(668.4) Establishing Neurospheres in vitro by a 3D Suspension Culture System and Evaluating Neurite Growth to Facilitate Nerve Repair

Poster Board Number: A346

Meaghan Harley-Troxell (University of Tennessee College of Veterinary Medicine), Madhu Dhar (University of Tennessee College of Veterinary Medicine), David Anderson (University of Tennessee College of Veterinary Medicine), Larry Millet (University of Tennessee)

The overall goal of this research is to promote nerve repair through applications of carbon-based nanoparticles and mesenchymal stem cells in a synthetic extracellular matrix. We hypothesize that this combination of nerve tissue engineering technologies will provide us with the optimal environment for neuronal growth. Towards this end, we assess the suitability of neurospheres to be used as an in vitro model to evaluate axonal outgrowth and for the production of cell-free extracellular matrices to facilitate neuronal growth. Here we employ a new commercial, high throughput 3D culture system to establish neurospheres. A single 3D bioreactor provides a stress-free environment to produce a 10-fold increase in the number of neurospheres compared to the conventional 96-well U-bottom plate. This in vitro method alleviates the challenges of a co-culture process, producing neurospheres with a more efficient and cost-effective approach. Neurospheres were established using primary neurons isolated from 1 to 3-day old rat hippocampi. Reactors were coated with no-stick reagents to prevent cell adhesion to the reactor chamber during neurosphere assembly. After 48 hours in the bioreactor, seeded neurospheres were transferred to substrate-coated (laminin + poly-D-lysine (PDL), PDL only) and tissue culture plates (tissue culture treated polystyrene (TCPS)). Immunochemistry and microscopy were used to identify and quantify the neurosphere size and neurite length. The data shows that laminin provides superior neurite outgrowth for neurospheres compared to non-biological (TCPS) and conventional (PDL) synthetic substrates. Our next steps are to incorporate mesenchymal stem cell and endothelial cell populations to develop an in vitro organoid for nerve repair studies with nanoparticles and synthetic extracellular matrices. The long-term goal of this project is to generate a regenerative medicine strategy to treat peripheral nerve injuries.

A portion of this research was supported by National Institute of Biomedical Imaging and Bioengineering of the National Institutes of Health under grant number R03EB028494 (LJM). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. MEH and a portion of this research was supported by grants from the University of Tennessee Office of Research (DEA), and National Institute of Arthritis, Musculoskeletal and Skin Diseases of the National Institutes of Health under grant number R15 AR070460-01 (MD).