Optimization of Albumin Secretion and Metabolic Activity of Cytochrome P450 1A1 of Human Hepatoblastoma HepG2 Cells in Multicellular Spheroids by Controlling Spheroid Size

Abstract

Key words albumin; CYP1A1; human hepatoblastoma HepG2 cell; size control; spheroid Three-dimensional multicellular spheroids possess greater in vivo tissue-like morphology and function than two-dimensional monolayered cells. Therefore, multicellular spheroids are considered useful in vitro systems for drug screening, including tests of drug metabolism and toxicity, [1][2][3][4][5][6] and for studying tumor metastasis. 7,8) Furthermore, such spheroids have attracted much attention for application in cell-based therapy [9][10][11] and as a building block for the construction of large tissues. 12,13) In a previous study, we demonstrated that spheroid formation greatly prolonged the survival of insulinsecreting NIT-1 cells after transplantation in mice. 9)Spheroid size is a key parameter to determine the functions and properties of spheroids. It is reported that oxygen concentration is decreased in the core of the spheroids of human hepatoblastoma HepG2 cells, hepatocytes, and tumor cells. [14][15][16][17] This affects the expression of various molecules, including hypoxia inducible factor (HIF)-1. In addition, paracellular or transcellular transport is required in order for the inner cells to take up extracellular materials and to release secretory products into the culture media. Despite their importance, details regarding the relationship between spheroid size and cellular functions remain to be elucidated. In particular, few studies have investigated the effect of spheroid sizes over 300 µm on their functions and properties.Investigating the effects of spheroid size requires the preparation of size-controlled, multicellular spheroids. Several techniques for spheroid formation have been reported, including the spinner flask, hanging drop, and liquid overlay methods. [18][19][20] However, a common problem with these techniques is the difficulty in controlling spheroid size. In contrast, microfabrication is suitable for the development of sizecontrolled microwells with narrow size distribution. 10,21,22) In a previous study, we demonstrated that polydimethylsiloxane (PDMS)-based microwells constructed using a microfabrication technique could be used to obtain spheroids with a very narrow size distribution. 23) We also found that coating the microwells with poly(N-isopropylacrylamide) (PNIPAAm), a thermoresponsive polymer, increased the quality of the spheroids, since the coating prevented the cells from adhering to the PDMS-based microwells. 23)In the present study, we developed PDMS-based microwells of different sizes in order to prepare variably sized multicellular spheroids. HepG2 cells were used for the study as they represent one of the most extensively used cell lines to evaluate drug metabolism and toxicity. 3,24,25) Production of various proteins is an index of liver function. Albumin is the most abundant serum protein produced by hepatocytes. 26)Another important liver function is the metabolism of various compounds, including drugs. CYP enzymes are the major enzymes involved in drug metabolism. CYP1A1 is ...

Development of 3D Hepatic Constructs Within Polysaccharide-Based Scaffolds with Tunable Properties

“…These albumin concentrations are in agreement with several others studies using HepG2 cells [48][49][50]. CYP3A4 activity (Figure 5b) exhibited a profile inversely proportional to albumin secretion, also in agreement with the literature [51][52][53]. In line with these data, we also observed cytoplasmic expression of albumin in HepG2 cells within both types of scaffolds, and nuclear expression of HNF-4α in most cells at day seven (Figure 5c).…”

“…We found a low CYP3A4 activity of HepG2 constructs in our scaffolds similar to the limited activity observed for small size cell spheroids of less than 200 µm [66]. For larger HepG2 spheroids, hypoxic areas in HepG2 3D constructs increases in the core and subsequently increases CYP3A4 activity [53]. In our study, no hypoxia or cell death was observed in 3D constructs which could explain the reduced activity of CYP3A4 overtime.…”

CXCL5 as an autocrine or paracrine cytokine is associated with proliferation and migration of hepatoblastoma HepG2 cells

“…HepG2 was originally thought to be a hepatocellular carcinoma cell line and was utilized to investigate hepatocellular carcinoma. However, previous research has shown that HepG2 is a HB-derived cell line (22), which has a crucial role in studying the underlying progression and mechanism of HB (23,24). In the present study, it was confirmed that CXCL5 and its receptor CXCR2 are expressed in HepG2 cells.…”

Effective Transplantation of 2D and 3D Cultured Hepatocyte Spheroids Confirmed by Quantum Dot Imaging

“…The expression levels of AFP and CYP3A4 in 50 µm spheroids were greater than those of the two larger spheroids (Figure d). Consistent with this result, it has been reported that the function of HepG2 cells is better for smaller spheroids than for larger spheroids . In spheroid culture, the upper limit of the viable size is 100–150 µm in diameter due to oxygen depletion .…”