Stiff-to-Soft Transition from Glass to 3D Hydrogel Substrates in Neuronal Cell Culture

Abstract

Over the past decade, hydrogels have shown great potential for mimicking three- dimensional (3D) brain architectures in vitro due to their biocompatibility, biodegradability, and wide range of tunable mechanical properties. To better comprehend in vitro human brain models and the mechanotransduction processes, we generated a 3D hydrogel model by casting photo-polymerized gelatin methacryloyl (GelMA) in comparison to poly (ethylene glycol) diacrylate (PEGDA) atop of SH-SY5Y neuroblastoma cells seeded with 150,000 cells/cm2 according to our previous experience in a microliter-sized polydimethylsiloxane (PDMS) ring serving for confinement. 3D SH-SY5Y neuroblastoma cells in GelMA demonstrated an elongated, branched, and spreading morphology resembling neurons, while the cell survival in cast PEGDA was not supported. Confocal z-stack microscopy confirmed our hypothesis that stiff-to-soft material transitions promoted neuronal migration into the third dimension. Unfortunately, large cell aggregates were also observed. A subsequent cell seeding density study revealed a seeding cell density above 10,000 cells/cm2 started the formation of cell aggregates, and below 1500 cells/cm2 cells still appeared as single cells on day 6. These results allowed us to conclude that the optimum cell seeding density might be between 1500 and 5000 cells/cm2. This type of hydrogel construct is suitable to design a more advanced layered mechanotransduction model toward 3D microfluidic brain-on-a-chip applications.

Analysis of Three-Dimensional Cell Migration in Dopamine-Modified Poly(aspartic acid)-Based Hydrogels

“…According to these results, most of the cavities are in connection with each other and only a few of them are separated. This may confirm that the cells, especially the SH-SY5Y cells, are organizing themselves into clusters at first with high cell density [ 61 ]. These clusters are clearly observable on the phase-contrast microscopic images ( Figure 3 , SH-SY5Y day 14).…”

“…We observed that the tumor cell line proliferated at a higher rate than PDLCs even under standard culture conditions in Petri dishes before seeding them for these experiments. SH-SY5Y cells usually form clusters on three-dimensional hydrogel matrices [ 42 , 56 , 61 ], which can also be seen in Figure 3 ; this behavior can explain the discrepancy between the viability and microscopic results. Clusters can cause insufficient nutrition supply and hampered diffusion, therefore the WST-1 reagent cannot be converted to formazane by the mitochondria since it cannot reach the inner region of the clusters [ 42 ].…”

“…Although the mechanism behind the migration of the SH-SY5Y cells has not been clarified yet, similar behavior has been described in several studies. G. Akcay and co-workers investigated the vertical migration of SH-SY5Y cells into photopolymerized poly(ethylene glycol)-diacrylate hydrogels [ 61 ]. According to their results, the migration of the cells was induced by the stiff-to-soft material transition, and the cells were able to migrate into the hydrogels up to 60 μm.…”

“…According to their results, the migration of the cells was induced by the stiff-to-soft material transition, and the cells were able to migrate into the hydrogels up to 60 μm. They concluded that SH-SY5Y cells in the 3D hydrogels exhibit a spherical morphology—unlike in 2D, where they have a flat shuttle-like shape with long pseudopods—due to the mechanical support and the limited space to spread [ 56 , 61 ]. Similar behavior of PDLCs has never been described in any type of hydrogel matrix.…”

Editorial for the Special Issue on Microfluidic Brain-on-a-Chip

“…For example, rat cortical cells and neuroblastoma cells (SH-SY5Y) [ 21 ] have already been investigated by us simply by adding hydrogels into the on-chip culture reservoir. To this end, we presented here our additional article on the design and testing of a straightforward concept to evaluate whether 2D seeded cells transition into 3D, when cultured within a hard–soft interface adding a hydrogel atop of the seeded cells [ 22 ]. This concept allows us to assess if neurons are susceptible in surviving in these materials and how they change their morphology and organization upon the change in mechanical cue, i.e., offering a far lower Young’s modulus compared to the glass and multitude of 3D spreading anchoring points for cell interactions within the porous 3D matrix of the hydrogel.…”