(K-439) VASCULARIZATION OPTIMIZATION OF IN VITRO NEURONAL MODELS

Introduction::
Introduction: One of the central challenges of neurobiology is modeling the complex, three-dimensional structure of the human brain in vitro. Organoids represent a promising strategy towards this end, allowing us to replicate intricate cellular properties and interactions. However, organoid models often suffer from necrosis when their size exceeds the capacity for oxygen diffusion. To address this issue, we previously induced vascularization in brain organoids using an angiogenesis assay co-culturing endothelial cells with fibroblasts or pericytes. In our current project, we sought to optimize the conditions for in vitro angiogenesis, including improving survival and proliferation of co-cultured cells. To that end, we investigated the effects of endothelial media mixed with ReN differentiation media on Human Brain Vascular Pericytes (HBVP), Human Umbilical Vein Endothelial Cells (HUVEC), and ReN cells. By concurrently addressing media and cellular interactions, we make headway towards the development of viable, complex brain models that incorporate self-organizing vasculature and open new avenues for studying human brain development and pathology.




Materials and Methods::
Methods and Materials: Cell Culture: Normal Human Lung Fibroblasts (NHLF), Human Umbilical Vein Endothelial Cells (HUVEC), Human Brain Vascular Pericytes (HBVP), and ReNcells were cultured individually in T25 flasks. Cells were maintained for 5-7 days until reaching approximately 80% confluency, with media changes conducted every other day. Plate Seeding: The cells were subsequently seeded into a 48-well plate. Each cell type was assigned to one row, ensuring each type had its own dedicated section of the plate. For the initial seeding, the native growth media specific to each cell type was used. Media Exchange: Following the seeding, the media in the first four wells of each row were replaced with their respective native media. The remaining four wells were switched to a media mixture composed of a 1:1 ratio of endothelial cell media and ReN differentiation media. Imaging and Immunocytochemical Staining: On day 7, bright-field and confocal microscopy was conducted to observe and record the morphology and behavior of the cells. Immunocytochemical staining was also performed at this stage. Markers used in staining included cluster of differentiation 31 (CD31) to visualize endothelial cells and assess vessel formation, cleaved caspase-3 (ASP175) to detect apoptosis, and beta-tubulin III (TUJ1) to identify differentiated neurons.




Results, Conclusions, and Discussions::
Results and Discussion: While some vascularization was observed in the HUVEC and NHLF model, it appeared much less developed at the same time point for the HUVEC and NHLF. CD31 staining supports this hypothesis because while (Fig.1B) HUVEC/HBVP shows some vascular-looking structures, the development of the constructions seen in the HUVEC/NHLF (Fig.1A) is further progressed. Fig 1.C show the beginnings of what appears to be a well differentiated network of neurons. Beta-tubulin III staining (Red) confirms that neurons are growing. Figure 1.D however, shows much less development of a visible network. Additionally, there is some ASP-175 (purple) present in these cells as well. Both groups have good visualization of dapi (blue), indicating that while both groups remained viable, the group that received the 1:1 media mixture (Fig.1D) remained undifferentiated. While the presence of ASP-175 was observed in Fig.1D, we hypothesize this was due to over-proliferation and high confluency.














Conclusions: Our central aim was to explore the influence of media compositions on endothelial cell proliferation and neuronal differentiation, in cocultures. The choice of media did not significantly impact the behavior of the Normal Human Lung Fibroblasts (NHLF), Human Umbilical Vein Endothelial Cells (HUVEC), and Human Brain Vascular Pericytes (HBVP). However, interestingly, the ReN cells remained in an undifferentiated state following the switch to a 1:1 ratio of endothelial cell media and ReN differentiation media. We hypothesize that factors in the endothelial cell media are inhibiting ReN maturation. Future efforts seek to identify these inhibitory factors and formulating a novel media mixture that can sustain both endothelial cell proliferation and the maturation of neurons. Through these efforts, we make steps towards a more physiologically representative, vascularized cerebral model.




Acknowledgements (Optional): :
Acknowledgments: This research was supported, in part, by the National Science Foundation (EEC-1852560).




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