Primary Hepatocyte Isolation and Cultures: Technical Aspects, Challenges and Advancements

Abstract

Hepatocytes are differentiated cells that account for 80% of the hepatic volume and perform all major functions of the liver. In vivo, after an acute insult, adult hepatocytes retain their ability to proliferate and participate in liver regeneration. However, in vitro, prolonged culture and proliferation of viable and functional primary hepatocytes have remained the major and the most challenging goal of hepatocyte-based cell therapies and liver tissue engineering. The first functional cultures of rat primary hepatocytes between two layers of collagen gel, also termed as the “sandwich cultures”, were reported in 1989. Since this study, several technical developments including choice of hydrogels, type of microenvironment, growth factors and culture conditions, mono or co-cultures of hepatocytes along with other supporting cell types have evolved for both rat and human primary hepatocytes in recent years. All these improvements have led to a substantial improvement in the number, life-span and hepatic functions of these cells in vitro for several downstream applications. In the current review, we highlight the details, limitations and prospects of different technical strategies being used in primary hepatocyte cultures. We discuss the use of newer biomaterials as scaffolds for efficient culture of primary hepatocytes. We also describe the derivation of mature hepatocytes from other cellular sources such as induced pluripotent stem cells, bone marrow stem cells and 3D liver organoids. Finally, we also explain the use of perfusion-based bioreactor systems and bioengineering strategies to support the long-term function of hepatocytes in 3D conditions.

Gelatin scaffold ameliorates proliferation & stem cell differentiation into the hepatic like cell and support liver regeneration in partial-hepatectomized mice model

“…Despite its potential for differentiation into mature HLC, there is batch-to-batch variability of the scaffold properties and decellularization process which involves various detergents that could restrict the use of this decellularized material in stem cell differentiation [60]. The biochemical differences in 2D with 3D, indicate the protection of differentiated hepatocytes from the loss of its polarity and functionality induced by culturing on the conventional 2D culture system [61]. Another possible description for this change may be the beneficial effect of gelatin scaffold communication with differentiated HLCs.…”

Hepatoblast iterative apicobasal polarization is regulated by extracellular matrix remodeling

“…However, the basic assumptions of the model have never been tested in vivo . Moreover, it has been shown that adult primary hepatocytes can form multiaxial polarity and thin tubular lumina in vitro (Mooney, 1992 29 ; Zeigerer, 2017 30 ; Kaur, 2023 31 ), which calls for a refinement of this cell division-centered model. Additionally, the key element for hepatocyte polarization in vitro is their contact to ECM (Dunn, 1989 32 ), which is required for polarization in multiple systems (Yu, 2005 33 ; Bryant, 2014 34 ) and sufficient to orient intracellular trafficking (Ojakian, 1997 35 ; Akhtar, 2013 36 ; Li, 2016 37 ).…”

On‐Chip Differentiation of Hepatic Cords with Radial Flow

“…They have been shown to possess increased sensitivity over hepatic spheroid models in predictive toxicity of small molecules and have been shown to have increased expression of genes representing Phase 1 and Phase II metabolizing enzymes [5]. While primary human hepatocytes remain the main model for preclinical studies, difficulties associated with batch-to-batch variability, loss in functionality over time, low post thaw viability, high cost and low availability can be circumvented by using established cell lines like HepaRG [6].…”