Thermal scanning probe lithography (t-SPL) is a nanofabrication technique in which an immobilized thermolabile resist, such as polyphthalaldehyde (PPA), is locally vaporized by a heated atomic force microscope tip. Compared with other nanofabrication techniques, such as soft lithography and nanoimprinting lithography, t-SPL is more efficient and convenient as it does not involve time-consuming mask productions or complicated etching procedures, making it a promising candidate technique for the fast prototyping of nanoscale topographies for biological studies. Here, we established the direct use of PPA-coated surfaces as a cell culture substrate. We showed that PPA is biocompatible and that the deposition of allylamine by plasma polymerization on a silicon wafer before PPA coating can stabilize the immobilization of PPA in aqueous solutions. When seeded on PPA-coated surfaces, human mesenchymal stem cells (MSC) adhered, spread, and proliferated in a manner indistinguishable from cells cultured on glass surfaces. This allowed us to subsequently use t-SPL to generate nanotopographies for cell culture experiments. As a proof of concept, we analyzed the surface topography of bovine tendon sections, previously shown to induce morphogenesis and differentiation of MSC, by means of atomic force microscopy, and then "wrote" topographical data on PPA by means of t-SPL. The resulting substrate, matching the native tissue topography on the nanoscale, was directly used for MSC culture. The t-SPL substrate induced similar changes in cell morphology and focal adhesion formation in the MSC compared to native tendon sections, suggesting that t-SPL can rapidly generate cell culture substrates with complex and spatially accurate topographical signals. This technique may greatly accelerate the prototyping of models for the study of cell−matrix interactions.

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One of the most crucial components of regenerative medicine is the controlled differentiation of embryonic or adult stem cells into the desired cell lineage. Although most of the reported protocols of stem cell differentiation involve the use of soluble growth factors, it is increasingly evident that stem cells also undergo differentiation when cultured in the appropriate microenvironment. When cultured in decellularized tissues, for instance, stem cells can recapitulate the morphogenesis and functional specialization of differentiated cell types with speed and efficiency that often surpass the traditional growth factor-driven protocols. This suggests that the tissue microenvironment (TME) provides stem cells with a holistic ''instructive niche'' that harbors signals for cellular reprogramming. The translation of this into medical applications requires the decoding of these signals, but this has been hampered by the complexity of TME. This problem is often addressed by a reductionist approach, in which cells are exposed to substrates decorated with simple, empirically designed geometries, textures, and chemical compositions (''bottom-up'' approach). Although these studies are invaluable in revealing the basic principles of mechanotransduction mechanisms, their physiological relevance is often uncertain. This review examines the recent progress of an alternative, ''top-down'' approach, in which the TME is treated as a holistic biological entity. This approach is made possible by recent advances in systems biology and fabrication technologies that enable the isolation, characterization, and reconstitution of TME. It is hoped that these new techniques will elucidate the nature of niche signals so that they can be extracted, replicated, and controlled. This review summarizes these emerging techniques and how the data they generated are changing our view on TME.

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