Semi-Automated Phenotypic Analysis of Functional 3D Spheroid Cell Cultures.

We present a protocol that describes the properties and advantages of using a standalone clinostat incubator for growing, treating, and monitoring 3D cell cultures. The clinostat mimics an environment where cells can assemble as highly reproducible spheroids with low shear forces and active nutrient diffusion. We demonstrate that both cancer and non-cancer hepatocytes (HepG2/C3A and THLE-3 cell lines) require 3 weeks of growth prior to achieving functionalities comparable to liver cells.

Mapping Proteome and Lipidome Changes in Early-Onset Non-Alcoholic Fatty Liver Disease Using Hepatic 3D Spheroids.

Non-alcoholic fatty liver disease affects one-fourth of the world's population. Central to the disease progression is lipid accumulation in the liver, followed by inflammation, fibrosis and cirrhosis. The underlying mechanism behind the early stages of the disease is poorly understood.

Method to Disassemble Spheroids into Core and Rim for Downstream Applications Such as Flow Cytometry, Comet Assay, Transcriptomics, Proteomics, and Lipidomics.

Cells cultured in a monolayer have been a central tool in molecular and cell biology, toxicology, biochemistry, and so on. Therefore, most methods for adherent cells in cell biology are tailored to this format of cell culturing. Limitations and disadvantages of monolayer cultures, however, have resulted in the ongoing development of advanced cell culturing techniques.

Clinostat 3D Cell Culture: Protocols for the Preparation and Functional Analysis of Highly Reproducible, Large, Uniform Spheroids and Organoids.

Growing cells as 3D structures need not be difficult. Often, it is not necessary to change cell type, additives or growth media used. All that needs to be changed is the geometry: cells (whether primary, induced pluripotent, transformed or immortal) simply have to be grown in conditions that promote cell-cell adhesion while allowing gas, nutrient, signal, and metabolite exchange.

Hepatocellular carcinoma (HepG2/C3A) cell-based 3D model for genotoxicity testing of chemicals.

The major weakness of the current in vitro genotoxicity test systems is the inability of the indicator cells to express metabolic enzymes needed for the activation and detoxification of genotoxic compounds, which consequently can lead to misleading results. Thus, there is a significant emphasis on developing hepatic cell models, including advanced in vitro three-dimensional (3D) cell-based systems, which better imitate in vivo cell behaviour and offer more accurate and predictive data for human exposures. In this study, we developed an approach for genotoxicity testing with 21-day old spheroids formed from human hepatocellular carcinoma cells (HepG2/C3A) using the dynamic clinostat bioreactor system (CelVivo BAM/bioreactor) under controlled conditions.