Perfusion of 3D encapsulated hepatocytes—A synergistic effect enhancing long‐term functionality in bioreactors

Abstract

Long-term primary cultures of hepatocytes are essential for bioartificial liver (BAL) devices and to reduce and replace animal tests in lead candidate optimization in drug discovery and toxicology tests. The aim of this work was to improve bioreactor cultures of hepatocyte spheroids by adding a more physiological perfusion feeding regime to these bioreactor systems. A continuous perfusion feeding was compared with 50% medium replacement (routinely used for in vitro tests) at the same dilution rate, 0.125 day(-1), for three operative weeks. Perfusion feeding led to a 10-fold improvement in albumin synthesis in bioreactors containing non-encapsulated hepatocyte spheroids; no significant improvement was observed in phase I drug metabolizing activity. When ultra high viscous alginate encapsulated spheroids were cultured in perfusion, urea synthesis, phase I drug metabolizing activity and oxygen consumption had a threefold improvement over the 50% medium replacement regime; albumin production was the same for both feeding regimes. The effective diffusion of albumin in the alginate capsules was 7.75.10(-9) cm(2) s(-1) and no diffusion limitation for this protein was observed using these alginate capsules under our operational conditions. In conclusion, perfusion feeding coupled with alginate encapsulation of hepatocyte spheroids showed a synergistic effect with a threefold improvement in three independent liver-specific functions of long-term hepatocyte spheroid cultures.

Nanostructured self-assembling peptides as a defined extracellular matrix for long-term functional maintenance of primary hepatocytes in a bioartificial liver modular device

“…Much encouraging effort has been made to optimize the capturing of the in vivo hepatocytes microenvironment for functional maintenance of primary hepatocytes in a micropatterned co-culture module, 2 bioreactor-based liver module, [3][4][5] microfluidic 3D chip, 6 and other innovative approaches. [7][8][9] The most encouraging reports relied on undefined extracellular matrices like Matrigel, rigid collagen, or serum supplementations, which are often problematic and unacceptable in preclinical and clinical applications.…”

“…The paracrine fashion secretion pattern of several soluble factors from feeder cells for primary cells cultures 10 is more suitable, but it is much more difficult to test the effect of the exogenous factors on the functional maintenance of primary hepatocytes. However, most experimental approaches aimed at the long-term functional maintenance of primary hepatocytes in a culture from 1 week to several weeks (42 days) have been reported by various investigators, [2][3][4] including us. [11][12][13] However, to the best of our knowledge, there is no report for the long-term functional maintenance of primary hepatocytes up to 90 days within a defined hepatic microenvironment to provide a better strategy as an alternative to in vivo animal experimentation.…”

“…We hope that this 3D interaction of growth factors and cytokines in nanostructured self-assembling peptides in a bioartificial liver modular device might add significant value to other existing toxicology screening devices as well as to the pharmaceutical industrial methodology, enabling more accurate toxicological assays and increasing the predictive accuracy during drug candidate screening. This 3D signaling can be useful in other existing micropatterned co-culture modules, 2 bioreactor based liver modules, [3][4][5] microfluidic 3D chips, 6 and other innovative approaches. It is a formidable challenge to establish an in vitro hepatocyte cellular model to allow the long-term functional maintenance of primary hepatocytes up to 90 days, which can be useful for basic studies of hepatocytes physiology, drug metabolism, enzyme induction, transplantation, viral hepatitis, hepatocyte regeneration, and other purposes.…”

State-of-the-art of 3D cultures (organs-on-a-chip) in safety testing and pathophysiology

“…the decision whether to use primary cell sources, cell lines and single type or co-culture models depends strongly on the the liver spheroids formed in stirrer bioreactors have been proposed to be suitable for toxicity studies Miranda et al, 2010;tostoes et al, 2011). the hanging drop liver spheroids have been extensively characterised for liver specific functions (Michailov et al, 2004) and applied in the assessment of toxicity (Gunness et al, 2013).…”

Arginine–glycine–aspartic acid–polyethylene glycol–polyamidoamine dendrimer conjugate improves liver-cell aggregation and function in 3-D spheroid culture

“…To maintain the activity and function of cells as far as possible, many research groups are committed to improving the methods of cell culture. Commonly used methods are coculture with other cells, 2 microencapsulated culture, 3 spheroidal aggregate culture, 4 and bioreactor culture. 5 Spheroidal aggregate culture makes hepatic cells aggregate into a sphere, in which the contact area is the largest.…”