No abstract

Controlled activation of hepatocyte aggregation is critical to three‐dimensional (3D) multicellular morphogenesis during native regeneration of liver as well as tissue reconstruction therapies. In this work, we quantify the stimulatory effects of two model hepatotrophic activators, epidermal growth factor (EGF) and hepatocyte growth factor (HGF), on the aggregation kinetics and liver‐specific function of hepatocytes cultured on organotypic substrates with differing mechanical resistivity. Substrate‐specific morphogenesis of cultured hepatocytes is induced on a tissue basement membrane extract, Matrigel, formulated at two distinct levels of mechanical compliance (storage modulus G′, at oscillatory shear rate 1 rad/s, was 34 Pa for basal Matrigel and 118 Pa for crosslinked Matrigel). Overall, we report that growth factor stimulation selectively promotes the kinetics of aggregation in the form of two‐dimensional corded aggregates on basal Matrigel and three‐dimensional spheroidal aggregates on crosslinked Matrigel. Our analysis also indicates that costimulation with EGF and HGF (20 ng/mL each) cooperatively maximizes the kinetics of aggregation in a substrate‐specific manner. In addition, we show that the role of growth factor stimulation on hepatocyte function is sensitively governed by the mechanical compliance of the substrate. In particular, on matrices with high compliance, costimulatory aggregation is shown to elicit a marked increase in albumin secretion rate, whereas on matrices with low compliance aggregation results in effective functional repression to basal, unstimulated levels. Thus, our studies highlight a novel interplay of physicochemical parameters of the culture microenvironment, leading to selective enhancement or repression of differentiated functions of hepatocytes, in concert with the activation of cellular morphogenesis. © 2000 John Wiley & Sons, Inc. Biotechnol Bioeng 69: 359–369, 2000.

A fed-batch process using concentrated medium was evaluated for its ability to improve cell culture longevity and final monoclonal antibody (MAb) titers for two monoclonal antibody producing cell lines. It was found to result in up to 7-fold increases in final antibody titers compared to batch culture controls. Although the development cell line specific fed-batch protocols is critical to the development of cost-efficient large-scale production processes, the use of complete medium concentrates provided us with a quick and simple method for producing large quantities of antibodies in the early stages of process development, thus accelerating early work on purification process development, analytical development, biochemical characterization, and safety studies. Insights gained from the concentrated medium fed-batch approach were valuable for the development of refined, cell line specific feeding strategies yielding final MAb titers on the order of 1-2 g/L. Process development data on the effects of inhibitory growth byproducts, medium osmolarity, and the mode of nutrient feed addition on culture longevity and MAb production and information on culture metabolic behavior were successfully incorporated in the development of the optimized fed-batch protocols.

The regulation of cell motility by ligand density on substrates with variable microtopography is not well understood. In this report, we studied the adhesion and motility behavior of HepG2 cells on microtextured poly(glycolic-co-lactic)acid (PGLA) copolymer substrates, whose surface bioactivity was differentially modified through the adsorption of 0-5.5 ng/cm(2) collagen. Microtextured PGLA substrates were fabricated as thin films with a uniform surface distribution of micropores of median size of 3.1 +/- 1.5 microm and three-dimensional root mean squared roughness of 0.253 microm. Even in the absence of collagen, cells on microtextured substrates responded to substrate topography by exhibiting a 200% increase in adhesion strength compared with untextured controls and ventral localization of the intracellular adhesion protein vinculin. Further enhancement in adhesion strength (420% over untextured, untreated substrates) was demonstrated with bioactivated, microtextured surfaces, indicating that cell adhesion responses to topography and surface ligand density were cooperative. Our motility studies of cells on untextured substrates adsorbed with different levels of collagen demonstrated that a classical biphasic relationship between the cell population averaged migration rate, mu, and the collagen ligand density was preserved. However, comparison of cell motility responses between untextured and microtextured substrates indicates that the motility versus ligand density curve shifted, such that equivalent levels of cell motility were achieved at lower ligand density on microtextured surfaces. Furthermore, the maximum mu values achieved on the microtextured substrates exceeded those on untextured substrates by twofold. Taken together, we show that the magnitude of subcellular scale microtexture of a polymer substrate can sensitize the cell motility responsiveness to substrate ligand concentration; we suggest that the underlying mechanisms involve alteration in the degree of cell-substrate adhesivity as well as changes in the nature of ligand-induced cell activation processes.