A novel ultrathin collagen nanolayer assembly for 3-D microtissue engineering: Layer-by-layer collagen deposition for long-term stable microfluidic hepatocyte culture

The creation of stable hepatocyte cultures using cell-matrix interactions has proven difficult in microdevices due to dimensional constraints limiting the utility of classic tissue culture techniques that involve the use of hydrogels such as the collagen double gel or overlay. To translate the collagen overlay technique into microdevices, we modified collagen using succinylation and methylation reactions to create polyanionic and polycationic collagen solutions, and deposited them layer-by-layer to create ultrathin collagen nanolayers on hepatocytes. These ultrathin collagen layers covered hepatocytes in microdevices and 1) maintained cell morphology, viability, and polarity, 2) induced bile canalicular formation and actin reorganization, and 3) maintained albumin and urea secretions and CYP activity similar to those observed in hepatocytes in collagen double gel hepatocytes in plate cultures. Beyond the immediate applications of this technique to create stable, in vitro microfluidic hepatocyte cultures for drug toxicity testing, this technique is generally applicable as a thin biomaterial for other 3D microtissues.