Induction of three-dimensional assembly of human liver cells by simulated microgravity

The establishment of long-term cultures of functional primary human liver c ells (PHLC) is formidable. Developed at NASA, the Rotary Cell Culture Syste m (RCCS) allows the creation of the unique microgravity environment of low shear force, high-mass transfer, and 3-dimensional cell culture of dissimil ar cell types. The aim of our study was to establish long-term hepatocyte c ultures in simulated microgravity;. PHLC were harvested from human livers b y collagenase perfusion and were cultured in RCCS. PHLC aggregates were rea dily formed and increased up to 1 cm long. The expansion of PHLC in bioreac tors was further evaluated with microcarriers and biodegradable scaffolds. While microcarriers were not conducive to formation of spheroids, PHLC cult ured with biodegradable scaffolds formed aggregates up to 3 cm long. Analys es of PHLC spheroids revealed tissue-like structures composed of hepatocyte s, biliary epithelial cells, and/or progenitor liver cells that were arrang ed as bile duct-like structures along nascent vascular sprouts. Electron mi croscopy revealed groups of cohesive hepatocytes surrounded by complex stro mal structures and reticulin fibers, bile canaliculi with multiple microvil li, and tight cellular junctions. Albumin mRNA was expressed throughout the 60-d culture. A simulated microgravity environment is conducive to maintai ning long-term cultures of functional hepatocytes. This model system will a ssist in developing improved protocols for autologous hepatocyte transplant ation, gene therapy and liver assist devices, and facilitate studies of liv er regeneration and cell-to-cell interactions that occur in vivo.