Cell Culture

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20 Lee, HK et al (2016). Three dimensional human neuro- spheroid model of Alzheimer's disease based on differentiated induced pluripotent stem cells. PLoS ONE 11:e0163072. 21 Raja, WK et al. Self- organizing 3D human neural tissue derived from induced pluripotent stem cells recapitulate alzheimer's disease phenotypes. PLoS ONE 11, e0161969 (2016). 22 Lee, J, Kotliarova, S, Kotliarov, Y, Li, A, Su, Q, Donin, NM, Pastorino, S, Purow, BW, Christopher, N, Zhang, W, et al (2006). Tumor stem cells derived from glioblastomas cultured in bFGF and EGF more closely mirror the phenotype and genotype of primary tumors than do serum-cultured cell lines. Cancer Cell 9, 391-403. 23 Linkous, A et al. Modeling Patient-Derived Glioblastoma with Cerebral Organoids. Cell Rep. 2019 Mar 19;26(12): 3203-3211. 24 Hay, DC, O’Farrelly, C. Designer human tissue: coming to a lab near you. Philos Trans R Soc Lond B Biol Sci. 2018;373(1750):20170212. 25 Zhang, YS, Duchamp, M, Oklu, R, Ellisen, LW, Langer, R, Khademhosseini, A. Bioprinting the Cancer Microenvironment. ACS Biomater Sci Eng. 2016;2(10):1710-1721. 26 Jo, J, Xiao, Y, Sun, AX et al. Midbrain-Like Organoids from Human Pluripotent Stem Cells Contain Functional Dopaminergic and Neuromelanin-Producing Neurons. Cell 2016, 19, 248-257. 27 Pasca, AM, Sloan, SA, Clarke, LE et al. Functional Cortical Neurons and Astrocytes from Human Pluripotent Stem Cells in 3D Culture. Nat. Methods 2015, 12, 671-678. 28 Mansour, AA, Gonçalves, JT, Bloyd, CW et al. An In Vivo Model of Functional and Vascularized Human Brain Organoids. Nat. Biotechnol. 2018, 36, 432-441.

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Figure 3: Flow cytometry analysis. Similar percentage of cells can be stained by NSCs marker (Nestin) in 3D spheroid than in 2D adherent culture. Higher percentage of cells can be stained by neuronal marker (MAP2) and glial markers (GFAP and A2B5) in 3D spheroid than in 2D adherent culture. Source: Corning Life Sciences

Advances in neurological research Nonetheless, the limitations of 2D cell culture are most clear in neurological research. Indeed, 2D cultures lack the complexity of the brain in terms of function and development18. While neural stem cells can differentiate success-

fully in both 2D and 3D cell culture formats, dif- ferentiated neural stem cell spheroids have been shown to present similar or stronger neuronal and glial signals than those grown in 2D cultures (Figure 2 and Figure 3). Progress has also been made in generating

HiPSC-derived neuronal organoids that display a level of self-organisation that is not recapitulated in 2D cell cultures, suggesting a promising advance in understanding neurodegenerative diseases, such as Parkinson’s and Alzheimer’s disease19-21. Figure 4 illustrates the classification of brain models, from less complex 2D neural aggregates to highly com- plex in vivo animal models. Three-dimensional cultures of HiPSC-derived

neuronal cells have been generated in large-scale arrays and imaging technologies used to measure neurite outgrowth. HiPSC-derived neurons from patients with familial Alzheimer’s disease mutations can be grown in 3D cultures for at least eight weeks. It was shown that diseased neurons expressed sever- al key proteins involved in Alzheimer’s disease neu- rodegeneration, including A and phosphorylated tau protein. These data may provide a screening for- mat to identify compounds acting to reduce the pro- duction of these disease-causing biomarkers20.

Another recent study monitored the expression

of A in 3D cell cultures of HiPSC-derived neurons from five different patients with familial Alzheimer’s disease. They measured the action of known inhibitors of - and -secretase (enzymes responsible for generating neuronal A), and cul- tured the neurons for up to nine weeks to generate more mature neurons for drug testing. The addi- tion of - and -secretase inhibitors lowered A levels in the neurons cultured in 3D, but their effi- cacy was markedly less than that in 2D cultures of the same cells – likely due in part to reduced bioavailability. It was also shown that neurons from a patient in which the -secretase inhibitors were ineffective lacked key proteins required for enzyme inhibition. These data, and other reports, suggest that drug

efficacy is affected by the variability in the cell genome and proteome of different patients. They also reiterate the advantage of 3D cell culture ver- sus 2D cell culture as a predictor of human drug efficacy in vivo. This is particularly relevant given that -secretase inhibitors have now all failed to show efficacy in late-stage clinical trials21. This also illustrates the importance of screening HiPSC- derived neurons from multiple patients to estimate variations in clinical efficacy.

Patient-derived glioblastoma model23 Glioblastoma multiforme (GBM) is the most lethal, as well as the most common, type of prima- ry brain tumor in adults. The research and drug

Drug Discovery World Spring 2019

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