The culture of primary hepatocytes as spheroids creates an efficient three-dimensional tissue construct for hepatic studies in vitro. Spheroids possess structural polarity and functional bile canaliculi with normal differentiated function. Thus, hepatocyte spheroids have been proposed as the cell source in a variety of diagnostic, discovery, and therapeutic applications, such as a bioartificial liver. Using a novel rocking technique to induce spheroid formation, kinetics of spheroid formation, cell-cell adhesion, gene expression, and biochemical activities of rat hepatocyte spheroids were tested over 14 days of culture. Evidence was provided that the formation of spheroids occurred faster and with fewer nonadherent hepatocytes in rocked suspension culture compared to a traditional rotational system. Hepatocyte spheroids in rocked culture showed stable expression of more than 80% of 242 liver-related genes including those of albumin synthesis, urea cycle, phase I and II metabolic enzymes, and clotting factors. Biochemical activity of rocked spheroid hepatocytes was superior to monolayer culture of hepatocytes on tissue culture plastic and collagen. N ovel systems are needed to facilitate short-term and long-term culture of hepatocytes for diagnostic, discovery, and therapeutic applications. 1 Traditional monolayer culture of primary hepatocytes on tissue culture plastic is problematic and has been associated with a rapid loss of differentiated function. 2 Although single-layer and double-layer surfaces of collagen or other biomatrix materials are associated with improved differentiated functions in vitro, 3 biological surfaces can pose manufacturing hurdles and do not support high cell density culture of primary hepatocytes (exceeding 1 ϫ 10 7 cell/mL). In contrast, spheroids, which are nonadherent multicell aggregates of greater than 40 m diameter, provide a three-dimensional tissue construct which form spontaneously and allow suspension culture of primary hepatocytes at high cell density under oxygenated bioreactor conditions. 4 Spheroid formation allows recapitulation of the cuboidal geometry of primary hepatocytes with relatively stable long-term differentiated function. 5,6 Reports of structural polarity and bile canaliculi formation by primary rat hepatocytes in spheroid aggregates provide further evidence that hepatic spheroids mimic the hepatocellular microanatomy of the liver. 7 Original observations of tissue-like aggregate formation from isolated cells was reported by Moscona in 1961, using fetal liver cells and a rotational technique. 8 The descriptive term "spheroid" was coined years later by Landry in 1985 when multicellular aggregates were formed from isolated rat hepatocytes after 3-5 days of culture on nonadherent plastic surfaces. 9 Later, spheroids

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