Photolithographic Fabrication of Pocket-like Hydrogel Microstructures for Novel Cell Culture System

Abstract

Various functional hydrogels have been developed, and can be applied to the fields of bioengineering, such as cell culture substrates and microfluidic systems. It is very important to provide these materials with some functional shapes in a scale of micrometers when these applications are examined. In this study, we proposed the fabrication of unique hydrogel microstructures through photolithography, and examined its application to novel cell culture system. They are expected to be applied to compose novel cell culture system which can maintain physiological activity of living cells, and to contribute to increasing precision of drug screening through providing totally different culture environment from conventional systems composed of rigid materials. First, we coated a solution containing a UV responsive photo acid generator (PAG) on polystylene (PS) surface, and then coated a methanol solution containing poly (acrylic acid) (PAAc). The micro-patterned light irradiation (wavelength: 365nm) was carried out to induce area-selective cross-linking. After flushing with water, ethanol and 1M NaOH aqueous solution, a micro-patterned cross-linked PAAc layer was obtained. Next, we prepared a pre-gel layer by coating a solution containing hydroxypropyl cellulose (HPC) 1,3,4,6-tetrakis (methoxymethyl) glycoluril, an acid catalyzed cross-linker, and PAG, on the cross-linked PAAc layer. The pre-gel layer was irradiated with the light in a different micro-pattern, and was baked at 85 ˚C to promote acid-catalyzed cross-linking of HPC. After flushing with water, a micro-patterned cross-linked HPC layer was obtained on the cross-linked PAAc layer. By soaking the patterned bilayer in 1M NaOH aqueous solution, HPC gel sheet lifted off of the substrate and swelled in the areas where the cross-linked PAAc had remained while the sheet was immobilized on the PS surface in the other areas. As a result, pocket-like semi-3D microstructures were obtained. To provided the hydrogel with the property of cell adhesion, we examined the coating of several polymer materials on HPC.