High-throughput spheroid screens using volume, resazurin reduction and acid phosphatase activity

Ivanov, Delyan Pavlov; Grabowska, Anna M.; Garnett, Martin C.

Abstract

© 2017, Springer Science+Business Media LLC. Mainstream adoption of physiologically relevant three-dimensional models has been slow in the last 50 years due to long, manual protocols with poor reproducibility, high price, and closed commercial platforms. This chapter describes high-throughput, low-cost, open methods for spheroid viability assessment which use readily available reagents and open-source software to analyze spheroid volume, metabolism, and enzymatic activity. We provide two ImageJ macros for automated spheroid size determination—for both single images and images in stacks. We also share an Excel template spreadsheet allowing users to rapidly process spheroid size data, analyze plate uniformity (such as edge effects and systematic seeding errors), detect outliers, and calculate dose-response. The methods would be useful to researchers in preclinical and translational research planning to move away from simplistic monolayer studies and explore 3D spheroid screens for drug safety and efficacy without substantial investment in money or time.

Citation

Ivanov, D. P., Grabowska, A. M., & Garnett, M. C. (2018). High-throughput spheroid screens using volume, resazurin reduction and acid phosphatase activity. In D. F. Gilbert, & O. Friedrich (Eds.), Cell Viability Assays (43-59). Humana Press (Springer). https://doi.org/10.1007/978-1-4939-6960-9_4

• Acceptance Date: Sep 1, 2016
• Online Publication Date: May 4, 2017
• Publication Date: 2018-06
• Deposit Date: Jun 16, 2017
• Publicly Available Date: Jun 16, 2017
• Peer Reviewed: Peer Reviewed
• Volume: 1601
• Pages: 43-59
• Series Title: Methods in Molecular Biology
• Book Title: Cell Viability Assays
• ISBN: 978-1-4939-6959-3
• DOI: https://doi.org/10.1007/978-1-4939-6960-9_4
• Keywords: alamar blue, viability assays, overlay culture, hanging drop, FiJi ImageJ, image analysis, three-dimensional cell culture, in vitro model, preclinical screening, drug sensitivity
• Public URL: https://nottingham-repository.worktribe.com/output/859158
• Publisher URL: https://link.springer.com/protocol/10.1007%2F978-1-4939-6960-9_4
• Additional Information: The publisher has agreed open access publication via email:; ; ; From: Suchy, Monica, Springer US ; Sent: 22 May 2017 16:58; To: Ivanov Delyan; AgnesFelema, Anbazhagan; Subject: RE: Springer BWF Book_Gilbert_385089_1_En_Cell Viability Assays//Introductory mail; ; Dear Delyan,; ; The book already published so I am going to have to check with Comp Copies and see if the order was processed yet. If it was I will request that they send out a new copy to your home address. ; ; I was unaware of the previous conversation, I spoke with Patrick and in this instance, you have our permission.; ; Best,; Monica; ; From: Delyan Ivanov [mailto:[e-mail]] ; Sent: Monday, May 22, 2017 3:13 AM; To: Suchy, Monica, Springer US; AgnesFelema, Anbazhagan; Subject: Re: Springer BWF Book_Gilbert_385089_1_En_Cell Viability Assays//Introductory mail; ; ; Hi Monica,; ; Thank you for the reply. I have moved house in the mean time - can the book go to my new address?; ; Delyan Ivanov; 3A Chapel Street; Buxton, SK176HX; UK; ; In my communication with Oliver and Daniel, they contacted Patrick Marton from Springer US and he mentioned that Springer may be willing to make an exemption for my chapter and allow me to self-archive earlier than the 12 month embargo as per the email below.; ; Would you or one of your colleagues be able to allow early self-archiving for my chapter?; ; Best wishes,; Delyan; ; ; From: Oliver Friedrich ; Sent: 17 July 2016 14:16; To: Delyan Ivanov; Dr. Daniel Gilbert; Subject: Re: Methods in Molecular biology book chapter; ; Dear Delyan,; ; thank you very much for submitting your manuscript. Daniel and I will be in due course of editing the chapters within the next four weeks and coe back to everyone for editorial feedback if changes are required.; Regarding your previous questions, I was in contact with Patrick Marton from Springer US and this is what he came back with:; For the first question about the supplementary files -- we can actually accept supplementary files -- we publish them on SpringerLink along with the book. The files are linked from the abstract page for the chapter. What I would like to do if the author is amendable is to have him do as he suggests (publish them on an open database with a link out) while also having him submit them with the chapter so we can publish them online. Doing it both ways provides all readers (electronic and print) easy access to the files.; ; ; The second question is a bit trickier as we don't have an open access option for single book chapters, only full volumes (which is cost prohibitive). We do have a self archiving policy in place for authors who are required to submit their chapters to repositories (such as PubMedCentral). The policy calls for a one year embargo on submitting the final author version of the chapter, but if that's too long for this author, I'd be happy to make an exception and grant permission to archive it post publication if that works?; ; I think this looks very accommodating and I suggest, we finalize thos issue on the 'exception' once we have the chapter in its final form, ok?; ; Cheers,; ; Oliver; ; ; From: Suchy, Monica, Springer US ; Sent: 16 May 2017 14:01; To: Ivanov Delyan; AgnesFelema, Anbazhagan; Subject: RE: Springer BWF Book_Gilbert_385089_1_En_Cell Viability Assays//Introductory mail; ; Dear Dr. Ivanov,; ; Here is out policy for self-archiving: http://www.springer.com/gp/open-access/authors-rights/self-archiving-policy/2124.; ; Per the agreements the authors signed, each corresponding author will receive a copy of the book. These generally arrive about 5-6 weeks after the book is published. All of the chapter authors are entitled to a 40% discount on everything Springer publishes. In order to get this, they need to go to the follow url and fill out the information:http://www.springer.com/authors/book+authors?SGWID=0-154102-12-99823-0.; ; Best,; Monica