Type of endothelial cells affects HepaRG cell acetaminophen metabolism in both 2D and 3D porous scaffold cultures

Abstract

Recent advances in developing in vitro tissue models show that function of hepatocytes is altered in when cultured in 3D configuration and co‐culturing with various non‐parenchymal cells. However, tissue source for such non‐parenchymal cells on viability and metabolic products of hepatocytes have not been explored. In this study, we evaluated the effect of 2D and 3D cultures either with HepaRG cells alone or in combination with liver sinusoidal endothelial cells (LSECs) and human umbilical vein ECs (HUVECs). For 3D cultures, we used chitosan‐gelatin porous structures formed by freeze‐drying. We cultured cells for 8 days before challenging with 1 mm acetaminophen (APAP) and assessed APAP, APAP‐sulfate and APAP‐glucuronide for 24 hours at 6‐hour time intervals using high‐performance liquid chromatography. We used multiple methods (phase contrast, confocal and scanning electron microscopy and histology via hematoxylin and eosin staining) to ensure cell distribution. We also measured total protein content and albumin secretion and viability. HUVEC 3D co‐cultures showed the lowest HepaRG cell viability, while both 2D and 3D LSEC co‐cultures had highest HepaRG cell viability. In addition, 3D cultures had significantly higher EC viability relative to 2D cultures. Further, HUVEC co‐cultures showed reduced total protein content and albumin expression as early as day 4. However, urea production on a total protein content basis did not change. In addition, LSEC 3D co‐cultures had the highest APAP conversion with reduced APAP‐sulfate and APAP‐glucuronide formation. CYP3A4 was higher in co‐culture with HUVEC for 2D and 3D cultures. In conclusion, HepaRG cells with EC co‐cultures demonstrated sensitivity to the EC line used.

Liver Tissue Engineering as an Emerging Alternative for Liver Disease Treatment

“…93 A chitosan-gelatin porous structure with a pore size of 84-150 mm has been a suitable environment for cell infiltration. 92 Another method for increasing the porosity, which in combination with other methods such as the microsyringe technique can provide smaller pores (10-30 mm), is particulate leaching with different particles such as glucose grains, which can be washed with deionized water. 109 Therefore, tailoring the pore size and porosity is an essential consideration in manufacturing scaffolds.…”

“…However, these methods had a significant role in the progress of the LTE field. 30,135 There are many methods used for TE approaches, including solvent-casting, [137][138][139][140] freeze-drying, 92,95,138,[141][142][143] electrospinning, [144][145][146][147] phase separation, 103,135 and gas foaming. 77 In this study, we represent some of these techniques that have been utilized in LTE.…”

“…It has been shown that embryonic cells survived more on the 3D porous scaffold. 92 Electrospinning is a well-known method for producing a nanostructure/microstructure with a high special surface. 144,148 A PLA-gelatin scaffold was made using the electrospinning method by Hoveizi et al and the formation of definitive endoderm (DE) from human iPSCs, needed for developing visceral organs such as lung, liver, and pancreas, was studied.…”

Regulated differentiation of stem cells into an artificial 3D liver as a transplantable source

“…76 On this basis, the researchers optimized the aforementioned scaffold and improved it through laminin incorporation; they found that the cells generated on this scaffold were strikingly similar to a standard hepatocyte cell line in terms of the expression of hepatocyte markers. 77 Moreover, gelatin was also employed to fabricate microcarriers with chitosan for hepatocyte culture, 78 and improved the performance of hepatocytes for as long as 2 months. 79 Chitosan is a linear polysaccharide which has good biocompatibility and wide applications in biomedical fields.…”

“…81 Rajendran et al 82 established a chitosan nanofiber scaffold via an electrospinning technique combined with a coculture system that consisted of hepatocytes and fibroblasts, and thus developed a new 3D liver model for the maintenance of long-term liver functions. To obtain better biocompatibility, chitosan can be further modified with other macromolecules, such as collagen, 83 gelatin, 78 alginate, 84 and heparin. 85 For example, Fan et al 86 proved that chitosan/galactosylated hyaluronic acid scaffolds were suitable for improving hepatocytes adhesion and maintaining liver function in a previous work.…”