Approximating in vivo physiology is not only a matter of allowing cells to grow and develop in three dimensions; it also requires cell lines that reliably act like differentiated tissues.
Creation and testing of improved cell lines were addressed by Christophe Lipps, a Ph.D. student at the Helmholtz Centre for Infection Research (HZI), and Joris Braspenning, Ph.D., a managing director of Medicyte.
Tobias May, Ph.D., speaking for the HZI spinoff company InSCREENeX, summarized the limitations of established cell-culture models. Primary cells taken directly from humans or animals exhibit realistic physiology but don’t proliferate well in culture, whereas immortal cell lines do proliferate well but don’t really reflect what goes on in vivo.
“We wanted to combine the advantages of both systems,” said Dr. May. So the group surveyed the literature, compiled a list of 33 genes known to be involved in proliferation, put these genes into a library, transduced the genes into primary cells, and let the best cells emerge via natural selection.
“The primary cells die off, and the cells that received advantageous gene combinations proliferate and become immortalized,” explained Dr. May. “And these immortalized cells are then further characterized for their phenotype.”
In the next steps of this work, HZI and InSCREENeX will increase the number of available cell lines to cover various species (mice, rats, pigs, cows, humans) and cell types (endothelial cells, fibroblasts, lung epithelial cells, hepatocytes, chondrocytes).
A similar approach has been taken by Medicyte, which tranduces proliferation-stimulating genes into primary human hepatocytes to create longer-living cells dubbed upcyte® hepatocytes. These cells are then grown in 3D cultures by seeding them onto alvetex scaffolds. Recent work has focused on the 3D cultures’ drug-metabolizing activity and application to cytotoxicity and drug interaction assays.
Compared to 2D monolayer cultures, the 3D cultures seem to demonstrate enhanced CYP1A2, CYP2B6, and CYP3A4 enzyme activities, both at baseline levels and when induced by drugs such as omeprazole, phenobarbitone, and rifampin, respectively.
Nicola Hewitt, Ph.D., a scientific consultant at Medicyte, is enthusiastic about the new opportunities made possible by 3D hepatocyte cultures.
“We are investigating the use of upcyte hepatocytes in bioartificial liver (BAL) models as part of two European projects (HemiBio and ReLiver),” said Dr. Hewitt. “In these projects, other ‘upcyted’ cells such as endothelial and stellate cells will be co-cultured with hepatocytes to form a much more in vivo-like culture structure. This gives us the possibility to extend this work to therapeutic areas.”
In addition, said Dr. Hewitt, “We are also looking into whether the 3D culture of hepatocytes—with and without co-culturing with other cell types—can result in a model that is more predictive than previous hepatotoxicity prediction models, which may be based on the less relevant hepatoblastoma cell line, HepG2. If we can produce a model that can pick up the hepatotoxicity of drugs that have been withdrawn from the market due to liver toxicity, then this would be a powerful tool to prevent such compounds reaching the market.”
As 3D cell cultures become more commonplace, there will be a corresponding need to verify the performance of assays originally developed for 2D cultures, said Terry Riss, Ph.D., a senior product specialist at Promega, who spoke at the Institute for Scientific Communications’ recent “Early Toxicity Screening” conference.
There are several reasons why an assay protocol might need tweaking when applied to 3D cultures. One is the fact that cells in the middle of large 3D structures may not be disrupted as easily as cells in 2D monolayers. To ensure complete disruption of all cells, researchers may need to use “higher concentrations or different types of detergents—as long as they are compatible with the assay chemistry,” noted Dr. Riss. Physical disruption by pipetting is another option, he said.
Promega offers numerous commercial kits for detecting early biochemical markers of various mechanisms of cytotoxicity. Many of the kits detect activation of pathways that respond to specific stresses such as DNA damage, heat shock, hypoxia, oxidative stress, and so forth.
Promega is currently checking these kits to verify their utility with 3D culture samples. In cases where it is hard to lyse the cells, Promega is developing new assays for specific use with 3D cultures, said Dr. Riss. He hopes to validate many of these assays with 3D model systems within the next year.