Cell Culture
weeks for clearing and the process severely reduces tissue integrity. This approach is also not compati- ble with immunolabelling techniques. Use of highly lipophilic organic solvents are not compatible with plastic well plates. Hydrogel embedding techniques are slow, requiring days to weeks to clear even small tissue samples. This approach uses a complex processing technique that is not amenable to a well plate format and thus not amenable to medium and high throughput applications. We have collaborated with Visikol to develop an
organic solvent approach (Visikol® HISTO-M™) that replaces the water in the cytosol and makes the cytosol match the refractive index of lipids and proteins causing the spheroid to become transpar- ent. Untreated, each cell acts as a lens and scatters light through the spheroid because the proteins and lipids have a high refractive index while the cytosol has a low refractive index. The reagent rapidly ren- ders tissue transparent, dramatically improving both wide field imaging and confocal microscopy and visualisation of the spheroid interior. A second challenge identified via our survey is
the need for novel tools and technologies to sup- port development of spheroid and organoid cul- tures. To address this challenge, many new and highly innovative products are reaching the lab bench and screening laboratories and enabling crit- ical activities such as bulk spheroid production, sorting and dispensing. For example, microcavity vessels enable growth of more than 3,000 uniform- sized spheroids per T-25 flask. The spheroids are confined to <400µm and maintain their shape up to 30 days in culture. Additional advances are making use of fluorescence to sort and dispense spheroids. A final challenge that must be addressed to
enable widespread adoption of 3D culture systems is the need for greater expertise combined with more training and support in establishing and opti- mising 3D cell cultures. The number of publications citing use of 3D cell
culture systems in the past eight years has grown five-fold and provides a robust resource for the research and discovery communities. At the same time, it is incumbent on companies such as Corning to continue development of methods and technologies such as those for 3D spheroid cultures of human hepatocytes and offer expert technical and applications support. We also listen to the needs of customers as they progress towards adop- tion of 3D cell culture systems and have developed products and protocols based on their input including those for immune cell invasion, the blood brain barrier and intestinal organoids.
Drug Discovery World Winter 2018/19
In summary, remarkable advances have been
made in the use of 3D cell culture systems and offer important advantages across the drug dis- covery workflow. As remaining hurdles are addressed, more widespread adoption of 3D approaches will improve productivity and lead to fewer costly late-stage failures. Better disease models will become available, including those for oncology, neuroscience and cardiovascular dis- eases. With incorporation of HiPSCs, these mod- els will allow researchers to discern differences among patients and guide development of more personalised therapeutics. Further along the workflow, 3D systems will enable more effective compound screening and deliver better hits. Compound libraries originally screened using 2D systems will be rescreened to find active hits; approved drugs will be screened in 3D to poten- tially reposition or expand indications. Finally, 3D systems will allow more accurate assessments of toxicity and metabolic liabilities. The end result of incorporation of 3D systems across the discovery workflow will be compounds entering the clinic with a better prediction of efficacy and tolerance.
DDW
Dr Richard M. Eglen is Vice President and General Manager of Corning Life Sciences. Eglen joined Corning in 2011 with more than 35 years’ experi- ence in the life sciences industry. He has authored more than 325 publications, book chapters and patents, and serves on numerous industry, academ- ic advisory and journal editorial boards.
Dr Feng Li is a Senior Development Scientist of Corning Life Sciences and expert cell biologist with more than 12 years’ industry experience. In recent years, he has focused on spheroid models using pri- mary human hepatocytes and Corning 3D tech- nologies for applications such as liver toxicity test- ing and disease modelling.
Dr Anthony G. Frutos is the Business Technology Director for Corning Life Sciences. He has 19 US patents and is an author on more than 39 technical publications. He holds a Bachelor’s degree in chemistry from Brigham Young University and a PhD in analytical chemistry from the University of Wisconsin, Madison.
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References 1 Li, Feng et al. 3D Primary Human Hepatocytes (PHH) Spheroids Demonstrate Increased Sensitivity to Drug- Induced Liver Injury in Comparison to 2D PHH Monolayer Culture.
www.corning.com/catalog/cls/d ocuments/application- notes/CLS-AN-514.pdf. 2 Vorrink, Sabine et al. Prediction of Drug-Induced Hepatotoxicity Using Long- Term Stable Primary Hepatic 3D Spheroid Cultures in Chemically Defined Conditions. Toxicological Sciences 163,2 (2018): 655-665. 3 Tulloch, Nathaniel L et al. Growth of engineered human myocardium with mechanical loading and vascular coculture. Circulation research vol. 109,1 (2011): 47-59. 4Takayama, Kazuo et al. Prediction of interindividual differences in hepatic functions and drug sensitivity by using human iPS-derived hepatocytes. Proceedings of the National Academy of Sciences of the United States of America vol. 111,47 (2014): 16772-7. 5 Oleaga, Carlota et al. Multi- Organ toxicity demonstration in a functional human in vitro system composed of four organs” Scientific reports vol. 6 20030. 3 Feb. 2016, doi:10.1038/srep20030.
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