crucial lymphatic function, directly linked to LE and influenced by numerous biological factors. Although animal models have contributed to major discoveries in the field, isolating and controlling biological factors that may contribute to LE in the animal models is challenging. Majorities of in vitro models that have attempted to solve these problems were two-dimensional (2D) cell culture models on a plastic dish or a transwell, which have not successfully recapitulated lymphatic cell behaviors in 3D environment as in in vivo settings. As such, detailed, mechanistic investigation of LE has remained limited. Thus, 3D biomimetic vascular models would be useful to study blood and lymphatic biology in many different contexts [11-13].
To study lymphatic drainage function and LE pathogenesis in 3D in vitro, we built a new biomimetic âhuman lymphatics-on-a-chip" model system by fabricating a microfluidics-based device that includes a poly-dimethyl siloxane (PDMS) housing and two parallel micro-channels within 3D collagen matrix. Employing physiological luminal flow in the micro-channels, human primary dermal lymphatic endothelial cells (LECs) seeded in the channels formed lumenized and perfusable LVs within 3D collagen, exhibiting physiologically relevant LV structure and function. For example, interstitial flow containing lymph molecules, such as albumins, fatty acids, phospholipids, and particles, demonstrated that LVs much better drain the lymph molecules than blood vessels (BVs) do. Surprisingly, we observed that LVs exhibited dilated lumens under interstitial flow, however, BVs were squeezed in the same condition. Immunostaining revealed that LVs have weak tight junctions and jagged adherens junctions with specialized portal-like structures to promote fluid transport. The dynamic LV lumens under interstitial pressure, weaker junctions, and preformed portals have been reported in mouse models and human lymphatic tissues, so we concluded that we have a functionally relevant LV model in 3D.
Based on the normal lymphatics-on-a-chip model, inflammatory cytokine treatment and Podoplanin (Pdpn) deficiency significantly impaired lymphatic drainage function by abnormally tightening LV junction. This is a notable finding because most of the inflammation conditions cause vessel leakages by loosening vascular junction. Mechanistically, we discovered that one of the integrin families is a new therapeutic target for LE. This integrin is normally inactivated, however, becomes highly activated in LE conditions, including both cytokine treated- and Pdpn deficient groups. The activated integrin dramatically tightens lymphatic junction, delays fluid, lipid, cell transport through the LVs, and ultimately impairs lymphatic drainage rate. Employing multiple inhibitors of this integrin and the integrin-related pathways, we could normalize pathological lymphatic junction, restoring almost 100% of lymphatic drainage function. Now, we are in validating these findings in mouse LE models.
In conclusion, our âlymphatics-on-a-chipâ model recapitulates physiological lymphatic drainage function and LV structure in nature. Moreover, the âlymphatics-on-a-chipâ provides an important in vitro platform for revealing previously unappreciated disease mechanisms of LE. Together, these data suggest that the âlymphatics-on-a-chipâ may serve as a new platform for mechanistic studies and drug screening for lymphatics associated disorders.
REFERENCES (*Equal contribution)
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ACKNOWLEDGEMENTS
This work was supported in part by grants from the National Institutes of Health (EB00262, UH3EB017103, UC4DK104196). Esak Lee acknowledges financial support from LE&RN postdoctoral grant from Lymphatic Education and Research Network (LE&RN), and BU-CTSI grant (TL1TR001410) from the National Center for Advancing Translational Sciences at the National Institutes of Health.Â