Cell Culture
chemical cell-based assays currently in use were designed with monolayers or single cell suspen- sion in mind. In 3D, many of the cells are not directly exposed to the cell culture medium and therefore do not or only slowly contribute to the detection of soluble factors. Tissue diffusion becomes an important factor not only for nutri- ents, oxygen and cellular products, but also for therapeutic antibodies or staining agents. For ter- minal endpoints, such as nucleic acid and protein extractions or fixing the tissues for immunola- belling, the stability of the 3D tissue versus simple 2D monolayers must be taken into consideration when dissociating the material. Achieving a good and complete lysis of in vitro 3D tissues often requires the harsher conditions required for their in vivo counterparts. This becomes especially challenging when single cell integrity needs to be preserved. Thus, single-cell isolations from tis- sues, regardless if in vivo or in vitro, necessitates specialised protocols. As the community of single- cell analytics users is growing rapidly, there is also an increasing number of protocols to perform such isolations. Genetically modifying cells, transiently or stably,
works most efficiently when applied to single cells. For 3D tissue models composed of adult cells, transfection or transduction is usually performed upstream (pre-aggregation) of tissue generation. Once aggregated, formed and matured, outermost cell layers or vascularised regions are usually still accessible to transduction or transfection methods. Accessing cells buried deeper within the tissue is more difficult. This leads to a significant bias on which cells are being modified. With organoid models, having genetically-stable modified precur- sor cells enables the growth of identically-modified progenitor cells, albeit epigenetic silencing can still be a challenge for transgenes containing non-con- stitutive promoters.
Technologies for fully unlocking 3D Now, let us consider a hypothetical gold standard: the ability to extract multi-omics information from a single 3D tissue cell with high spatial and tempo- ral resolution. This would enable a researcher to understand, at the single cell level, what is happen- ing within the tissue over time through multiple omics endpoints. While 3D cell-based technologies are evolving quickly, we are not quite there as all existing solutions have specific constraints. Do not be discouraged by that – current technologies can provide a wealth of meaningful data, so much so the bottleneck is usually the analysis and interpre- tation, rather than the amount of data.
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Transcriptomics are routinely performed with 3D tissues. Ensuring a good lysis of the tissue is impor- tant, and methods or kits used for lysing tissues from biopsies are often best suited as a starting point. When working with smaller microtissues as, for example spheroids encompassing a few thou- sand cells, it is also possible to lyse these tissues directly in the cell culture well, using the lysate for downstream NGS library generation, bypassing classical RNA extraction.
3D volume imaging. Just as pathologists use thin tissue sections as clinical endpoints, the same is routinely used for in vitro 3D models. Classical histochemical stains, even though they can involve some processing, circumvent the challenge of tissue thickness and provide important information about the structure and composition of the tissue at a specific point in time. The trade-off (and an inherent limitation) is that it looks only at slices of the whole tissue model. If the tissues exhibit signif- icant heterogeneity, then laborious acquisition, staining and analysis of many slices will be required. Whole mount stainings, facilitated by the advent of optimised clearing reagents, offer signif- icantly more flexibility and information, with the disadvantage of requiring upfront optimisations (clearing, antibody penetration in 3D) and more advanced microscopic technologies and solutions for data storage, processing and analysis. Especially for larger 3D models (>100um in diam- eter), imaging becomes a major challenge due to light scattering of the tissue. Advanced imaging technologies such as two-photon microscopy can solve this problem but often go with the cost of reduced throughput.
4D live imaging. While following cellular events in 3D over time is conceptually the top-tier endpoint, current challenges, limitations and required investments do not permit routine use of this tech- nology. Lightsheet microscopy, with its fast acqui- sition and short exposure to strong light, is emerg- ing as an approach to track 3D tissues or even small organisms over time. For doing so, one needs to have cells labelled with fluorescent mark- ers compatible with little or no impact on their viability over long periods of time. These are usu- ally fluorescent proteins expressed by transduced cells within the tissue, or live chemical stains, per- mitting to distinguish single cells or even organelles. Current systems require laborious sample preparation and can only handle a few specimens in parallel due to tight space constrains to ensure high optical image quality.
Drug Discovery World Winter 2018/19
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