Maintenance of Liver Functions in Rat Hepatocytes Cultured as Spheroids in a Rotating Wall Vessel

Abstract

Rat hepatocytes were cultured initially as spheroids on culture plates and then transferred into a rotating wall vessel (high-aspect ratio vessel [HARV]) for further culturing. Morphological evaluation based on electron microscopy showed that hepatocyte spheroids cultured for 30 d in the HARV had a compact structure with tight cell-cell junctions, numerous smooth and rough endoplasmic reticulum, intact mitochondria, and bile canaliculi lined with microvilli. The viability and differentiated properties of the hepatocytes cultured in the HARV were further substantiated by the presence of both phase I oxidation and phase II conjugation drug-metabolizing enzyme activities, as well as albumin synthesis. Homogenates prepared from freshly isolated hepatocytes and hepatocytes cultured in the HARV showed similar cytochrome P450 2B activities measured as pentoxyresorufin-O-dealkylase and testosterone 16beta-hydroxylase. Further, intact hepatocytes cultured in the HARV were found to metabolize chlorzoxazone to 6-hydroxychlorzoxazone; dextromethorphan to dextrorphan, 3-methoxymorphinan, and 3-hydroxymorphinan; midazolam to 1-hydroxymidazolam and 4-hydroxymidazolam; and 7-hydroxycoumarin to its glucuronide and sulfate conjugates. In conclusion, we found that hepatocyte spheroids could be cultured in a HARV to retain cellular and physiological properties of the intact liver, including drug-metabolizing enzyme activities, plasma protein production, and long-term (1 mo) maintenance of viability and cellular function.

Is There a Space-Based Technology Solution to Problems with Preclinical Drug Toxicity Testing?

“…At present, primary human hepatocytes represent the “gold standard” for preclinical in vitro metabolism and toxicity studies (13). Hepatocytes grown as single cell suspensions lose polarity, integrity and differentiation (8,9,14). Primary hepatocytes adherent to plastic dishes are the most commonly used model but these also begin to de-differentiation within 24–48 hours.…”

“…Three dimensional culture systems are being extensively explored as a means of extending differentiation and function of hepatocytes to better reproduce the microenvironment of the intact liver (14–19). A variety of initiatives have explored the use of automated, microfluidic, and organ-on-a-chip approaches (14–20).…”

“…A variety of initiatives have explored the use of automated, microfluidic, and organ-on-a-chip approaches (14–20). Hepatocytes grown in gels or on various bead/fiber scaffoldings display increased and sustained functionality (21–23).…”

“…However, the inability to adequately oxygenate the cells continues to be a major limiting factor (2). This is where culture techniques, originally designed for space flight studies, may offer a distinct advantage (14,24–30). Hepatocytes grown on Earth in NASA’s rotating wall vessel suspension culture vessels form three-dimensional colonies that maintain their function for at least several weeks (14,15,24).…”

“…This is where culture techniques, originally designed for space flight studies, may offer a distinct advantage (14,24–30). Hepatocytes grown on Earth in NASA’s rotating wall vessel suspension culture vessels form three-dimensional colonies that maintain their function for at least several weeks (14,15,24). These cell colonies closely resemble and function like natural cells in the human body, which makes them excellent candidates for preclinical drug testing (14,24–30).…”