Designer Self-Assembling Peptide Nanofiber Scaffolds for Adult Mouse Neural Stem Cell 3-Dimensional Cultures

TLDR

Conventional 2‑D culture systems fail to recapitulate the 3‑D microenvironment, gradient diffusion, cell migration, and cell‑cell interactions essential for studying cell biology, cancer biology, and neurobiology. The authors aim to create a fully defined 3‑D culture system in which every component is known, enabling accurate study of cell behavior in a body‑like context. They engineered a self‑assembling peptide scaffold based on RADA16 functionalized with adhesion, differentiation, and bone‑marrow‑homing motifs that form nanofibers resembling Matrigel and fully embed mouse adult neural stem cells. The bone‑marrow‑homing motifs markedly increased neural cell survival without added growth factors, produced cell populations and gene expression profiles comparable to Matrigel, and demonstrate that these pure, modular peptide scaffolds offer a versatile, controlled 3‑D culture platform for diverse tissue cells.

Abstract

Biomedical researchers have become increasingly aware of the limitations of conventional 2-dimensional tissue cell culture systems, including coated Petri dishes, multi-well plates and slides, to fully address many critical issues in cell biology, cancer biology and neurobiology, such as the 3-D microenvironment, 3-D gradient diffusion, 3-D cell migration and 3-D cell-cell contact interactions. In order to fully understand how cells behave in the 3-D body, it is important to develop a well-controlled 3-D cell culture system where every single ingredient is known. Here we report the development of a 3-D cell culture system using a designer peptide nanofiber scaffold with mouse adult neural stem cells. We attached several functional motifs, including cell adhesion, differentiation and bone marrow homing motifs, to a self-assembling peptide RADA16 (Ac-RADARADARADARADA-COHN2). These functionalized peptides undergo self-assembly into a nanofiber structure similar to Matrigel. During cell culture, the cells were fully embedded in the 3-D environment of the scaffold. Two of the peptide scaffolds containing bone marrow homing motifs significantly enhanced the neural cell survival without extra soluble growth and neurotrophic factors to the routine cell culture media. In these designer scaffolds, the cell populations with β-Tubulin+, GFAP+ and Nestin+ markers are similar to those found in cell populations cultured on Matrigel. The gene expression profiling array experiments showed selective gene expression, possibly involved in neural stem cell adhesion and differentiation. Because the synthetic peptides are intrinsically pure and a number of desired function cellular motifs are easy to incorporate, these designer peptide nanofiber scaffolds provide a promising controlled 3-D culture system for diverse tissue cells, and are useful as well for general molecular and cell biology.