(G-260) Effects of Culture Environment on Nanoparticle Uptake in Primary Brain-Derived Astrocytes and Pericytes

Introduction:: The blood-brain barrier (BBB) is a collection of astrocytes, pericytes, endothelial cells, and proteins, which restricts transport of more than 98% of drugs from the blood to the parenchyma. Because of the challenge to drug delivery, in vitro BBB models are commonly used for first pass evaluations of potential therapeutics. It has been established that both endothelial cells and astrocytes display phenotypic and genetic differences when cultured in 2-dimensional and 3-dimensional environments, as well as in mono-culture and co-culture. Though it is known that pericytes contribute to BBB tightness, the effects of culture conditions on their morphology and interactions with other glial cells such as astrocytes has not been well characterized. Moreover, primary cells have shown the ability to replicate BBB permeability in vitro, but extensive use and characterization of these cells in microfluidic devices has been limited due to low cell yields in isolations. Finally, our previous work in brain slices has assessed nanoparticle uptake in parenchymal cells, but has not captured nanoparticle interaction with the BBB. Here, we present methodology and critical design aspects to obtain high yields of primary pericytes and astrocytes as well as document differences in nanotherapeutic uptake in these cells in 2D mono-culture, 3D microfluidic monoculture under flow, and a 3D microfluidic co-culture under flow. This work provides a methodological roadmap for use of primary cells in microfluidic BBB models and a foundation for optimizing targeting and uptake strategies for drug delivery to astrocytes and pericytes.


Materials and Methods:: Primary astrocytes are isolated from healthy postnatal (P) day 5 to P7 rats using mechanical dissociation methods, and purified by removing surface vessels, cell straining, and selective plating on poly-D-lysine coated flasks. Primary pericytes are isolated from healthy P56 rats and purified through mechanical and enzymatic dissociation, isopycnic 20% bovine serum albumin (BSA) and 33% Percoll separations, and selective plating on non-coated dishes with low-glucose media. Importantly, cell clusters were not disturbed during mechanical dissociation and enzyme exposure time was reduced from values previously reported. We confirm the isolation of these cell types via immunohistochemical staining and imaging using anti-GFAP for astrocytes and anti-NG2 for pericytes as seen in Figure 1. Cells are only used at Passage 2 or below for uptake experiments. The microfluidic device is a commercially fabricated SynBBB from SynVivo®. After treating with poly-D-lysine, 1.5 x 10^6 cells are loaded into the basolateral channel of the device as seen in Figure 1. Co-culture was established by seeding astrocytes and pericytes in a ratio of 2:1. After the culture is established, a polystyrene polyethylene glycol (PS-PEG) nanoparticle solution, representative of a model drug delivery platform, is introduced. These fluorescently labeled nanoparticles are formulated according to previously established protocols in our lab¹. The rate of diffusion, retention, and distribution in the cells and device is visualized with confocal microscopy at 2h, 6 h, and 24 h.




Results, Conclusions, and Discussions:: Our isolation procedures consistently resulted in confluent T-75 flasks of GFAP+ astrocytes every 5-7 days and confluent T-75 flasks of NG2+ pericytes every 2 weeks. Though culture time is longer, we show the ability to obtain sufficient numbers of primary pericytes to run characterization experiments within a microfluidic device. Our procedure, with reduced enzyme exposure and gentle mechanical dissociation, resulted in the isolation of pure pericytes and maximized yield by reducing cell death. Particularly, we show that incomplete removal of myelin-associated cells and tissue during the BSA step prevents full separation at the Percoll gradient step which can also reduce cell yield. We also show that in this case, pericytes can be recovered by including one additional centrifugation step downstream. Our work, optimizing primary cell isolation procedures, identifying key steps and troubleshooting options, and maximizing cell viability within microfluidic devices, can permit primary cells to be used more widely in microfluidic BBB models to investigate the effects of disease, environmental, and genetic history on therapeutic transport across the BBB.

In microfluidic co-culture, we saw that astrocytes prefer to make connections with astrocytes, while pericytes position themselves to align alongside astrocyte networks in addition to connecting with other pericytes. Importantly, we demonstrate that pericytes and astrocytes respond differently to PS-PEG nanoparticles, and their response is model-dependent. Finally, we identified that culture environment has a strong effect on astrocyte and pericyte uptake. Cells co-cultured in the microfluidic model under flow showed the most “in-vivo-like” response and PS-PEG nanoparticle uptake.

Navigating through endothelial cells is only one hurdle that therapeutics must overcome prior to uptake by target cells. Thus, in vitro drug modeling cannot be limited to endothelial permeability studies alone. Additionally, because astrocytes and pericytes play significant roles in modulating the BBB in healthy and pathological conditions, these cells should be considered as target cells for neurotherapeutic intervention. Our work investigates astrocyte and pericyte interactions in a physiologically relevant architecture in the absence of endothelial cells to establish baseline uptake efficiency in these cells for use in designing therapeutics for brain disease.


Acknowledgements (Optional): :

References (Optional): : Reference:
1. Xu, N., Wong, M., Balistreri, G., & Nance, E. (2023). Neonatal Pharmacokinetics and Biodistribution of Polymeric Nanoparticles and Effect of Surfactant. Pharmaceutics, 15(4), 1176.