Toward the Construction of a Vascularized, Hydrogel-Based Lymph Node Model for In Vitro and In Vivo Therapeutic Applications

Abstract

Clinical trials for drugs and vaccines often suffer from the use of culture or animal models that do not accurately recreate the microenvironment of human tissues, including the lymph nodes. Furthermore, insufficient immune function resulting from genetic deficits, cancer or auto-immune disease, and the loss of lymph nodes due to surgical resection or radiation therapy may benefit from the implantation of therapeutic immune cells. These needs could be served through the development of a biocompatible, vascularized, 3D hydrogel scaffold that supports leukocytes and recreates lymph node function by providing a biomimetic microenvironment. While other lymph node models exist, their complexity and function are limited in that they incorporate few of the bioactive molecules from the lymph node microenvironment and do not contain vasculature. Preliminary viability studies were performed to determine the optimal choice of hydrogel type and density for the 3D culture of peripheral blood mononuclear cells (PBMCs). Fibrin hydrogels were found to better maintain PBMC viability over three days compared to gelatin methacrylate hydrogels. The inclusion of ECM from decellularized porcine lymph nodes into fibrin hydrogels was met with technical challenges with regard to solubilization and peripheral blood cell (PBC) viability assessment, indicating that additional steps or different approaches were required. However, successful decellularization was demonstrated by the sufficient removal of DNA content as determined by DNA quantification and histological staining. Cytokine and growth factor analysis showed significant depletion of many of the analytes but retention of IFNα2, IL-3, IL-9, IL-13, IL-17A, and VEGF. To construct a perfusable model, pin molds were used to create channels that would undergo eventual endothelialization and angiogenesis. Few of these channels maintained structural integrity. While continued experimentation is required to implement all of the features desired for the construction of a biomimetic and functional human lymph node model, these results indicate that it is feasible to improve upon previous designs by incorporating decellularized ECM and introducing vascularization in fibrin hydrogels.