Developing a multiplex microscale screening platform by multiphoton biofabrication technology for maintenance of cell phenotype

Abstract

Multiphoton biofabrication is an emerging technology that makes use of photochemical crosslinking to immobilize natural biological molecules with sub-micron spatial resolution without involving harsh reagents and fabrication conditions. Our group previously demonstrated the capability of the technology in constructing micro-patterns with various controllable mechanical properties and extracellular matrix (ECM) immobilization. Moreover, we have successfully made use of the ultra-high spatial resolution to construct various topological features including both 2D and 3D ones such as pillar array, convex, concave, cave and niche. The excellent controllability makes the technology a pertinent platform for screening purposes. The microscale fabrication technique also enables the investigation of primary cells or other less-readily available cell types. This study aims to develop such a platform to screen for the optimal microenvironment for phenotype maintenance of cells types that transform spontaneously during in vitro culture, like spontaneous differentiation of mesenchymal stem cells and the dedifferentiation of nucleus pulposus cells (bNPCs). This study investigates the effect of mechanical properties (elastic modulus, stiffness), ECM composition (fibrinogen, fibronectin, laminin, vitronectin, collagen type I) and topological features (both 2D and 3D) in phenotype maintenance. Micropatterns were fabricated by a femto-second laser, in the presence of photosensitizer. The marker expressions were evaluatedand quantified by immunofluorescent staining and intensity quantification after certain days of culture. The effect of different factors was examined and scored. Take bNPC as an example. High level of fibrinogen was found to favor the expression of collagen type II (one of the bNPC markers), while the effects of fibronectin were less significant. Low modulus and stiffness were also demonstrated to favor the phenotype maintenance.