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

Abstract

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.

Fabrication and Characterization of Heparin/Collagen Sponge for in Vitro Differentiation of Wharton’s Jelly-Derived Mesenchymal Stem Cells into Hepatocytes

“…The hepatocytes lose their functions and phenotype when they are isolated from the natural niche (8,9). Many in vitro models have been introduced to improve the hepatocyte activities and morphology (43).…”

Microengineered cell and tissue systems for drug screening and toxicology applications: Evolution ofin-vitroliver technologies

“…While many potential polymers could be utilized for good results in terms of hepatocyte morphology, function, co-culture with LSECs, and CYP 1A1/2 activity have been reported for up to 12 days using chitosan and hyaluronic acid 85,207 . We have recently been able to translate this approach to a microfluidic device for hepatocyte culture using charge modified collagen strands creating a ECM barrier that is on the order of ~100 nm and have also shown long-term stability of important hepatic functions 208 .…”

“…We have also developed a new ultrathin collagen coating 208 solution for microfluidic applications which circumvents problems that arise when translating hydrogel methodology to the microfluidic realm. We will use this technique to further reduce dimensions in our device and approach more realistic scenarios for interaction between different cell types.…”

Effects of nitric oxide on ammonia decomposition by hepatocytes under shear stress

“…McCarty et al also reported that albumin secretion by cultured hepatocytes increases steadily if an ultrathin, pure collagen nanolayer is deposited on top of the cells, reaching a plateau on day 10 of culture; without this top matrix, albumin secretion by hepatocytes apparently remains low [23]. Moreover, the actin filaments in seeded hepatocytes are diffuse or radially oriented around the nucleus in the absence of a top matrix layer; however, actin fibers are reorganized along the cell-to-cell borders in hepatocytes covered with the ultrathin collagen layer [23].…”

“…Moreover, the actin filaments in seeded hepatocytes are diffuse or radially oriented around the nucleus in the absence of a top matrix layer; however, actin fibers are reorganized along the cell-to-cell borders in hepatocytes covered with the ultrathin collagen layer [23].…”