Modeling Structural Elements and Functional Responses to Lymphatic-Delivered Cues in a Murine Lymph Node on a Chip.

Lymph nodes (LNs) are organs of the immune system, critical for maintenance of homeostasis and initiation of immune responses, yet there are few models that accurately recapitulate LN functions in vitro. To tackle this issue, an engineered murine LN (eLN) has been developed, replicating key cellular components of the mouse LN; incorporating primary murine lymphocytes, fibroblastic reticular cells, and lymphatic endothelial cells. T and B cell compartments are incorporated within the eLN that mimic LN cortex and paracortex architectures. When challenged, the eLN elicits both robust inflammatory responses and antigen-specific immune activation, showing that the system can differentiate between non specific and antigen-specific stimulation and can be monitored in real time. Beyond immune responses, this model also enables interrogation of changes in stromal cells, thus permitting investigations of all LN cellular components in homeostasis and different disease settings, such as cancer. Here, how LN behavior can be influenced by murine melanoma-derived factors is presented. In conclusion, the eLN model presents a promising platform for in vitro study of LN biology that will enhance understanding of stromal and immune responses in the murine LN, and in doing so will enable development of novel therapeutic strategies to improve LN responses in disease.

Deployable extrusion bioprinting of compartmental tumoroids with cancer associated fibroblasts for immune cell interactions.

Realizing the translational impacts of three-dimensional (3D) bioprinting for cancer research necessitates innovation in bioprinting workflows which integrate affordability, user-friendliness, and biological relevance. Herein, we demonstrate 'BioArm', a simple, yet highly effective extrusion bioprinting platform, which can be folded into a carry-on pack, and rapidly deployed between bio-facilities. BioArm enabled the reconstruction of compartmental tumoroids with cancer-associated fibroblasts (CAFs), forming the shell of each tumoroid. The 3D printed core-shell tumoroids showedde novosynthesized extracellular matrices, and enhanced cellular proliferation compared to the tumour alone 3D printed spheroid culture. Further, thein vivophenotypes of CAFs normally lost after conventional 2D co-culture re-emerged in the bioprinted model. Embedding the 3D printed tumoroids in an immune cell-laden collagen matrix permitted tracking of the interaction between immune cells and tumoroids, and subsequent simulated immunotherapy treatments. Our deployable extrusion bioprinting workflow could significantly widen the accessibility of 3D bioprinting for replicating multi-compartmental architectures of tumour microenvironment, and for developing strategies in cancer drug testing in the future.

Neuroserpin Inclusion Bodies in a FENIB Yeast Model.

FENIB (familial encephalopathy with neuroserpin inclusion bodies) is a human monogenic disease caused by point mutations in the SERPINI1 gene, characterized by the intracellular deposition of polymers of neuroserpin (NS), which leads to proteotoxicity and cell death. Despite the different cell and animal models developed thus far, the exact mechanism of cell toxicity elicited by NS polymers remains unclear. Here, we report that human wild-type NS and the polymerogenic variant G392E NS form protein aggregates mainly localized within the endoplasmic reticulum (ER) when expressed in the yeast S. cerevisiae. The expression of NS in yeast delayed the exit from the lag phase, suggesting that NS inclusions cause cellular stress. The cells also showed a higher resistance following mild oxidative stress treatments when compared to control cells. Furthermore, the expression of NS in a pro-apoptotic mutant strain-induced cell death during aging. Overall, these data recapitulate phenotypes observed in mammalian cells, thereby validating S. cerevisiae as a model for FENIB.

Bio-assembling Macro-Scale, Lumenized Airway Tubes of Defined Shape via Multi-Organoid Patterning and Fusion.

Epithelial, stem-cell derived organoids are ideal building blocks for tissue engineering, however, scalable and shape-controlled bio-assembly of epithelial organoids into larger and anatomical structures is yet to be achieved. Here, a robust organoid engineering approach, Multi-Organoid Patterning and Fusion (MOrPF), is presented to assemble individual airway organoids of different sizes into upscaled, scaffold-free airway tubes with predefined shapes. Multi-Organoid Aggregates (MOAs) undergo accelerated fusion in a matrix-depleted, free-floating environment, possess a continuous lumen, and maintain prescribed shapes without an exogenous scaffold interface. MOAs in the floating culture exhibit a well-defined three-stage process of inter-organoid surface integration, luminal material clearance, and lumina connection. The observed shape stability of patterned MOAs is confirmed by theoretical modelling based on organoid morphology and the physical forces involved in organoid fusion. Immunofluorescent characterization shows that fused MOA tubes possess an unstratified epithelium consisting mainly of tracheal basal stem cells. By generating large, shape-controllable organ tubes, MOrPF enables upscaled organoid engineering towards integrated organoid devices and structurally complex organ tubes.

Stromal regulation of tumor-associated lymphatics.

Recent advances have identified a growing array of roles played by lymphatics in the tumor microenvironment, from providing a route of metastasis to immune modulation. The tumor microenvironment represents an exceptionally complex, dynamic niche comprised of a diverse mixture of cancer cells and normal host cells termed the stroma. This review discusses our current understanding of stromal elements and how they regulate lymphatic growth and functional properties in the tumor context.