Publication: The Mini‐Organo: A rapid high‐throughput 3D coculture organotypic assay for oncology screening and drug development

Thomas R. Cox, Aug 2019

The Mini‐Organo: A rapid high‐throughput 3D coculture organotypic assay for oncology screening and drug development

Just published in Cancer Reports is our new protocol paper detailing the development of a rapid high-throughput (96wp) 3D organotypic coculture assay that is optimised for screening cancer cell and cancer-associated fibroblast response to drugs in physiologically relevant matrices.

Mini-Organo workflow

In this paper we present a method for the rapid and scalable production of 3D physiologically relevant coculture assays suitable for dissecting both stromal remodelling and cancer cell invasion. One of the major goals in designing 3D physiologically relevant in vitro approaches is to capture the complexity of intercellular communication, nutrient and oxygen gradients, as well as cell polarity and interactions with the extracellular matrix (ECM) that is lacking in more traditional 2D monolayer cultures. The Mini‐Organo offers a simple, straightforward, scalable, and cost‐effective way of doing this.

Given the enormous number of new anticancer drugs and the accompanying exponentially increasing number of possible drug combinations, the use of in vivo models is prohibitively expensive. Therefore, approaches such as the “Mini‐Organo” offer a bridge between the two that allows for higher throughput screening of molecules and interventions within the standard laboratory environment for implementation into the clinic, not only in the cancer field but also many other diseases.


The use of in vitro cell cultures is a powerful tool for obtaining key insights into the behaviour and response of cells to interventions in normal and disease situations. Unlike in vivo settings, in vitro experiments allow a fine‐tuned control of a range of microenvironmental elements independently within an isolated setting. The recent expansion in the use of three‐dimensional (3D) in vitro assays has created a number of representative tools to study cell behaviour in a more physiologically 3D relevant microenvironment. Complex 3D in vitro models that can recapitulate human tissue biology are essential for understanding the pathophysiology of disease.

The development of the 3D coculture collagen contraction and invasion assay, the “organotypic assay,” has been widely adopted as a powerful approach to bridge the gap between standard two‐dimensional tissue culture and in vivo mouse models. In the cancer setting, these assays can then be used to dissect how stromal cells, such as cancer‐associated fibroblasts (CAFs), drive extracellular matrix (ECM) remodelling to alter cancer cell behaviour and response to intervention. However, to date, many of the published organotypic protocols are low‐throughput, time‐consuming (up to several weeks), and work‐intensive with often limited scalability. Our aim was to develop a fast, high‐throughput, scalable 3D organotypic assay for use in oncology screening and drug development.

Here, we describe a modified 96‐well organotypic assay, the “Mini‐Organo,” which can be easily completed within 5 days. We demonstrate its application in a wide range of mouse and human cancer biology approaches including evaluation of stromal cell 3D ECM remodelling, 3D cancer cell invasion, and the assessment of efficacy of potential anticancer therapeutic targets. Furthermore, the organotypic assay described is highly amenable to customisation using different cell types under diverse experimental conditions.

The Mini‐Organo high‐throughput 3D organotypic assay allows the rapid screening of potential cancer therapeutics in human and mouse models in a time‐efficient manner.


View the article on the Cancer Reports website (Open Access)
Download a copy of the PDF here


Chitty JL et al.  The Mini‐Organo: A rapid high‐throughput 3D coculture organotypic assay for oncology screening and drug development
Cancer Reports (2019) | doi: 10.1002/cnr2.1209

Key Words

3D model, Cancer-associated Fibroblast, coculture, drug screening, extracellular matrix, organotypic


This work was supported by the NHMRC, a Cancer Institute NSW (CINSW) fellowship, Cancer Council NSW, and a Susan G Komen Career Catalyst. T.R.C. is a recipient of an NHMRC RD Wright Biomedical Career Development Fellowship. C.D.M is supported by the Ragnar Söderberg Foundation, BioCARE, Cancerfonden, the Åke Wiberg foundation, Swedish Research Council, and the Crafoord Foundation. P.T. is a recipient of an NHMRC Senior Research Fellowship and NHMRC project grant. C.V. is a recipient of a post‐doctoral HFSP fellowship. This project was made possible by an Avner Pancreatic Cancer Foundation Grant.