Design and Fabrication of an organ-on-a-chip technology as a physiologically relevant in vitro model of the outer Blood-Retinal Barrier

Jun 30, 2022, 3:50 PM
20m
Room: S3 B

Room: S3 B

Speaker

Vozzi, Giovanni (Research Center “E. Piaggio”, University of Pisa)

Description

Age-related macular degeneration (AMD) is a chronic eye disease and the leading cause of irreversible vision loss in millions of elderly people world-wide. The ocular system implicated in this disease is the outer blood-retinal barrier (oBRB) comprised of the retinal pigment epithelium (RPE), the Bruch’s membrane (BrM), and the choriocapillaris (CC). The oBRB plays a pivotal role in maintaining the eye homeostasis by regulating the transport of nutrients and metabolic wastes from choroid to the sub-retinal space. In AMD patients, several morphological and structural changes occur in the oBRB resulting in its disfunction and a failure of such homeostasis. At present, due to the multifactorial nature of the AMD, the exact disease pathogenesis remains poorly understood. As a result, although palliative cares exist for only some forms of AMD, no effective treatments exist. In order to investigate the pathophysiological process underlying AMD and to validate novel drug candidates, several in vivo and in vitro models have been proposed. However, none of these have proven to be reliable to mimic the complex cellular interactions in the oBRB with physiological realism and great predictive value. Therefore, herein we present a novel oBRB-on-a-chip model as a biomimetic platform for AMD understanding and for new therapeutic agents development. The device is a 3D microfluidic platform consisting of a biomimetic blood vessel network mimicking the CC and of a novel BrM-mimetic bio-membrane both housed within a single-chamber which resembles the intraocular space and enables the co-culture of human RPE and endothelial cells above the BrM and inside the CC respectively. The microfluidic network, designed starting from Optical Coherence Tomography (OCT) scans , was fabricated from polydimethylsiloxane (PDMS) through a novel manufacturing method established to provide a time-saving and cost-effective alternative to the common lithographic-based techniques. The interior surfaces of the microfluidic channels were subsequently coated with chemically crosslinked gelatin to promote cell adhesion and long-term culture. The engineered BrM was fabricated from chemically crosslinked gelatin by electrospinning process to get porous, ultrathin and nanofibrous membranes mimicking the mechanical, chemical and physical properties of the native substrate. The co-culture chamber with a common internal footprint with the wells in standard 24-well plates was fabricated from PDMS via demoulding process. Perfusion tests were successfully performed for validating the overall microfluidic platform. Human embryonic stem cell-derived RPE cells and HUVECs cells were cultured on the engineered BrM and on PDMS-gelatin substrates respectively to evaluate cells adhesion and proliferation under static conditions. Immunofluorescence microscopy demonstrated that engineered BrMs supported functional RPE monolayer formation, while HUVECs cells shown good adhesion and proliferation on the PDMS-gelatin substrates. Tests of dynamic seeding and static/dynamic co-culture of the cellular species involved are in progress to set out standard protocols. Taken together our findings are encouraging, showing that we successfully designed a physiologically relevant oBRB model to study AMD in vitro, in which patient-derived cells could be used for the identification new drugs paving the way towards personalized medicine.

62903407479

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