HIGH-RESOLUTION TWO-PHOTON POLYMERIZATION OF ENGINEERED CELL MICROENVIRONMENTS FOR FUNDAMENTAL NEURO-MECHANOBIOLOGY AND BRAIN CANCER PROTON RADIOTHERAPY

Jun 29, 2022, 3:50 PM
20m
Room: S3 B

Room: S3 B

Speaker

Accardo, Angelo (Delft University of Technology )

Description

Introduction
Nowadays, great attention is devoted to the development of 3D in-vitro neuronal models both for fundamental neuro-mechanobiology applications as well as for disease modelling. Typical approaches include either scaffold-free or scaffold-based strategies. Although the first ones, based on cell self-assembly mechanisms, lead to the formation of tissue-like structures called neuro-spheroids or neuro-organoids, they often suffer from batch-to-batch variability and development of early-stage necrotic cores. Scaffold-based approaches involve instead the use of manufacturing techniques such as fused deposition modelling, bioprinting and electrospinning. These methods however lack the possibility of creating precise micrometric or sub-micrometric geometries able to guide cell fate. In this presentation, I will highlight two recent investigations where we employed a high-definition light-based technology to fabricate for the first time: 1) 2.5D and 3D nanostructures cultured in presence of primary microglia extracted from the brain of rhesus macaque; 2) 3D engineered glioblastoma microenvironments in the context of proton radiobiology studies.
Methodology
All the 3D structures were fabricated by two-photon laser assisted polymerization (2PP), exploiting the two-photon absorption of near-infrared radiation by focusing infrared femtosecond laser pulses onto an organic pre-polymer material. This non-linear mechanism is tuned in order to induce the photopolymerization of the exposed material in extremely confined volumes of sub-micrometric size. In the first study, primary microglia were derived from isolated brain tissue (white matter) of adult rhesus macaque (Macaca mulatta) donors that were free from neurological diseases and cultured both on flat substrates, 2.5D micro/nano-pillars and 3D micro/nano-decorated scaffolds. The morphology of the microglia was then assessed by immunofluorescence and scanning electron microscopy. In the second study, human glioblastoma (GBM) U-251 cells were cultured on 3D architectures mimicking the brain blood vessel geometry and its vascular branching points where GBM cells naturally cluster and proliferate. The engineered glioblastoma microenvironments were then exposed to different proton radiation doses (2 Gy and 8 Gy) and the amount of DNA damage was assessed by using the fluorescence Gamma-H2AX marker.
Results
The combination of sub-micrometric topographies (close to the dimensions of cell filopodia) and mechanical cues (represented by a low effective shear modulus approaching the stiffness of brain tissue), induced a substantial increase in the numbers of microglia characterized by a ramified resting phenotype as compared to cells cultured on flat stiff substrates, mostly featuring an amoeboid morphology. Concerning the second study, upon proton irradiation, GBM cells consistently showed lower DNA damage in the 3D engineered microenvironments compared to 2D GBM cell monolayers, which correlates with the response of GBM cells in-vivo where a greater radioresistance is observed. We hypothesize that this difference in the formation of the number of foci is directly connected to the differences in terms of cytoskeletal properties, cell-matrix interactions and repair kinetics between 2D and 3D cell culture configurations.
Conclusions
We demonstrate how 2PP can be employed to create: 1) 2.5D and 3D micro- and nano-structures able to guide the fate of primary microglia towards ramified phenotype; 2) 3D engineered glioblastoma microenvironments, which can be used as a reliable benchmark tool for proton radiobiology.

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