Fabrication of thin-layer matrigel-based constructs for three-dimensional cell culture

Extracellular matrix-based hydrogels such as Matrigel are easy-to-use, commercially available, and offer environments for three-dimensional (3-D) cell culture that mimic native tissue. However, manipulating small volumes of these materials to produce thin-layer 3-D culture systems suitable for analysis is difficult because of air–liquid-substrate interfacial tension effects and evaporation. Here, we demonstrate two simple techniques that use standard liquid-handling tools and nontreated 96-well plates to produce uniform, thin-layer constructs for 3-D culture of cells in Matrigel. The first technique, the floating 3-D cell culture method, uses phase-separating polymers to form a barrier between the dispensed Matrigel, air, and cultureware surface to generate consistently thin hydrogels from volumes as low as 5 μL. These unanchored gels provide a useful assay for investigating airway smooth muscle cell contraction and may have future applications in studying asthma pathophysiology. The second technique, the fixed 3-D cell culture method, provides an anchored gel system for culturing noncontractile cells (e.g., neurons) where 20 μL of Matrigel is dispensed into the bottom of a well filled with culture medium to form a thin gel containing embedded cells. This technique has potential widespread applications as an accessible 3-D culture platform for high-throughput production of disease models for evaluation of novel drug therapies.