Abstract
Frontotemporal lobar degeneration (FTLD) is a severe neurodegenerative disorder that is diagnosed with increasing frequency in clinical setting. Currently, no therapy is available and in addition the molecular basis of the disease are far from being elucidated. Consequently, it is of pivotal importance to develop reliable and cost-effective in vitro models for basic research purposes and drug screening. To this respect, recent results in the field of Alzheimer’s disease have suggested that a tridimensional (3D) environment is an added value to better model key pathologic features of the disease. Here, we have tried to add complexity to the 3D cell culturing concept by using a microfluidic bioreactor, where cells are cultured under a continuous flow of medium, thus mimicking the interstitial fluid movement that actually perfuses the body tissues, including the brain. We have implemented this model using a neuronal-like cell line (SH-SY5Y), a widely exploited cell model for neurodegenerative disorders that shows some basic features relevant for FTLD modeling, such as the release of the FTLD-related protein progranulin (PRGN) in specific vesicles (exosomes). We have efficiently seeded the cells on 3D scaffolds, optimized a disease-relevant oxidative stress experiment (by targeting mitochondrial function that is one of the possible FTLD-involved pathological mechanisms) and evaluated cell metabolic activity in dynamic culture in comparison to static conditions, finding that SH-SY5Y cells cultured in 3D scaffold are susceptible to the oxidative damage triggered by a mitochondrial-targeting toxin (6-OHDA) and that the same cells cultured in dynamic conditions kept their basic capacity to secrete PRGN in exosomes once recovered from the bioreactor and plated in standard 2D conditions. We think that a further improvement of our microfluidic system may help in providing a full device where assessing basic FTLD-related features (including PRGN dynamic secretion) that may be useful for monitoring disease progression over time or evaluating therapeutic interventions.
Highlights
Neurodegenerative diseases share common pathological features, such as abnormal protein aggregation, mitochondrial dysfunction, and disease-specific neuronal degeneration
To evaluate the optimal seeding conditions in 3D scaffolds, DNA content was measured about 24 h after seeding, the number of SH-SY5Y cells in 3D scaffolds was estimated as reported in Methods
As expected, when 1·106 SHSY5Y cells were seeded, a greater number of cells was obtained in 3D scaffolds (∗∗p-value < 0.01) with respect to the 2D condition (∗p-value < 0.05), confirming that the procedure did not lead to major cell loss
Summary
Neurodegenerative diseases share common pathological features, such as abnormal protein aggregation, mitochondrial dysfunction, and disease-specific neuronal degeneration. To provide structural and biochemical cues together with a suitable stiffness and a better control of the cellular context, 3D matrices such as hydrogels, spheroid-based systems or porous materials have been exploited as 3D microenvironments (Yamada and Cukierman, 2007; Zhang et al, 2014) Their main limitation is related to oxygen and nutrient diffusion. Dynamic culture systems can overcome limited mass transport, contributing to the extension of the culturing time In these systems, cells are cultured under a continuous flow of growing medium, mimicking the interstitial fluid movement that perfuses the body tissues, including the brain. They have suggested that astrocytes are targets of lipoxidative damage, glial fibrillary acidic protein is modified by oxidation and astrocytes play a key role in oxidative stress response
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