Browsing by Subject "3D cell model"

In vitro liver cell models are important systems to study for example hepatotoxicity, which is one of the major causes for safety-related failures of drug candidates. 2D cell culture-based tests for compound screening are standard procedures in drug discovery, but reliable data for in vivo studies is hard to obtain because cells in a monolayer are in unnatural microenvironment. In turn, cells in 3D culture systems have more natural interactions with other cells and extracellular matrix, and their responses to drugs resemble more in vivo responses. In drug discovery and development, automation of the cell culture processes and compound screening saves time and costs, and improves the consistency and sterility of the procedures. As 3D cell culture systems are becoming more compatible with automation, they are also more promising to be used in drug discovery and development. The aim of the study was to develop and optimize automated processes for preparing 3D cell cultures into 96-well plates. HepG2, a human liver cancer cell line, cultures in nanofibrillar cellulose were prepared into well plates manually or by using automated liquid handling system. To our knowledge, this was the first time that automated processes for cell seeding into NFC were used to prepare 3D cell cultures. Cell seeding steps that could be automated were identified and optimized based on visual analysis of the wells and viability of the cells after seeding. After optimization, manual and automated processes were compared by studying cell viability, morphology and functionality. Alamar blue assay, Live/Dead assay and fluorescence-activated cell sorting were used to study cell viability, and F-actin staining, differential interference contrast microscopy and light microscopy were used to investigate cell morphology. Cell functionality was analyzed by studying albumin secretion. Cells seeded by using automation secreted normal amounts of liver-specific albumin. Cells maintained viability, morphology and functionality for four days after seeding although the results of viability varied. Alamar blue assays showed decreased development of viability although viability of manually seeded cells increased, but in other experiments the results from cultures seeded manually or by using automation were more similar. For example, lower viscosity of nanofibrillar cellulose and longer waiting time of cells at room temperature before automated processes are possible explanations for differences, as well as the natural variability in cell studies. In the future, automated high-throughput screening of compounds could be performed in 3D cell cultures prepared by using automation. That would save time and costs, and increase the correlation between in vitro and in vivo studies.