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      High-Content Assays for Characterizing the Viability and Morphology of 3D Cancer Spheroid Cultures

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          Abstract

          There is an increasing interest in using three-dimensional (3D) spheroids for modeling cancer and tissue biology to accelerate translation research. Development of higher throughput assays to quantify phenotypic changes in spheroids is an active area of investigation. The goal of this study was to develop higher throughput high-content imaging and analysis methods to characterize phenotypic changes in human cancer spheroids in response to compound treatment. We optimized spheroid cell culture protocols using low adhesion U-bottom 96- and 384-well plates for three common cancer cell lines and improved the workflow with a one-step staining procedure that reduces assay time and minimizes variability. We streamlined imaging acquisition by using a maximum projection algorithm that combines cellular information from multiple slices through a 3D object into a single image, enabling efficient comparison of different spheroid phenotypes. A custom image analysis method was implemented to provide multiparametric characterization of single-cell and spheroid phenotypes. We report a number of readouts, including quantification of marker-specific cell numbers, measurement of cell viability and apoptosis, and characterization of spheroid size and shape. Assay performance was assessed using established anticancer cytostatic and cytotoxic drugs. We demonstrated concentration–response effects for different readouts and measured IC 50 values, comparing 3D spheroid results to two-dimensional cell cultures. Finally, a library of 119 approved anticancer drugs was screened across a wide range of concentrations using HCT116 colon cancer spheroids. The proposed methods can increase performance and throughput of high-content assays for compound screening and evaluation of anticancer drugs with 3D cell models.

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          Most cited references26

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          Recent advances in three-dimensional multicellular spheroid culture for biomedical research.

          Many types of mammalian cells can aggregate and differentiate into 3-D multicellular spheroids when cultured in suspension or a nonadhesive environment. Compared to conventional monolayer cultures, multicellular spheroids resemble real tissues better in terms of structural and functional properties. Multicellular spheroids formed by transformed cells are widely used as avascular tumor models for metastasis and invasion research and for therapeutic screening. Many primary or progenitor cells on the other hand, show significantly enhanced viability and functional performance when grown as spheroids. Multicellular spheroids in this aspect are ideal building units for tissue reconstruction. Here we review the current understanding of multicellular spheroid formation mechanisms, their biomedical applications, and recent advances in spheroid culture, manipulation, and analysis techniques.
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            High-throughput 3D spheroid culture and drug testing using a 384 hanging drop array.

            Culture of cells as three-dimensional (3D) aggregates can enhance in vitro tests for basic biological research as well as for therapeutics development. Such 3D culture models, however, are often more complicated, cumbersome, and expensive than two-dimensional (2D) cultures. This paper describes a 384-well format hanging drop culture plate that makes spheroid formation, culture, and subsequent drug testing on the obtained 3D cellular constructs as straightforward to perform and adapt to existing high-throughput screening (HTS) instruments as conventional 2D cultures. Using this platform, we show that drugs with different modes of action produce distinct responses in the physiological 3D cell spheroids compared to conventional 2D cell monolayers. Specifically, the anticancer drug 5-fluorouracil (5-FU) has higher anti-proliferative effects on 2D cultures whereas the hypoxia activated drug commonly referred to as tirapazamine (TPZ) are more effective against 3D cultures. The multiplexed 3D hanging drop culture and testing plate provides an efficient way to obtain biological insights that are often lost in 2D platforms.
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              Microscale technologies for tissue engineering and biology.

              Microscale technologies are emerging as powerful tools for tissue engineering and biological studies. In this review, we present an overview of these technologies in various tissue engineering applications, such as for fabricating 3D microfabricated scaffolds, as templates for cell aggregate formation, or for fabricating materials in a spatially regulated manner. In addition, we give examples of the use of microscale technologies for controlling the cellular microenvironment in vitro and for performing high-throughput assays. The use of microfluidics, surface patterning, and patterned cocultures in regulating various aspects of cellular microenvironment is discussed, as well as the application of these technologies in directing cell fate and elucidating the underlying biology. Throughout this review, we will use specific examples where available and will provide trends and future directions in the field.
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                Author and article information

                Journal
                Assay Drug Dev Technol
                Assay Drug Dev Technol
                adt
                Assay and Drug Development Technologies
                Mary Ann Liebert, Inc. (140 Huguenot Street, 3rd FloorNew Rochelle, NY 10801USA )
                1540-658X
                1557-8127
                01 September 2015
                01 September 2015
                : 13
                : 7
                : 402-414
                Affiliations
                [ 1 ]Molecular Devices , LLC, Sunnyvale, California.
                [ 2 ]Protein Fluidics, Inc. , Palo Alto, California.
                Author notes
                Address correspondence to: Oksana Sirenko, PhD, Molecular Devices, LLC 1311 Orleans Drive, Sunnyvale, CA 94089, E-mail: oksana.sirenko@ 123456moldev.com
                Article
                10.1089/adt.2015.655
                10.1089/adt.2015.655
                4556086
                26317884
                7ffda3a8-7642-4c42-a263-e137e4521c91
                © Sirenko et al. 2015; Published by Mary Ann Liebert, Inc.

                This Open Access article is distributed under the terms of the Creative Commons Attribution Noncommercial License ( http://creativecommons.org/licenses/by-nc/4.0/) which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited.

                History
                Page count
                Figures: 8, Tables: 3, References: 38, Pages: 13
                Categories
                Original Articles

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