Multiphoton fabrication of functionalized protein micropatterns - An in vitro model for study on cell-matrix interactions in human mesenchymal stem cells

Abstract

Micropatterning technique is a powerful tool in cell niche studies. Multi-photon-based micropattern fabrication is a promising technology able to incorporate bioactive molecules into process as well as precisely control the geometry and stiffness of micropatterns fabricated. In the present study, we aim to engineer fibronectinfunctionalized micropatterns with different stiffness based on multiphoton fabrication technique, and test the validity of this versatile tool on investigation of the cell-matrix adhesion formation and matuaration in human mesenchymal stem cells (hMSCs). Firstly, the fibronectin-functionalized protein micropatterns were fabricated by photo-crosslinking Bovine Serum Albumin (BSA) (100 mg/ml) and various concentrations of fibronectin (0 μg/ml, 50 μg/ml, 150 μg/ml and 350 μg/ml) in the presence of photosensitizer, Rose Bengal (RB) (0.1%, w/v), which was activated by femto-second laser with output power of 60 mW at wavelength of 800nm. Then the cross-linked fibronectin was verified by immunofluorescence staining. Afterwards, fibronectinfunctionalized protein micropatterns with different dimensions (length × width × height: 1 × 1 × 5 μm, 3 × 1 × 5 μm and 10 × 1 × 5 μm) were fabricated as described above using different region of interest (ROI). Finally, hMSCs were cultured on these micropatterns for 3 days to determine the maturation of 3D-matrix adhesions and fibrillar adhesions using immunofluorescence staining. Results show that immobilization of fibronectin by multiphoton photo-crosslinking on the protein micropatterns is dose-dependent of the initial amount of fibronectin incorporated. Fibronectin-functionalized micropatterns with longer length could induce the maturation of cell-matrix adhesions in terms of formation of fibrillar adhesion (FBA) in hMSCs. The current platform presents a good in vitro model for cell niche studies.