Stacked hydrogels to elicit neural mechanotransduction processes in 3D

IntroductionMany different methods of patterning hydrogels are currently investigated for their advances in 3D tissue engineering [1]. Nanogrooves, demonstrating aligned neural processes to extend into 3D hydrogels in a reductionist and primary CTX brain-on-chip model, have been also reported by us [2]. Here, we aim to combine these concepts to elicit neural mechanotransduction processes in a 2-layer stack of two distinct hydrogels, of which the top hydrogel can be fined-tuned to an astrocyte feeding layer for neuronal cells in the bottom hydrogel. Experimental procedure, results and discussion Stacks were fabricated using photo-polymerizing GelMA (900496, Sigma Aldrich) and red-colored thermoset gelatin (Dr. Oetker, strawberry jelly) to demonstrate our new mechanotransduction model, Figure 1a. For proofof-principle, we performed SH-SY5Y cell line (94030304, Sigma Aldrich) cultures in a PDMS-ring confinement and added a droplet of 10µl of GelMA, which was photo-polymerized at the defined thickness of 1 mm of the ring by putting a COC (cyclic olefin copolymer) foil atop to flatten the surface. When peeling-off the COC, SH-SY5Y culture protocol was carried out according to our experience with 3D cultures of SH-SY5Y in Matrigel [2] by differentiating the cells with 10 µM retinoic acid (RA; R2625, Sigma Aldrich) to initiate neuronal differentiation at DIV 0 and adding growth medium with 50 ng/mL brain-derived neurotrophic factor (B2795, Sigma Aldrich) at DIV 3, and then with growth medium being refreshed halfway for next 48 hours. After differentiation, cells were kept in growth medium with growth medium being refreshed every other day until DIV 5, Figure 1b, c. To provide a proof-of-principle for our mechanotransduction model, next we will perform the same SH-SY5Y culture using GelMA on a nanogrooved PDMS substrate and add astrocytes in Matrigel atop. We expect that the difference in ConclusionStacks of photo-polymerized and thermoset patterned hydrogels can be consistently achieved by pipetting small droplets of the pre-cursor hydrogels into a PDMS ring confinement on glass. SH-SY5Y cells showed successful proliferation and differentiation within the interface of glass and GelMA, forming an extensive neuronal cellnetwork. Migration of cells from and to the different layers of the stack needs to be further investigated Young’s module within the distinct culture regions will influence the network formation of the neuronal cells similar to our previous results in CTX 2D cultures [3]