Characterisation of a functional rat hepatocyte spheroid model

Abstract

Many in vitro liver cell models, such as 2D systems, that are used to assess the hepatotoxic potential of xenobiotics suffer major limitations arising from a lack of preservation of physiological phenotype and metabolic competence. To circumvent some of these limitations there has been increased focus on producing more representative 3D models. Here we have used a novel approach to construct a size-controllable 3D hepatic spheroid model using freshly isolated primary rat hepatocytes (PRH) utilising the liquid-overlay technique whereby PRH spontaneously self-assemble in to 3D microtissues. This system produces viable spheroids with a compact in vivo -like structure for up to 21 days with sustained albumin production for the duration of the culture period. F-actin was seen throughout the spheroid body and P-glycoprotein (P-gp) and multidrug resistance-associated protein 2 (MRP2) transporters had polarised expression on the canalicular membrane of hepatocytes within the spheroids upon formation (day 3). The MRP2 transporter was able to functionally transport 5 μM 5-chloromethylfluorescein diacetate (CMFDA) substrates into these canalicular structures. These PRH spheroids display in vivo characteristics including direct cell-cell contacts, cellular polarisation, 3D cellular morphology, and formation of functional secondary structures throughout the spheroid. Such a well-characterised system could be readily exploited for pre-clinical and non-clinical repeat-dose investigations and could make a significant contribution to replace, reduce and refine the use of animals for applied research.

Multiscale modelling of drug transport and metabolism in liver spheroids

“…Spheroids represent an intermediate experimental model that allows for increased physiological relevance over two-dimensional monolayers due to the three-dimensional environment, as well as more appropriate cell morphology and functionality while remaining cost-effective, consistent and easy to use [1]. The subsequent prevalence of liver spheroid cultures for studying hepatocyte behaviour in vitro is evident and represents a key component of drug development such that drug candidates can be tested for efficacy and toxic potential in a three-dimensional environment with physiological gradients [31,[33][34][35]. Data-driven multiscale mathematical models provide an ideal platform from which to try and enhance mechanistic understanding of new biotechnologies by simulating the underlying physical processes.…”

“…Primary rat hepatocyte spheroids with an initial seeding density of 5000 cells were produced using the liquid-overlay technique as described by Kyffin et al [31]. After 11 days in culture, the spheroids were washed in phosphate-buffered saline, fixed in 4% paraformaldehyde and subjected to routine histological processing before staining with haematoxylin or processed for TEM analysis.…”

“…After 11 days in culture, the spheroids were washed in phosphate-buffered saline, fixed in 4% paraformaldehyde and subjected to routine histological processing before staining with haematoxylin or processed for TEM analysis. For TEM imaging, spheroids were fixed in 3% glutaraldehyde and processed as previously described [31]. Ultrathin (approx.…”

Advanced biomedical applications based on emerging 3D cell culturing platforms

“…3,4 Since the knowledge on preparation methods, imaging, different assays and maintenance of spheroids has greatly improved, major attempts have been made to investigate the in vivo implantation of spheroids in various animal models, including rats and rabbits to study tissue regeneration. 5 Cell spheroids, also known as multicellular spheroids (MCS), are the simplest in vitro model of solid cell clusters. Spheroids are generated due to the production of several key proteins including fibronectin and collagen to form intercellular adhesions and self-assemble into compact aggregates.…”

“…15,27,51,[64][65][66] The results of the co-culture are mainly determined by the types of cells and the patterns of the co-culture. For example, different cells including endothelial cells (ECs), human adult keratinocytes (HaCaT), and mesenchymal stem cells have been co-cultured with hepatocytes, 5,[66][67][68][69][70][71] which either increased the liver-specific functionality such as albumin production or constructed angiogenesis tissue. In hepatic coculture/tri-culture spheroids, the hepatocytes are: (i) pre-mixed with another type of cells to allow cell self-sorting in the obtained spheroids.…”