To achieve the advancing models of neural development, the optimization of different parameters such as cell seeding density, extracellular matrix (ECM)ECM type and duration of differentiation, was assessed. This was performed in standard 96-well plates. BIONi010-C iPSCs embedded in various hydrogel (Matrigel, Collagen, HyStem-C and Polyvinyl alcohol (PVA)-HA gel with RGD peptide) at different concentrations were screened. At different time points during the 15 to 45 days of cell culture, we performed immunofluorescence procedure to test stem cell proliferating and differentiation potential. At day 15 cells were fixed and stained for Oct4, Pax6, Nestin; at day 30 cells were stained for Pax6, Nestin, TBR2; at day 45 cells were stained for Map2, Ctip2 and Pax6. iPSCs embedded in different hydrogel could proliferate and differentiate in 45 days of culture. However, matrigel and collagen shrinked over time and cells form dense 3D structures (round) in these conditions, which is big disadvantage for long-term functional neuro culture. Cells seeded into PVA-HA and HyStemC at higher density demonstrated better neural differentiation showed more rosette-like structures. To demonstrate iPSCs embedded in PVA-HA and HyStem hydrogel could survive, proliferate and differentiate on chip, we tested the cell differentiation in the MIMETAS OrganoPlate® 3-lane 40 Plate. iPSCs at high concentration were loaded into the cell chamber of the chips for 45 days of culture. We also tested 2 differentiation protocols (dorsally patterned forebrain organoids from Arlotta group and cortical spheroids from Pasca group). Immunohistochemistry was used to confirm the successful differentiation of iPSCs towards a forebrain neuronal fate within the hydrogels. At day 15 cells were fixed and stained for Sox2, Ki-67 and Vimentin; at day 30 cells were stained for Nestin, TBR2 and Pax6; at day 45 cells were stained for Map2, Ctip2 and Tbr2. Our results show that iPSCs seeded in hydrogels survive, proliferate, and differentiate in a 3D matrix on-chip. Testing of different gel types and seeding densities revealed best differentiation success using high initial cell seeding densities and HyStem-C as well as PVA-HA gels. After 45 days of on-chip differentiation, 3D networks of neurons were observed. Importantly, Ctip2 staining indicates that cells were differentiated toward deep layer neocortical fate analogously to differentiation in conventional organoid protocols.
To recreate this controlled gradient of morphogens and necessary polarity, the brain on chip design utilized in this project consists of three concentric circular chambers separated by hydrogel barriers permissive to signalling molecules and growth factors inside which hiPSCs can be seeded in between two distinct apical equivalent and basal equivalent media. The apical basal axis thus takes a toroidal shape inside which hiPSCs can be cultured in three dimension. The axis allows for the cells to recreate the layered cortex, and, contrary to brain organoids, does not lead to core necrosis in later stages of development as this design provides a consistent flow of media with the necessary nutrient s for cell survival on both sides of the cell chamber. With the spatiotemporal control over both media flow provided by this design, the study of various environmental effects on the developing brain are possible, as well as studies of drug application at different stages of development. With good optical transparency in the chip materials, long term live monitoring of any subtle physiological and phenotypical changes during development is also possible while keeping the cells in a sterile, closed, environment. A combination of microfluidic chip fabrication technologies and hydrogel designs were used for the fabrication of the chip. The material s used in this chip consist of a synthetic thermoplastic polymer p olymethylmethacrylate PMMA), and an elastomeric polymer (polydimethylsiloxane PDMS ), both biocompatible, chemically stable, and optically transparent. The entire design consists of seven layers, the first of which is a connective layer of the rest of the chip to the perfusion system. The hydrogel barriers in the chip chamber were created using a new photo curable hydrogel technology using a photoinitiator Lithium Phenyl 2,4,6 trimethylbenzoyl phosphinate (LAP) to make the polymerization dependent on UV light; photopolymerization can thus be spatiotemporally controlled. NPCs and iPSCs were seeded into this system and successfully differentiated into neurons.
Furthermore, mesh microelectrode arrays (MEAs) are integrated into brain-on-chip module to develop a platform for studying neuronal activities in vitro. The established microfluidic chip with bonded mesh MEA could be perfused for 25 days while staying leakage free, proving the robust fabrication process. Neurons and organoids were successfully seeded into chips. The established system is promising to record the electrophysiological activity of cells.
