Based on the decellularization of lymph nodes with a wide range of sizes, histological analyses revealed that some of the larger lymph nodes still retained a cellular core. We hypothesize that this incomplete decellularization is due to the larger size of the lymph nodes, which prevented the SDS from completely diffusing into the node to remove cells from the center. Based on analysis of the size and cellularity of the scaffolds, future experiments using this protocol should only utilize lymph nodes with a minimum feret diameter of less than 2 mm to ensure complete decellularization. Further research on decellularizing larger sample sizes of lymph nodes can be done to establish proper SDS concentrations and wash times to accommodate a wider range of lymph nodes.

The artificial lymph node has shown success in activating an antigen-specific T cell response after being seeded with dendritic cells in vitro. This is highly promising as it suggests that the scaffold is biocompatibile, functional, and can facilitate immune activity. As such, future research should aim to implant the artificial lymph node into mice to analyze the scaffold’s biocompatibility and interaction with the host immune system in vivo. This will ultimately allow for investigation into the artificial lymph node’s ability to recreate the lymph node microenvironment to rescue the response to anti-CTLA4 in the context of head and neck cancer. Furthermore, implantation of the artificial lymph node will allow the lymphatic mapping subproject to compare the physical impacts of the design solution to those of the cancerous and wild type mouse models. The lymphatic mapping techniques using IMARIS 3D image processing software established here can be used to evaluate the scaffold’s ability to reconnect to native lymphatic vasculature. This will help in elucidating the mechanism of action of the artificial lymph node in vivo by visualizing changes in the lymphatic system post-implantation.

To improve the efficacy of the artificial lymph node scaffold, further improvements can be made to improve immune cell homing in vivo (perhaps through inclusion of cytokines into the scaffold) and to characterize potential metastasis into the scaffold post implantation (utilizing the 4MOSC2 model - a more aggressive version of the 4MOSC1 orthotopic cell line used for this project). These efforts will determine whether an artificial lymph node will be successful at rescuing the antitumor immune response, or whether it will prevent metastasis to disparate parts of the body.