A three-dimensional biomimetic peripheral nerve model for drug testing and disease modelling

Abstract

In vitro peripheral nerve models provide valuable tools to study neurobiology questions and assess drug performance, in a regenerative or pathology context. To this end, we have developed a representative model of the peripheral nerve that displays three-dimensional (3D) neural anisotropy and myelination, which we showcase here as a simple and low-cost platform for drug screening. The model is composed of three main parts, including rat primary Schwann cells (SCs) seeded onto an electrospun scaffold to create bands of Büngner (BoB), primed PC12 cells as neuronal cell population, and a fibrin hydrogel to provide three-dimensionality. We also validated the use of primed PC12 as a neuron population by comparing it to rat dorsal root ganglions (DRGs) neurons. In both models we could obtain well aligned neurites and mature myelin segments. In short term cultures (7 days), we found that the addition of exogenous SCs enhanced neurite length and neurite growth area, compared to scaffolds with a laminin coating only. Addition of fibrin also lead to increased outgrowth of DRG and primed PC12 neurites, compared to 2D cultures. Moreover, neurite outgrowth in fibrin cultures was simultaneously multiplanar and anisotropic, suggesting that the SC-seeded scaffold can direct not only the growth of adjacent neurites, but also those growing above it. These results highlight the feasibility of the combination of a SC pre-seeded scaffold with a fibrin hydrogel, to direct and improve neurite growth in 3D. To demonstrate the model potential, we tested our platform at an immature (7 days in vitro) and mature state (28 days in vitro) of development. At the immature stage we could inhibit neurite growth through protein blocking (via antibody binding) and show suramin (200 μM) neurotoxicity on cells. At the mature stage, when myelin is compact, we exposed cells to hyperglycemic conditions (45 mM glucose) to mimic diabetic conditions and showed that myelin deforms consequently. Moreover, we demonstrated that by supplementing cultures with epalrestat (1 μM), myelin deformation can be partly prevented. In sum, we developed a biomimetic nerve platform using an affordable and accessible cell line as neuronal population, which displays similar results to primary neurons, but does not require recurrent animal sacrifice. This platform holds great promise as it can be used to conveniently and inexpensively perform drug screenings on peripheral nerve-like tissue, in a normal or pathological state.

Keywords: 3D; Bands of Bungner; Biomimetic; In vitro model; PC12 cells; Peripheral nerve.