Morphological and Functional Analysis of Hepatocyte Spheroids Generated on Poly-HEMA-Treated Surfaces under the Influence of Fetal Calf Serum and Nonparenchymal Cells.

Ali Acikgöz,Augustinus Bader,Man-Gi Cho,Shibashish Giri

doi:10.3390/biom3010242

Abstract

Poly (2-hydroxyethyl methacrylate) (HEMA) has been used as a clinical material, in the form of a soft hydrogel, for various surgical procedures, including endovascular surgery of liver. It is a clear liquid compound and, as a soft, flexible, water-absorbing material, has been used to make soft contact lenses from small, concave, spinning molds. Primary rat hepatocyte spheroids were created on a poly-HEMA-coated surface with the intention of inducing hepatic tissue formation and improving liver functions. We investigated spheroid formation of primary adult rat hepatocyte cells and characterized hepatic-specific functions under the special influence of fetal calf serum (FCS) and nonparencymal cells (NPC) up to six days in different culture systems (e.g., hepatocytes + FCS, hepatocytes – FCS, NPC + FCS, NPC – FCS, co-culture + FCS, co-culture – FCS) in both the spheroid model and sandwich model. Immunohistologically, we detected gap junctions, Ito cell/Kupffer cells, sinusoidal endothelial cells and an extracellular matrix in the spheroid model. FCS has no positive effect in the sandwich model, but has a negative effect in the spheroid model on albumin production, and no influence in urea production in either model. We found more cell viability in smaller diameter spheroids than larger ones by using the apoptosis test. Furthermore, there is no positive influence of the serum or NPC on spheroid formation, suggesting that it may only depend on the physical condition of the culture system. Since the sandwich culture has been considered a “gold standard” in vitro culture model, the hepatocyte spheroids generated on the poly-HEMA-coated surface were compared with those in the sandwich model. Major liver-specific functions, such as albumin secretion and urea synthesis, were evaluated in both the spheroid and sandwich model. The synthesis performance in the spheroid compared to the sandwich culture increases approximately by a factor of 1.5. Disintegration of plasma membranes in both models was measured by lactate dehydrogenase (LDH) release in both models. Additionally, diazepam was used as a substrate in drug metabolism studies to characterize the differences in the biotransformation potential with metabolite profiles in both models. It showed that the diazepam metabolism activities in the spheroid model is about 10-fold lower than the sandwich model. The poly-HEMA-based hepatocyte spheroid is a promising new platform towards hepatic tissue engineering leading to in vitro hepatic tissue formation.

Highlights

In the past few decades, hepatic tissue engineering has focused on the improvement of normal hepatocyte cell culture models to develop an organ-specific multicellular cell culture model to restore the stability of the adult hepatocyte’s functions in vitro for pharmacological research and hepatocyte research, including bioartificial liver supports
Our findings reveal that fetal calf serum (FCS) has a negative influence on the mortality of hepatocytes in the spheroid model (Figure 1A and 1B), likely due tothe presence of FCS, which contained some substances that are probably not influenced for spheroid formation
Numerical values of probability smaller than 0.05 were considered as statistically significant. This establishing of hepatocyte spheroids generated on poly-HEMA-treated surfaces, with its capability of forming hetero-cell aggregates with other liver cells, will offer an efficient experimental avenue for biological research, drug screening and regenerative medicine

Summary

Introduction

In the past few decades, hepatic tissue engineering has focused on the improvement of normal hepatocyte cell culture models to develop an organ-specific multicellular cell culture model to restore the stability of the adult hepatocyte’s functions in vitro for pharmacological research and hepatocyte research, including bioartificial liver supports. The main disadvantage is that primary cells lose their state of metabolic function in the conventional monolayer due to the lack of a proper multicellular three-dimensional microenvironment like in vivo To overcome this situation, researchers have developed two widely accepted advanced cellular culture models, such as the sandwich and spheroid models, to restore the metabolic functions over extended periods of long-term cultures, with further stimulation by adding suitable growth factors and using advanced media. Researchers have developed two widely accepted advanced cellular culture models, such as the sandwich and spheroid models, to restore the metabolic functions over extended periods of long-term cultures, with further stimulation by adding suitable growth factors and using advanced media Both models have been widely used to study the vast range of basic and clinical research and provide a broad spectrum of liver-specific functions

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