Mechanochemical manipulation of hepatocyte aggregation can selectively induce or repress liver-specific function

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.

Three-dimensional polarization sensitizes hepatocytes to Fas/CD95 apoptotic signalling

“…In the present work, we have extended these characterizations by showing that culture in an appropriate 3D environment led to a strong apico-basolateral polarization and the maintenance of liver-specific functions such as a high level of albumin secretion and accumulation of bilirubin. In agreement with previous reports on long-term culture of primary hepatocytes (LeCluyse et al, 1996;Moghe et al, 1996;Semler and Moghe, 2001;Semler et al, 2000), we found that the microenvironment of the Matrigel basalmembrane extract provided the best spatial and biochemical cues for mhAT3F polarization and function. It is noteworthy that Matrigel's chemical composition is similar both to the basal-membrane components of the liver in vivo (Stamatoglou and Hughes, 1994) and to the endogenous matrix deposited by aggregates of primary liver cells in suspension culture (Landry et al, 1985).…”

“…Thus, hepatocytes in polarized, functional organoids modulate an intracellular signal transduction pathway, allowing the recapitulation of their physiological response to an apoptotic stimulus. membrane extracellular matrix, provides an especially appropriate environment for hepatocyte culture, as judged by the cells' morphology and metabolism (LeCluyse et al, 1996;Semler and Moghe, 2001;Semler et al, 2000). Interestingly, in addition to the chemical composition of the matrix, the physical properties of the gels (especially the pore size and the pliability) profoundly influence both the spatial organization and the differentiation of the cells (Moghe et al, 1996;Ranucci et al, 2000;Semler and Moghe, 2001).…”

“…Although the influence of culture conditions on hepatocyte viability and function has been extensively studied (Allen et al, 2001;Block et al, 1996;Dunn et al, 1989;Khalil et al, 2001;LeCluyse et al, 1996;Michalopoulos and Pitot, 1975;Richert et al, 2002;Rojkind et al, 1980;Semler et al, 2000), little is known about the effect of hepatocyte morphology and physiology on the modulation of apoptosis. One well-described pathway of apoptosis induction originates from the engagement of death receptors, such as tumour necrosis factor receptor (TNFR) or Fas (CD95), expressed on the surface of many cell types (Debatin and Krammer, 2004).…”

Minimally invasive cell-seeded biomaterial systems for injectable/epicardial implantation in ischemic heart disease

“…Most of the biomaterials employed have unmatched mechanical properties to the infarcted myocardium. The stiffness of a range of possible injectable materials is in the range of 10 Pa to 20 kPa, such as fibrin (50 Pa), 224 Matrigel (30-120 Pa), 225 type I collagen gels (20-80 Pa for 1-3 mg/mL), 226 bioactive hydrogels based on N-isopropylacrylamide (100-400 Pa), 227,228 alginate (100 Pa to 6 kPa), 229 and PEG (1-3 kPa). 230 These biomaterials are significantly softer than the human cardiac muscle at the end of diastole, the stiffness of which is 50 kPa in normal hearts or 200-300 kPa in congestive heart-failure hearts.…”

Mechanical and Biological Properties of Bio-Inspired Nano-Fibrous Elastic Materials from Collagen

“…There was a lag time before steady increase in cell number on the e-BC gel. Semler et al reported that cells shows high growth rate on matrices with high mechanical compliance such as TCP, whereas the cells aggregate three-dimensionally on matrices with low mechanical compliance such as collagen gel [39]. Therefore, the difference in the mechanical properties of the surface of the culture substrate might affect the initial rate of cell growth.…”