Reprinted from: Publication of this reprint collection is supported by paid advertising SLAS Technology 27 (2022) 32–38 Contents lists available at ScienceDirect SLAS Technology journal homepage: www.elsevier.com/locate/slast Enabling high throughput drug discovery in 3D cell cultures through a novel bioprinting workflow Martin Engel a,∗ , Lisa Belfiore a , Behnaz Aghaei a , Margareta Sutija b,∗ a Inventia Life Science Operations Pty Ltd, Alexandria, NSW 2015, Australia bPerkinElmer Pty Ltd, Glen Waverley, VIC 3150, Australia a r t i c l e i n f o Keywords: AlphaLISA AlphaScreen Immunoassay cytokine biomarker ELISA kinase cell culture cell-based assay cancer small molecule HTS screening 3D cell culture high throughput screening a b s t r a c t Advanced three dimensional cell culture techniques have been adopted in many laboratories to better model in vivo tissue by recapitulating multi-cellular architecture and the presence of extracellular matrix features. We describe here a 3D cell culture platform in a small molecule screening workflow that uses traditional biomarker and intracellular kinase end point assay readouts. By combining the high throughput bioprinter RASTRUM with the high throughput screening assay AlphaLISA, we demonstrate the utility of the protocol in 3D synthetic hydrogel cultures with breast cancer (MDA-MB-231 and MCF-7) and fibroblast cells. To establish and validate the workflow, we treated the breast cancer cultures with doxorubicin, while fibroblast cultures were stimulated with the pro-inflammatory lipopolysaccharide. 3D and 2D MDA-MB-231 cultures were equally susceptible to doxorubicin treatment, while showing opposite ERK phosphorylation changes. Doxorubicin readily entered embedded MCF-7 spheroids and markedly reduced intracellular GSK3 phosphorylation. Furthermore, quantifying extracellular interleukin 6 levels showed a very similar activation profile for fibroblasts in 2D and 3D cultures, with 3D fibroblast networks being more resistant against the immune challenge. Through these validation experiments we demonstrate the full compatibility of the bioprinted 3D cell cultures with several widely-used 2D culture assays. The efficiency of the workflow, minimal culture handling, and applicability of traditional screening assays, demonstrates that advanced encapsulated 3D cell cultures can be used in 2D cell culture screening workflows, while providing a more holistic view on cell biology to increase the predictability to in vivo drug response. Introduction In the context of high throughput screening (HTS) for biological research, 2D cell cultures are the widespread model of choice for in vitro testing of novel compounds [1]. This is in large part due to the existence of countless highly optimised workflows for the generation and analysis of 2D cell culture models, making the use of 2D cell cultures practical and cost-effective in HTS [2]. However, growing evidence indicates that cells cultured in 2D do not sufficiently model the complex biology of in vivo tissues to reliably predict in vivo drug responses [3]. This fundamental limitation of 2D cell culture models arises primarily from lacking the 3D tissue cytoarchitecture and tissue microenvironment, and hence do not model the numerous intercellular interactions, proliferative behaviours and metabolic gradients characteristic of in vivo tissues [4]. Critically, these features of in vivo tissues have been demonstrated to influence cell behaviour and responses to drug treatments [5]. Therefore, utilising in vitro models that represent these crucial aspects of in vivo tissues is essential for reducing the failure rates of translating HTS data to clinical outcomes. ∗ Corresponding authors. E-mail addresses: martin.engel@inventia.life (M. Engel), Margareta.Sutija@perkinelmer.com (M. Sutija). 3D cell cultures represent critical features of in vivo tissues better than 2D cell cultures, meaning they may be able to more accurately predict therapeutic efficacy and drug responses [6–9]. Such findings have significant implications not only for HTS, but fundamental biological research more generally. This understanding has facilitated a gradual movement towards the use of 3D cell cultures over 2D cell cultures for biological studies [10]. However, the current lack of established workflows to produce large quantities of biologically relevant 3D cell cultures in an efficient and reproducible way, that are compatible with routinely usedin vitro assays, remains a practical limitation to the widespread use of 3D cell culture models in HTS applications. Many different methods have been developed over the past decades to create 3D cell cultures for biomedical research [11], but their technical limitations have largely restrictedin vitro 3D cell culture to spheroid models, which have very little resemblance to the in vivo extracellular environment due to their free-floating nature [12]. While spheroids enable a richer cell-to-cell interaction compared to 2D cultures, the highly influential role of the extracellular matrix in healthy and diseased tissue [13] is not addressed by this in vitro model option. Emerging 3D bioprinting technologies are addressing this shortcoming by generathttps://doi.org/10.1016/j.slast.2021.10.002 2472-6303/© 2021 Inventia Life Science Operations Pty Ltd. Published by Elsevier Inc. on behalf of Society for Laboratory Automation and Screening. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)
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