Scaling Down of a Clinical Three-Dimensional Perfusion Multicompartment Hollow Fiber Liver Bioreactor Developed for Extracorporeal Liver Support to an Analytical Scale Device Useful for Hepatic Pharmacological In Vitro Studies

Abstract

Within the scope of developing an in vitro culture model for pharmacological research on human liver functions, a three-dimensional multicompartment hollow fiber bioreactor proven to function as a clinical extracorporeal liver support system was scaled down in two steps from 800 mL to 8 mL and 2 mL bioreactors. Primary human liver cells cultured over 14 days in 800, 8, or 2 mL bioreactors exhibited comparable time-course profiles for most of the metabolic parameters in the different bioreactor size variants. Major drug-metabolizing cytochrome P450 activities analyzed in the 2 mL bioreactor were preserved over up to 23 days. Immunohistochemical studies revealed tissue-like structures of parenchymal and nonparenchymal cells in the miniaturized bioreactor, indicating physiological reorganization of the cells. Moreover, the canalicular transporters multidrug-resistance-associated protein 2, multidrug-resistance protein 1 (P-glycoprotein), and breast cancer resistance protein showed a similar distribution pattern to that found in human liver tissue. In conclusion, the down-scaled multicompartment hollow fiber technology allows stable maintenance of primary human liver cells and provides an innovative tool for pharmacological and kinetic studies of hepatic functions with small cell numbers.

The use of bioreactors as in vitro models in pharmaceutical research

“…As well as being used clinically [21], they are being utilised as in vitro models, like the scaling down of a 3D perfusion multicompartment hollow fibre liver bioreactor for use in in vitro pharmacological studies [22]. This bioreactor consists of three interwoven sets of hollow fibre, two for counter current medium perfusion and one for gas supply (Fig 1).…”

“…However non-uniform cell distribution along the fibres is a common problem, and although being beneficial in terms of support and improved cell growth and performance if adapted to the specific cell type [29], membranes can present a physical barrier against the transport of nutrients and metabolites. As mentioned previously, development from standard hollow fibre bioreactors to multi-compartmental devices such as that in Fig 1 has been a natural progression considering the native architecture of the liver and has been utilised for both clinical and pharmacological applications [22].…”

“…However, the use of complex chemically defined media has allowed hepatocytes to go through a number of cycles in culture as well as stabilize hepatocyte morphology, survival, and liver-specific functions [45] [38] [46] [43]. Co-culture with non-parenchymal cells has also shown to preserve liver specific functions including initiating physiological reorganisation of cells and the distribution of canalicular transporters similar to the pattern found in vivo [22]. The mechanisms behind these changes are not fully understood, but highly conserved signalling pathways are thought to be involved [9].…”

“…However the number of cells needed for bioreactor based models are approx. 3 x 10 8 per device [22], which could be attained with minimal culture in vitro.…”

“…When investigating the metabolism of diclofenac, freshly isolated human hepatocytes were found to produce 90% less hydroxydiclofenac, the hydroxylated product of diclofenac generated directly by the cytochrome P450 enzyme CYP2C9, after a 3-day culture in 2D conditions compared to a high level of metabolites from human hepatocytes in the multi-compartmental bioreactor described above [35] [22]. The major intermediary diclofenac metabolite, diclofenac acyl glucuronide (produced from the glucuronidation of diclofenac by UDPglucuronosyltransferase (UGT) enzymes before hydroxylation by the P450 enzyme CYP2C8), was found to be absent in human hepatocyte suspensions, but present in the hepatocyte seeded bioreactor, possibly due to the bioreactor system mimicking the in vivo environment and allowing the efflux of metabolites away from the cells, thereby avoiding further metabolism.…”