Automated High‐Throughput Drug Discovery in Peptide Hydrogel‐Based 3D Cell Cultures

Abstract3D in vitro cancer models are important therapeutic and biological discovery tools, yet formation of multicellular spheroids in a throughput and highly controlled manner to achieve robust and statistically relevant data, remains challenging. Here, we developed an enabling technology consisting of a bespoke drop-on-demand 3D bioprinter capable of high-throughput printing of 96-well plates of spheroids. 3D-multicellular spheroids are embedded inside a tissue-like matrix with precise control over size and cell number. Application of 3D bioprinting for high-throughput drug screening was demonstrated with doxorubicin. Measurements showed that IC50 values were sensitive to spheroid size, embedding and how spheroids conform to the embedding, revealing parameters shaping biological responses in these models. Our study demonstrates the potential of 3D bioprinting as a robust high-throughput platform to screen biological and therapeutic parameters.Significance StatementIn vitro 3D cell cultures serve as more realistic models, compared to 2D cell culture, for understanding diverse biology and for drug discovery. Preparing 3D cell cultures with defined parameters is challenging, with significant failure rates when embedding 3D multicellular spheroids into extracellular mimics. Here, we report a new 3D bioprinter we developed in conjunction with bioinks to allow 3D-multicellular spheroids to be produced in a high-throughput manner. High-throughput production of embedded multicellular spheroids allowed entire drug-dose responses to be performed in 96-well plate format with statistically relevant numbers of data points. We have deconvoluted important parameters in drug responses including the impact of spheroid size and embedding in an extracellular matrix mimic on IC50 values.

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Automated screening by 3D light-sheet microscopy with high spatial and temporal resolution reveals mitotic phenotypes

10.1101/2020.01.20.912659 ◽

2020 ◽

Author(s):

Björn Eismann ◽

Teresa G Krieger ◽

Jürgen Beneke ◽

Ruben Bulkescher ◽

Lukas Adam ◽

...

Keyword(s):

Cell Cultures ◽

High Throughput ◽

Light Exposure ◽

Epigenetic Modification ◽

Fluorescent Microscopy ◽

Light Sheet ◽

Drug Candidate ◽

Light Sheet Microscopy ◽

Phenotype Classification ◽

3D Cell Cultures

Abstract3D cell cultures enable the in vitro study of dynamic biological processes such as the cell cycle, but their use in high-throughput screens remains impractical with conventional fluorescent microscopy. Here, we present a screening workflow for the automated evaluation of mitotic phenotypes in 3D cell cultures by light-sheet microscopy. After sample preparation by a liquid handling robot, three-dimensional cell spheroids are imaged for 24 hours in toto with a dual inverted selective plane illumination (diSPIM) microscope with a much improved signal-to-noise ratio, higher imaging speed, isotropic resolution and reduced light exposure compared to a spinning disc confocal microscope. A dedicated high-content image processing pipeline implements convolutional neural network based phenotype classification. We illustrate the potential of our approach by siRNA knock-down and epigenetic modification of 28 mitotic target genes for assessing their phenotypic role in mitosis. By rendering light-sheet microscopy operational for high-throughput screening applications, this workflow enables target gene characterization or drug candidate evaluation in tissue-like 3D cell culture models.

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A Simple Vacuum-Based Microfluidic Technique to Establish High-Throughput Organs-On-Chip and 3D Cell Cultures at the Microscale

Advanced Materials Technologies ◽

10.1002/admt.201800319 ◽

2018 ◽

Vol 4 (1) ◽

pp. 1800319 ◽

Cited By ~ 3

Author(s):

Roberta Visone ◽

Giovanni Stefano Ugolini ◽

Vladimir Vinarsky ◽

Miriam Penati ◽

Alberto Redaelli ◽

...

Keyword(s):

Cell Cultures ◽

High Throughput ◽

3D Cell Cultures ◽

On Chip ◽

Microfluidic Technique

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Enabling high throughput target-based drug discovery in 3D cell cultures through novel bioprinting workflow

10.1101/2021.04.21.440768 ◽

2021 ◽

Author(s):

Martin Engel ◽

Lisa Belfiore ◽

Behnaz Aghaei ◽

Margareta Sutija

Keyword(s):

Breast Cancer ◽

Cell Culture ◽

Cell Cultures ◽

High Throughput ◽

Cell Biology ◽

Holistic View ◽

3D Cell Cultures ◽

High Throughput Screening Assay ◽

Mcf 7

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 workflow 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 GSK3β 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.

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High-Throughput Cancer Cell Sphere Formation for Characterizing the Efficacy of Photo Dynamic Therapy in 3D Cell Cultures

Scientific Reports ◽

10.1038/srep12175 ◽

2015 ◽

Vol 5 (1) ◽

Cited By ~ 54

Author(s):

Yu-Chih Chen ◽

Xia Lou ◽

Zhixiong Zhang ◽

Patrick Ingram ◽

Euisik Yoon

Keyword(s):

Cancer Cell ◽

Cell Cultures ◽

High Throughput ◽

Dynamic Therapy ◽

3D Cell Cultures ◽

Sphere Formation ◽

Photo Dynamic Therapy

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Three-Dimensional Cell Cultures in Drug Discovery and Development

SLAS DISCOVERY Advancing Life Sciences ◽

10.1177/1087057117696795 ◽

2017 ◽

Vol 22 (5) ◽

pp. 456-472 ◽

Cited By ~ 106

Author(s):

Ye Fang ◽

Richard M. Eglen

Keyword(s):

Drug Discovery ◽

Cell Cultures ◽

Disease Modeling ◽

Three Dimensional ◽

3D Cell Culture ◽

Compound Identification ◽

3D Cell Cultures ◽

Dimensional Cell ◽

Early Drug

The past decades have witnessed significant efforts toward the development of three-dimensional (3D) cell cultures as systems that better mimic in vivo physiology. Today, 3D cell cultures are emerging, not only as a new tool in early drug discovery but also as potential therapeutics to treat disease. In this review, we assess leading 3D cell culture technologies and their impact on drug discovery, including spheroids, organoids, scaffolds, hydrogels, organs-on-chips, and 3D bioprinting. We also discuss the implementation of these technologies in compound identification, screening, and development, ranging from disease modeling to assessment of efficacy and safety profiles.

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