Peptide- and collagen-based hydrogel substrates for in vitro culture of chick cochleae

The overall goal of this work is to improve the culture of the auditory organ of birds for the dual use of developing a hair cell regeneration model and charting a pathway to the eventual replacement of the hearing organ. In doing so, we develop a protocol for removing the auditory organ from its basement membrane in the inner ear, attach the organ to a series of artificial basement membranes, and conduct qualitative and quantitative analysis of how cell morphology, viability and function change with time. Native matrix cultures, where the epithelium was floating in media with the basement membrane and accessory structures attached, were used as a basis of comparison. PuraMatrix™, collagen I, collagen I/chondroitin-sulfate and Matrigel™ were chosen to encompass a diverse range of mechanical properties and macromolecule moieties. Surprisingly, we find that PuraMatrix™ outperformed the other matrices as a scaffold for sensory organ culture. PuraMatrix™, a self-assembled peptide hydrogel, is a biochemically specific culture substrate that contains none of the extracellular matrix (ECM) molecules and growth factors contained in the inner ear's basement membrane. Rheological measurements reveal that PuraMatrix™ may be a closer approximation to the stiffness of the soft tissue supporting the auditory organ. Cell density on the PuraMatrix™ substrate is comparable to that of the native matrix cultures, despite the absence of the basement membrane and accessory structures. Further studies show that PuraMatrix™ supports the culture of functional hair cells over a 72 h period, with a significant increase in the number of functional hair cells in comparison to the organ cultured without a matrix. This is the first example of adhesion of the adult auditory epithelium to a biomaterial for an extended period of time. With further optimization, this system will enable the performance of many novel biophysical and pharmacological studies involving hair cells and supporting cells.