The Importance of Coating Surface and Composition for Attachment and Survival of Neuronal Cells Under Mechanical Stimulation.

Cell culture of neuronal cells places high demands on the surface for these cells to adhere to and grow on. Native extracellular matrix (ECM) proteins are often applied to the cell culture surface. The substrate is even more important when mechanical strain is applied to the cells in culture. These cells will easily detach and die, precluding the study of how mechanical factors affect these cells. Mechanical factors are, for example, important in the eye disorder glaucoma, which is characterized by the loss of the retinal ganglion cells (RGCs), the retinal neurons that transfer the visual information from the retina via the optic nerve to the brain. High intraocular pressure is the main risk factor of glaucoma. Here, we aimed to find an optimal coating formulation for mechanical testing of the two cell types that are often used for in vitro studies on glaucoma: primary rat retinal ganglion cells (RGCs) and the neuronal PC-12 cell line. Glass and polymer coverslips as well as well plate wells were coated with various substrates: fibronectin, collagen 1, RGD peptide, polyethyleneimine (PEI), poly-D-lysine (PDL), and laminin. We used a thermomixer for 1 min at 500RPM and 37°C to apply mechanical strain and test cell attachment in medium throughput during mechanical stimulation. Cell density, morphology, and cell death were measured to evaluate the coatings. First, a screen of various surfaces and coatings was performed using PC-12 cells, after which a selection of coating strategies was tested with RGCs. For PC-12 cells, the best results were obtained using a coating with a mixture of 10 μg/mL PDL with 2 or 50 μg/mL laminin in PBS (M2). This resulted in the highest cell density, with and without mechanical stimulation. Many other coating strategies failed to provide an effective substrate for adherence and growth of PC-12 cells. Coating composition as well as coating strategy influenced cell attachment and survival. Contrary to PC-12 cells, RGCs performed better in a sequential coating of first 10 μg/mL PDL and then 2 μg/mL laminin (S2). With this protocol, RGCs showed best neurite growth and highest cell density. Based on this difference between PC-12 cells and RGCs, we conclude that the optimal coating depends on the cell type. When reporting cell culture studies, it is important to fully specify culture surface, surface treatment, and coating protocol since all these factors influence cell attachment, growth, and survival.