An In vitro Vascularised Liver Organotypic Model for the Testing of Nanomedicines

Jun 28, 2022, 11:50 AM
Room: S1

Room: S1


Santin, Matteo (Centre for Regenerative Medicine and Devices / University of Brighton )


Nanomedicine, where therapeutic or diagnostic agents are coupled with nanoparticles (NP), offers opportunities for more efficacious treatments and accurate diagnosis of severe clinical conditions. Polymeric, lipidic, metallic or ceramic NP have been considered as carriers of these agents, but their translation into clinics requires a thorough assessment of their biocompatibility and biodistribution. However, the existing regulatory frameworks not always address the specific biocompatibility issues associated to nano-biomaterials, including cell response upon NP internalisation and accumulation within tissues. Currently available in vitro tests based on very simplistic 2D cell cultures are not suitable to mimic the clinical conditions. At the same time, in vivo animal models do not reproduce the host conditions of humans and limit the ability of tracking the NP and their interactions with cells in an accurate manner. The present paper aims to contribute to the developing of new in vitro models that can fill the gap between conventional 2D cell cultures and animal experimentation. The objective was to develop a 3D organotypic model resembling the histological features of the liver vascularised lobuli, while preserving the operator-friendly features typical of 2D cultures. Human hepatocyte carcinoma cell lines (HepG2) were co-cultured with human umbelical endothelial vascular cells (HUVEC) at a 60/40 ratio. The deriving cell suspension was seeded at a density of 105 cells/mL in a serum-free medium into tissue culture wells previously coated with a thin and completely transparent film of a synthetic biomaterial substrate, PhenoDrive-Y (Tissue Click Ltd, UK), mimicking the natural basement membrane of tissues. Lobuli-like structure formations were allowed to form over 4 days of culture in serum-free medium to limit cell proliferation, cell cycle synchronisation and cell migration. After 4 days, the cell culture was either stopped or challenged with increasing concentrations of polymeric or lipidic NP for an additional 24h. The formed lobuli-like structures were fixed in formalin and characterised for their morphology, extracellular matrix production, cytotoxicity, pro-inflammatory phenotype both in absence and presence of a challenge with NP at increasing concentrations. Uncoated tissue culture plate and Matrigel were used as controls.
Time-lapse microscopy showed that, when adhering on PhenoDrive-Y, cells gradually migrated towards each other to form lobuli-like structures throughout the well surface. Confocal microscopy and scanning electron microscopy showed that angiogenic sprouting intercalated the spheroids forming each lobulus as well as covered their surface with branching. These spheroidal formations were joined together not only by angiogenic sprouting, but also by an extracellular matrix. The incubation with relatively low concentrations of either polymeric or lipidic NP (e.g. 5 micrograms/mL) showed a preferential binding of NP to the angiogenic sprouting. When challenged by increasing concentrations of NP, these lobuli-like structures appeared to lose their architecture showing clear sign of cytotoxicity.
The present work shows that 3D organotypic cultures, using liver carcinoma cells and endothelial cells and driven by a synthetic biomimetic substrate, are able to resemble the histological features of the liver vascularised lobuli thus providing valuable data of toxicity and biodistribution of NP at cellular levels.


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