Enhanced Hepatic Functions of Genetically Modified Mouse Hepatoma Cells by Spheroid Culture for Drug Toxicity Screening

Abstract

While hepatic cell lines are mainly used for in vitro drug induced toxicity studies, they exhibit limited functionalities. To overcome this, the authors have employed genetically engineered mouse hepatoma cells, Hepa/8F5, wherein expression of liver enriched transcription factors is induced by doxycycline leading to increased functionality. Further enhancement in functionality is achieved by spheroid culture in a previously developed 3D cell culture platform. Cells are seeded in presence of temperature-responsive poly(N-isopropylacrylamide) on poly(N-isopropylacrylamide--co-gelatin) cryogel scaffold based high throughput platform. Cells seeded in presence of poly(N-isopropylacrylamide) and induced with doxycycline exhibited highest functionalities. There is an increase of ≈26, 36, and 39% in albumin secretion, ammonia removal, and CYP3A4 activity, respectively. Morphological analysis showed arrest in cell proliferation and enlarged nucleus in presence of doxycyline and spheroid formation in presence of poly(N-isopropylacrylamide). Drug induced liver toxicity studies revealed that cells induced with doxycycline are resistive to tamoxifen but sensitive to acetaminophen whereas, cultures initiated in presence of poly(N-isopropylacrylamide) are resistive to both the drugs which is indicative of diffusional barrier of the spheroids. The authors conclude that Hepa/8F5 cells show enhanced functionality in cryogel based spheroid culture platform which can be successfully used for high throughput screening of hepatic toxicity in vitro.

Liver Cancer: Current and Future Trends Using Biomaterials

“…Spheroids are artefacts obtained in cell culture leading to compact cellular microspheres with a broad size range (20-1000 µm) which have become largely used in cancer research [142,155]. In order to improve the reliability of the results obtained with the spheroid model, many 3D cultures of liver tumor cells are availing themselves of hydrogels based on collagen, alginate or gelatin, containing molecules or growth factors immobilized in the structure via different chemical processes, even though also synthetic hydrogels are recently gaining attention for their peculiar properties, such as stimuli responsiveness [143,[156][157][158][159][160][161]. Within these hydrogel structures, spheroidal masses or cell aggregates are formed under non-adherence conditions, in which the ECM-cell interactions are very narrow and grant for a better communication of intracellular transduction signals.…”

“…There are several works reporting on hepatic cells that better express their function in spheroid culture, and different synthetic hydrogels have been used to better control shape, size and formation of spheroids due to their stimuli-responsiveness to specific external triggers, therefore reacting to dynamic microenvironments [159,160]. For example, a redox responsive hydrogel based on PEG allowed increased liver-related functionalities, in terms of albumin and urea synthesis, in spheroids of the hepatoma cell line HepG2 [159].…”

“…Cell recovery was also possible thanks to the polymer biodegradation under mild reducing conditions exerted by cysteine ( Figure 11). Another study has reported on the use of a temperature responsive hydrogel based on poly(Nisopropylacrylamide) (pNIPAAm)-co-gelatin for spheroid formation, in which the hepatoma cell line Hepa/8F5 derived from a genetically engineered mouse model showed higher function with respect to the 2D cell counterpart, according to albumin synthesis and other metabolic activities [160]. In addition, Hepa/8F5 cell spheroids grown in presence of pNIPAAm after doxycycline induction become drug-resistant, not only to tamoxifen but also to acetaminophen.…”

A minimally‐invasive cryogel based approach for the development of human ectopic liver in a mouse model

“…The cell pellet was then resuspended in DMEM containing Poly-NIPAAm (PNIPAAm, 0.05% w/v). PNIPAAm was synthesized by free radical polymerization using NIPAAm as described elsewhere (Shakya, Sharma, & Kumar, 2010).…”

“…Scaffolds prepared using various polymers can be used to support cells and also facilitate its integration with the host tissue (Jain et al, ; Mazza et al, ). Polymeric hydrogels and cryogel scaffolds with structures similar to native soft tissue can be fabricated and used as 3D scaffolds for tissue engineering applications (Gupta et al, ; Kumari & Kumar, ; Sarkar, Kumari, Tonello, Kamihira, & Kumar, ; Van Vlierberghe, Dubruel, & Schacht, ). Previously, we have synthesized the poly( N ‐isopropylacrylamide) (PNIPAAm) incorporated poly(ethylene glycol)‐alginate‐gelatin (PAG) cryogel which was employed in in vitro hepatic tissue development (Kumari, Karande, & Kumar, ).…”