Design and testing of a centrifugal fluidic device for populating microarrays of spheroid cancer cell cultures

Abstract

Background: In current cancer spheroid culturing methods, the transfer and histological processing of specimens grown in 96-well plates is a time consuming process. A centrifugal fluidic device was developed and tested for rapid extraction of spheroids from a 96-well plate and subsequent deposition into a molded agar receiver block. The deposited spheroids must be compact enough to fit into a standard histology cassette while also maintaining a highly planar arrangement. This size and planarity enable histological processing and sectioning of spheroids in a single section. The device attaches directly to a 96-well plate and uses a standard centrifuge to facilitate spheroid transfer. The agar block is then separated from the device and processed. Results: Testing of the device was conducted using six full 96-well plates of fixed Pa14C pancreatic cancer spheroids. On average, 80% of spheroids were successfully transferred into the agar receiver block. Additionally, the planarity of the deposited spheroids was evaluated using confocal laser scanning microscopy. This revealed that, on average, the optimal section plane bisected individual spheroids within 27% of their mean radius. This shows that spheroids are largely deposited in a planar fashion. For rare cases where spheroids had a normalized distance to the plane greater than 1, the section plane either misses or captures a small cross section of the spheroid volume. Conclusions: These results indicate that the proposed device is capable of a high capture success rate and high sample planarity, thus demonstrating the capabilities of the device to facilitate rapid histological evaluation of spheroids grown in standard 96-well plates. Planarity figures are likely to be improved by adjusting agar block handling prior to imaging to minimize deformation and better preserve the planarity of deposited spheroids. Additionally, investigation into media additives to reduce spheroid adhesion to 96-well plates would greatly increase the capture success rate of this device. Background Cell culturing is a key experimental tool in the study of solid tumor biology, pharmacology, and the search for more effective cancer treatments. Two-dimensional (2D) in vitro cell culturing, in which cells are grown on flat glass or plastic substrates (Fig. 1a), gained widespread acceptance after its introduction in the early twentieth century, and remains the most common choice for drug screening studies partly due to its well-developed compatibility with highthroughput and automated methods. Unfortunately, several factors limit the accuracy with which 2D cultures model in vivo tissues, leading to the development of phenotypes in 2D cultures that vary significantly from cells in vivo [1, 2]. These factors include: differences in strain distributions for cells grown on 2D rigid substrates versus 3D environments [3-6]; differences in mass transport, which limits cellular access to oxygen, nutrients, and soluble factors; and the lack of molecular gradients [7-9]. For these reasons, drug screens b...

A method for reproducible high‐resolution imaging of 3D cancer cell spheroids

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Coplanar embedding of multiple 3D cell models in hydrogel towards high-throughput micro-histology

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