Multiphoton microfabrication and micropatternining (MMM)-based screening of multiplex cell niche factors for phenotype maintenance - Bovine nucleus pulposus cell as an example.

Abstract

Upon monolayer cultures on flat and rigid plastic dishes, many cells de-differentiate and lose their native phenotype. Technologies able to identify and reconstitute the cell niche factors that best maintain the physiological cellular phenotype in cultures are critical. We have developed a multiphoton microfabrication and micropatterning (MMM) technology, a robust 3D micro-printing platform capable to fabricate protein microstructures and micropatterns with quantitative, spatial and independent control of the mechanical, topological and extracellular matrix properties. Here, using bovine nucleus pulposus cells (bNPCs) as an example, we aim to reconstitute a spectrum of individual cell niche factors (2 mechanical, 9 topological and 4 matrices) in vitro for multiplex cell niche factor screening, and fabricate the optimal combinations of a series of shortlisted cell niche factors that best maintain the bNPC phenotype. Among all factors screened, two topological (micropillar array; fiber-bead structure) and two matrix (laminin; vitronectin) factors were shortlisted and the combinatory cell niche factors reconstituted from the shortlisted factors were found to synergistically augmented the expression of selected bNPC phenotype markers (Col II, SNAP25 and Keratin 8) and maintained their morphology and phenotype. These optimal cell niches can be micro-printed on culture dishes for physiologically relevant cultures and contribute to biomimetic scaffold design for intervertebral disc tissue engineering.