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12 | JULY 2016 | GENengnews.com | Genetic Engineering & Biotechnology News New Dimensions in 3D Cell Culture See 3D Cell Culture on page 14 technologies for high-content phenotypic screening and vali- dation. Applications discussed at the meeting ranged from drug design to safety testing, and from cancer therapeutics to regenerative medicine. Bioprinting 3D Liver Tissue For many years, scientists have struggled to capture the form and replicate the function of native tissues. Although native tissues possess subtleties that still elude 3D culture sys- tems, scientists are making progress. For example, scientists at Organovo have utilized bioprinting technology to create a new 3D model of human liver tissue and move beyond the limitations of animal models and 2D cell culture. "Although animal models help determine the acute, high- dose toxicity of a drug being evaluated, the problem is the un- predicted toxicity that is not detected in animals," commented Sharon Presnell, Ph.D., Oranovo's CSO. "Another problem is that conventional 2D cell culture models do not accurately refect the complex microenvironment of liver tissue." The company's exVive3D™ human liver tissue model is constructed from three basic cellular elements: primary hepa- tocytes, primary endothelial cells, and primary stellate cells. "Once a tissue design is established and a protocol is put in place, the multicellular 'bio-ink' building blocks are then dispensed from a bioprinter, using a layer-by-layer approach that is scaled for the target output," Dr. Presnell said. "The automated bioprinting process allows spatial control such that tissue-specifc patterns or compartments can be quickly produced that mimic key aspects of in vivo native tissues." It takes about 30 minutes to bioprint the beginnings of tiny livers into a 24-well plate. These mature into liver-like tissue in about 60 hours and can be maintained for at least six weeks. "They maintain a tissue-like density," asserted Dr. Presnell. "They have highly organized cellular features, such as intercellular tight junctions and microvascular networks." To examine drug-induced liver injury, Organovo em- ployed its model while using valproic acid (VPA), a com- pound known to induce liver injury in humans. The com- pany found that tissues treated with VPA daily for 14 days showed clear evidence of dose-dependent oxidative stress and tissue death as assessed by glutathione and adenosine triphosphate assays, respectively, and clinically relevant his- tological changes including the deposition of fat. Organovo concluded that its exVive3D human liver tissues effectively modeled multiple modes of VPA-induced liver injury. Organovo reports that it is working on additional models. "We expect," Dr. Presnell revealed, "that our kidney proxi- mal tubule model will be available the third quarter of 2016." Constructing Human Mini-Brains With the increasing incidence of neurodegenerative dis- orders such as Alzheimer's disease, multiple sclerosis, and Parkinson's disease, researchers are testing 3D cellular mod- els that could be superior to animal studies because the cells are derived from humans instead of rodents. Researchers at Johns Hopkins Bloomberg School of Public Health created "mini-brain" organoids composed of neurons and glia cells that closely mimic the brain's cellular composition. "We used induced pluripotent stem cells (iPSCs) derived from the skin of healthy adults," explained Lena Smirnova, Ph.D. "Then we genetically programmed them to an embry- onic stem cell–like state. "These cells are then grown over a period of eight weeks into very small mini-brains of about 350 microns in diameter consisting of different types of neurons as well as astrocytes and oligodendrocytes (to serve as supports). Oligodendro- cytes produce myelin that helps insulate the axons of the neu- rons to allow for rapid communication." As evidence that the cells were recapitulating real brain tis- sue, the Johns Hopkins team monitored electrophysiological activity with electrodes. The team, which used a setup similar to that used by an electroencephalogram, found spontaneous electrical activity. "Obviously, the system could be useful in drug develop- ment," said Dr. Smirnova. In support of this contention, she referenced the system's reproducibility. "Hundreds to thousands of exact copies of the organoids can be derived in each batch providing a highly standardized model. However, there are many other applications including studying central nervous system mechanisms, neurotoxicity reactions, and brain development." Further, mini-brains can be developed from patients as well as from healthy individuals. "We can generate a mini- brain organoid bank by reprogramming fbroblasts from the skin of patients with different neurological diseases," assert- ed Dr. Smirnova. The leader of the Johns Hopkins team, Thomas Hartung, M.D., Ph.D., is applying for a patent. Besides serving as a professor of evidence-based toxicology at the Bloomberg school, Dr. Hartung is a cofounder of Organome, which aims to produce mini-brains for distribution. Optimizing the 3D Environment Investigators setting up a 3D cell culture system have many decisions to make as to which culture conditions pro- vide the most accurate biology for their particular cell type. "One of the frequent questions investigators ask is what ma- trix and coating concentration is optimal for their cell type," noted Marshall Kosovsky, Ph.D., global scientifc support manager, Corning Life Sciences. "The frst advice I can give them is to do a thorough literature search regarding the biol- ogy of their cell type." "They need to understand the key requirements for cell behavior—including interactions at the level of extracellular DRUG DISCOVERY Continued from page 1 Human neural stem cell–derived neurospheres generated in the 96-well Corning Spheroid Microplate (A) exhibit multipotency and diferentiate into neurons (B), oligodendrocytes (C), and astrocytes (not shown).