Cell Culture

development work focused on GBM has been lim- ited by pre-clinical models that relay on cell autonomous in vitro models, or in vivo models that poorly simulate human disease. Tumours are complex systems that are greatly influenced by their host. While several 3D models of cancers have been developed, all are fundamentally limited and only address some aspects of GBM. Patient-derived glioma stem cells (GSCs) are the

accepted standard for studying GBM biology22. This subpopulation of cells is the most phenotypi- cally-relevant to the parental tumour, and is imper- ative for tumour initiation, maintenance and inva- sion. In addition, GSCs demonstrate an increased resistance to cytotoxic drugs and ionising radiation. GSCs are not cell autonomous, however, but are

instead greatly influenced by tumour host-cell interactions. Efforts to generate 2D co-cultures of GSCs with neurons, glia and other brain-specific cell types often result in disorganised cellular struc- tures that are not representative of the human brain. Furthermore, while tumour organoids allow GSCs to grow within a 3D extracellular matrix, they do not address the criticality of the tumour- host tissue microenvironment interactions. A pow- erful tool has recently been developed for mod- elling human GBM, using human embryonic stem cell (hESC)- or iPSC-derived cerebral organoids and patient-derived GSCs23. Linkous et al estab- lished GLICO (cerebral organoid glioma) models to retro-engineer patient-specific GBMs, resulting in tumours that closely mimic the patient’s original brain tumour.

Using Corning®Matrigel® as a 3D extracellular

matrix, hESCs were differentiated into fully- formed cerebral organoids. GFP-labelled GSCs were then co-cultured with individual cerebral organoids for 24 hours. The tumour take rate was 100% for all GSC lines, and considerable tumour growth was detected one week after co-culture of the tumour-infiltrated organoids. Additionally, the tumour-bearing organoids recapitulated the tumour morphology found in human patient GBMs. The interrogation of the growth patterns of six patient-derived GSC lines in the model revealed dramatically differentiated patterns and degrees of tumour cell invasion and proliferation between the different lines. This differentiation reflects the het- erogeneity of the invasive phenotypes clinically observed in patients. The GLICO model addresses several limitations

presented by prior models, allowing researchers to study patient-specific GBMs within a microenvi- ronment like that of a human brain. Because it is grown ex vivo, the model can be used for experi- mental drug treatment. In addition, the model is scalable for high throughput drug screening, open- ing the possibility for successfully screening tumour cells for clinically active drugs and other interventions.

Summary and outlook The culturing of human-derived tissues in 3D organoid systems presents a major advance in drug discovery and, specifically, the ability to successful- ly screen tumour cells in vitro for clinically active

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29 Medda, X, Mertens, L, Versweyveld, S et al. Development of a Scalable, High-Throughput-Compatible Assay to Detect Tau Aggregates Using iPSC-Derived Cortical Neurons Maintained in a Three-Dimensional Culture Format. J. Biomol. Screen. 2016, 21, 804-815. 30 Choi, SH, Kim, YH, Quinti, L et al. 3D Culture Models of Alzheimer’s Disease: A Road Map to a Cure-in-a-Dish. Mol. Neurodegener. 2016, 11, 75-90. 31 Jorfi, M, D’Avanzo, C,Tanzi, RE et al. Human Neurospheroid Arrays for In Vitro Studies of Alzheimer’s Disease. Sci. Rep. 2018, 8, 2450. 32 Zhong, X, Guiterrez, C Xue, T et al. Generation of Three-Dimensional Retinal Tissue with Functional Photoreceptors from Human iPSCs Nat. Commun. 2015, 5, 4047-4053. 33Tzatzalos, E, Abeliz, OJ, Shukla, P et al. Engineered Heart Tissues and Induced Pluripotent Stem Cells: Macro- and Microstructures for Disease Modeling, Drug Screening, and Translational Studies. Adv. Drug Deliv. Rev. 2016, 96, 234-244. 28.

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Figure 4:Classification of brain models. Classification of brain models, from monolayers to in vivo animal models. Source: Modified from Poli et al, ‘Experimental and Computational Methods for the Study of Cerebral Organoids: A Review’

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