Optimization of Biomaterial Microenvironment for Motor Neuron Tissue Engineering

Injury and disease in the nervous system results in loss of sensory and motor neurons. Tissue engineering strategies have great potential to develop more effective therapeutics by combining appropriate biological cues with responsive biomaterial architectures. We have recently shown that more in vivo-like sensory neuron responses are found in 3D culture vs 2D flat substrates. Unfortunately, primary motor neurons are harder to isolate and culture compared to sensory neurons. Moreover, PC-12 cells are a well-characterized sensory neuron-like cell line and provide an alternative to primary sensory neurons. An analogous motor neuron-like cell line, NSC-34, is available but has not yet been characterized for tissue engineering applications. Thus, our goal was to systematically characterize NSC-34 cells and optimize biomaterial microenvironment and culture conditions for tissue engineering applications. For 2D studies, we cultured NSC-34 cells on coverslips coated with laminin or type I collagen in DMEM with fetal bovine serum (FBS; 3%, 10%), or N2 supplement. In our 3D studies, cells were cultured in gels composed of type I collagen with or without laminin or type IV collagen. We found that laminin substrates were associated with a higher percent of cells expressing neurites and that the neurites were significantly longer than for type I collagen substrates, with greatest results at 3% serum. Medium with N2 was associated with lower cell proliferation vs serum-containing medium regardless of substrate. Although proliferation results showed low percent proliferation for N2 medium, no significant difference was found between type I collagen and laminin. Preliminary results in 3D gels indicate that type IV collagen provides a favorable environment for neurite expression. Ongoing work focuses on rigorously characterizing neuronal morphologies and neurite lengths in 3D cultures.