Formation of Porcine Hepatocyte Spheroids for use in a Bioartificial Liver

Abstract

Xenogeneic hepatocytes have recently been used in a bioartificial liver device as a potential short-term extracorporeal support of acute liver failure. Scaling up the system requires large quantities of viable and highly active cells. Hepatocytes grown as spheroids manifest higher metabolic activities for longer time periods as compared to those in monolayer cultures. Use of hepatocyte spheroids for application in a bioartificial liver can possibly alleviate the need of scaling up. Porcine hepatocytes when cultured under stirred conditions, from multicellular spheroids in a defined culture medium. Spheroids were formed 24 h after cell inoculation with an efficiency of 80-90% and a mean diameter of about 135 microns. Scanning electron microscopy revealed numerous microvilli projecting from the entire surface of the spheroids. Transmission electron microscopy revealed differentiated hepatocytes which displayed well-developed cytoplasmic structures separated by bile canaliculus-like structures. The morphological studies show a resemblance between cells in the spheroids and in the liver in vivo. Urea-genesis by spheroids was twice as active and was sustained for a longer culture period than that by hepatocytes cultured as monolayers. Preparation of porcine hepatocyte spheroids in an agitated vessel is simple efficient and reproducible. It will allow for preparation of large quantities of spheroids to be employed in a bioartificial liver device as well as in liver metabolism studies.

Dynamic analysis of hepatoma spheroid formation: roles of E-cadherin and β1-integrin

“…Spheroids have been widely used in tissue engineering, bioartificial livers, and embryoid body formation in stem cell research as they have been shown to improve the viability, function, and tissue-specific gene expression of several cultured primary cells (Abu-Absi et al 2002;Fukuda et al 2004;Kale et al 2000;Kelm and Fussenegger 2004;Korff and Augustin 1998;Landry et al 1985;Lazar et al 1995;Narayanan et al 2002;O'Brien et al 2002). All these applications demand reliable culture conditions for the efficient production of large quantities of spheroids with a uniform size, high cell viability, and proper cell-cell organization.…”

Direct Self-assembly of Hepatocytes Spheroids within Hollow Fibers in Presence of Collagen

“…Numerous mitochondria and lipid droplets were observed in the cytoplasm of several cells, similar to the interior structure of spheroids formed by attachment (Peshwa et al 1996) or by stirring in a spinner flask (Lazer et al 1995). Morphological characteristics predominant in spheroids were junctional complexes such as bile-canaliculus like structures between hepatocytes.…”

“…Similar to the spheroids formed by surface attachment culture (Peshwa et al 1996) or suspension culture (Lazer et al 1995), spheroids formed by addition of collagen possess an intricate continuous bile canalicular-like network, which serves as open structures to facilitate diffusion of nutrients and waste products. These hepatocyte spheroids maintained not only viabilities but also liver-specific functions for at least 8 days.…”

Hepatic Tissue Engineering on 3-Dimensional Biodegradable Polymers within a Pulsatile Flow Bioreactor

“…With this pre-transplant cultivation the freshly isolated hepatocytes are stimulated to form neotissue. Therefore, the liver tissue specific hepatocytes assembly will be already initiated in vitro [30, 54]. As a biodegradable polymer scaffold, we chose PLLA, with its highly porous structure offering a favorable environment for the hepatocytes.…”