Hierarchical hydrogel microarrays fabricated based on a microfluidic printing platform for high-throughput screening of stem cell lineage specification.

2D cell cultures are suitable for rapid exploration of the factors in the extracellular matrix affecting the development of cells. The technology of the micrometre-sized hydrogel array provides a feasible, miniaturized, and high-throughput strategy for the process. However, current microarray devices lack a handy and parallelized methodology in sample treatment, which makes the process of high-throughput cell screening (HTCS) expensive and inefficient. Here, based on the functionalization of micro-nano structures and the fluid control capability of microfluidic chips, we build a microfluidic spotting-screening platform (MSSP). The MSSP can print 20000 microdroplet spots within 5 min, coupled with a simple strategy for parallel addition of compound libraries. Compared with open microdroplet arrays, the MSSP can control the evaporation rate of nanoliter droplets, providing a stable fabrication platform for hydrogel-microarray-based materials. As a proof-of-concept demonstration, the MSSP successfully controlled the adhesion, adipogenic, and osteogenic differentiation behavior of mesenchymal stem cells by rationally designing the substrate stiffness, adhesion area, and cell density. We anticipate that the MSSP may provide an accessible and promising tool for hydrogel-based HTCS. STATEMENT OF SIGNIFICANCE: High-throughput screening of cells is a common approach to improve the efficiency of biological experiments, and one challenge of the existing technologies is to achieve rapid and precise cell screening with a low-cost and simple strategy. Through the integration of the microfluidic and micro-nanostructure technologies, we fabricated a microfluidic spotting-screening platforms. Benefiting from the flexible control of the fluids, the device can print 20000 microdroplet spots within 5 min, coupled with a simple procedure for parallel addition of compound libraries. High-throughput screening of stem cell lineage specification has also been achieved by the platform, which provides a high-throughput, high-content information extraction strategy for cell-biomaterial interaction research.