Session: Rising Stars of Mechanical Engineering Celebration & Showcase

Paper Number: 149903

149903 - An Ex Vivo Microfluidic Whole Human Lymph Node Model to Uncover Mechanisms of Drug Transport and Immune Function 

Introduction: In addition to serving as the primary site for the initiation of the adaptive immune response, the architecture of the lymph node (LN), ironically, makes it an ideal sanctuary site for invasive agents capable of penetrating the lymph node, such as lymphoma and metastatic tumor cells, mycobacteria, and HIV. However, there are no models of the LN in which transport through the whole organ can be mechanistically studied, which has stifled the design of therapeutics to increase drug penetration into lymphoid tissue. The current standard for investigating lymph node drug transport and cellular interactions is murine or non-human primate models; however, these models do not reliably replicate the specific immune responses seen in humans. Existing organ-on-a-chip models can use human-derived immune cells but do not replicate important physiological components such as biophysical forces, 3D spatial organization, and native extracellular matrix composition. Furthermore, replicating the physiological balance of effector vs. regulatory cells and cytokines presents a “chicken and egg” problem, as these components are not easily modulated in vivo, making validation of these in vitro models difficult. Therefore, we developed an integrated ex vivo whole human lymph node-on-a-chip (hLNChip) model in conjunction with a predictive computational model that would create a novel system enabling the study of the immune system and close the gap between drug discovery and human clinical trials.

Methods: Our hLNChip model consists of independently controlled lymphatic and vascular perfusion, enabling precise control of cells, antigens, and signaling molecules entering the lymph node through the blood or lymphatic circulation. Lymph nodes are sourced from human organ donors. The lymphatic and vascular systems of the lymph node were cultured in a complete medium bath that was periodically recirculated from a larger volume medium chamber exposed to the incubator atmosphere (5% CO₂, normoxic) for gas equilibration to serve as a chemostat. Flow rate out of the efferent lymph and venous channels was continuously measured, and artery and afferent lymphatic pump volumetric flow rates were independently regulated via closed-loop control to enforce a balanced flow rate (zero net fluid flow between lymphatic and vascular pathways). Based upon the preliminary lymph node culture data and the fact that the Womersley number (0.34) is small, we did not need to reproduce the pulsatile flows in the vascular fluidic circuit for successful maintenance of ex vivo organ culture.

Preliminary Results and Conclusions: Following 48 hours of culture, the lymph node was assessed for cell viability. A human lymph node cultured in a medium bath without perfusion (control) resulted in widespread cell death throughout the lymph node following 4 hours of culture, as expected. However, a perfused human lymph node demonstrated significant cell viability with modest staining for dead cells over 48 hours of culture. Further, in our model, immune cell drainage can be observed in a lymph node cultured for 24 versus 48 hours. Immunostaining for CD3 revealed significant depletion of T cells over 48 hours when the lymph node was perfused only with complete medium, consistent with previously reported immune cell migration rates. Close examination revealed that the population of cells in the CD3-positive region is alive, and DAPI staining demonstrates the presence of additional non-CD3⁺ cells. Experimental results are coupled to our novel predictive computational model of a lymph node that captures the kinetics and dynamic interactions of cells, antigens, and signaling molecules spatially within the lymph node lobules. This work represents a new class of microphysiological device platforms capable of supporting the in vitro culture of whole human immune organs, enabling mechanistic interrogation of immune system function that is not possible in vivo. These advances will improve upon the current gold standard by including the impact of transport and cell-cell interactions in the context of the complete LN architecture and will drive understanding of the adaptive immune system function, chemotherapeutic and antiretroviral drug delivery strategies, and immunotherapy design.

Presenting Author: Jason Gleghorn University of Delaware

Presenting Author Biography: Dr. Jason Gleghorn earned a Ph.D. in soft tissue mechanics and tissue engineering from Cornell University and subsequently completed postdoctoral fellowships in cancer biology and molecular and developmental biology at Cornell University and Princeton University, respectively. He is currently an Associate Professor at the University of Delaware, leads a large interdisciplinary research laboratory, and holds faculty appointments in the Departments of Biomedical Engineering, Mechanical Engineering, Bioinformatics and Data Science, and Biological Sciences. His lab develops and uses microfluidic technologies and artificial intelligence algorithms to develop experimental drug screening models and therapeutics for conditions involving the immune system, women’s health, maternal-fetal health, and preterm infants. He has pioneered the development of a new class of microfluidic systems enabling the culture and perfusion of whole human organs on a chip. Dr. Gleghorn is the recipient of national and international recognition for his work including the March of Dimes Basil O’Connor Award, the ORAU Ralph E. Powe Award, and the University of Delaware Bernard Canavan Early Career Research Award. He has been named a Cellular and Molecular Bioengineering Rising Star from the Biomedical Engineering Society and a Young Innovator in Cellular and Molecular Bioengineering from the CMBE journal. Additionally, Dr. Gleghorn received the prestigious National Science Foundation CAREER Award and the Francis Alison Society’s Gerard J. Mangone Young Scholar Award, the highest young faculty research award at the University of Delaware.

Authors:

Jason Gleghorn University of Delaware