Abstract
We describe and share a device, methodology and image analysis algorithms, which allow up to 66 spheroids to be arranged into a gel-based array directly from a culture plate for downstream processing and analysis. Compared to processing individual samples, the technique uses 11-fold less reagents, saves time and enables automated imaging. To illustrate the power of the technology, we showcase applications of the methodology for investigating 3D spheroid morphology and marker expression and for in vitro safety and efficacy screens. First, spheroid arrays of 11 cell-lines were rapidly assessed for differences in spheroid morphology. Second, highly-positive (SOX-2), moderately-positive (Ki-67) and weakly-positive (βIII-tubulin) protein targets were detected and quantified. Third, the arrays enabled screening of ten media compositions for inducing differentiation in human neurospheres. Last, the application of spheroid microarrays for spheroid-based drug screens was demonstrated by quantifying the dose-dependent drop in proliferation and increase in differentiation in etoposide-treated neurospheres.
Highlights
We describe and share a device, methodology and image analysis algorithms, which allow up to 66 spheroids to be arranged into a gel-based array directly from a culture plate for downstream processing and analysis
We present a device which overcomes this issue by allowing users to arrange up to 66 spheroids in a single plane for high-throughput downstream analysis of three-dimensional cell cultures
We have developed and share the designs of a device which creates spheroid microarrays by transferring previously-fixed spheroids from the cell culture plate to a precast agarose mold (Fig. 1b)
Summary
We describe and share a device, methodology and image analysis algorithms, which allow up to 66 spheroids to be arranged into a gel-based array directly from a culture plate for downstream processing and analysis. The methods for their culture were low-throughput and resulted in large variation in spheroid size This has recently been overcome with the introduction of high-throughput plate-based platforms for 3D culture. We present a device which overcomes this issue by allowing users to arrange up to 66 spheroids in a single plane for high-throughput downstream analysis of three-dimensional cell cultures. Spheroid morphology and single cell protein/gene analysis can identify spatial patterns in expression due to nutrient and oxygen gradients and the phenotype of small populations of cells resistant to drug therapy. These properties can be examined using www.nature.com/scientificreports/
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