Preliminary Study on Derivation of Sympathetic-like Tissue Using 3D Hydrogel Cell Culture

The peripheral nervous system, composed of the autonomic and somatic nervous subsystems, is responsible for relaying information between the brain and body. Despite efforts to optimize nerve regeneration, peripheral nerve injuries have poor recovery. Researchers rely on in vitro nerve models to better understand nerve injury, however these models often fail to simulate in vivo structure and function, as they incorporate animal cells and lack 3D culture environments. Human 3D in vitro models, such as organ-on-chip devices, can address these shortcomings, especially if they incorporate the use of human stem cell-derived neurons to better reflect human physiology. Previously, our lab has cultured neonatal rat autonomic neurons in 3D (Soucy et al., Advanced Biosystems, 2020); this preliminary study expands this work toward the development of a 3D human sympathetic-like tissue model. To our knowledge, this is the first attempt to differentiate human sympathetic neurons in 3D.

Induced pluripotent stem cells were differentiated to generate autonomic sympathetic-like neurons following a previously published method (Takayama et al.,Scientific Reports, 2020). In contrast to this method, we re-plated our cells at an earlier stage (Day 13 vs. Day 6, respectively) into a 3D gelatin methacryloyl (GelMA) hydrogel to allow for growth into the gel before development into post-mitotic mature neurons. Cells were re-plated on Matrigel-coated plates (2D) or encapsulated in GelMA hydrogels (3D), and neurite outgrowth was compared to a 2D group which was not replated. The number of neurites per overall cell count was evaluated in brightfield images taken on Days 7, 10, and 13. For the 2D cultures, there was an increase in neurites per total cell count, though most neurites for the re-plated group had developed prior to Day 10 whereas for the non-re-plated group more neurites grew post Day 10. Contrary to our hypothesis, the neurites per total cell count in 3D dropped from Day 10 to 13 and had less neurite growth than 2D cultures. These results indicate that the cells are maturing after re-plating, but are having difficulty growing into the GelMA matrix. Future experiments will incorporate the use of supportive glial cells to further encourage neurite formation in 3D, and morphology will be compared to rat sympathetic neurons in the same matrix to optimize growth conditions. Overall, this approach has shown promising results and can be further developed to become a robust 3D-cell-culture method for better understanding the relationship between autonomic nerves and their surrounding environments.