Fluorescence hyperspectral imaging for live monitoring of multiple spheroids in microfluidic chips
ABSTRACT
Tumor spheroids represent a realistic 3D in vitro cancer model because they provide a missing link between monolayer cell culture and live tissues. While microfluidic chips can easily form and assay thousands of spheroids simultaneously, few commercial instruments are available to analyze this massive amount of data. Available techniques to measure spheroid response to external stimuli, such as confocal imaging and flow cytometry, are either not appropriate for 3D cultures, or destructive. We designed a wide-field hyperspectral imaging system to analyze multiple spheroids trapped in a microfluidic chip in a single acquisition. The system and its fluorescence quantification algorithm were assessed using liquid phantoms mimicking spheroid optical properties. Spectral unmixing was tested on three overlapping spectral entities. Hyperspectral images of co-culture spheroids expressing two fluorophores were compared with confocal microscopy and spheroid growth was measured over time. The system can spectrally analyze multiple fluorescent markers simultaneously and allows multiple time-points assays, providing a fast and versatile solution for analyzing lab on a chip devices.
INTRODUCTION
Interest in multicellular tumor spheroids (MCTS, or spheroids) as a 3D in vitro cancer model has been steadily growing in the past decade.1 They represent a realistic 3D cell culture model with properties that bridge the gap between monolayer cell culture and live tissues, including human biopsies, surgical specimens, or mouse xenografts.2,3 MCTS are 3D constructs made of cells that aggregate together to form spheres of varying compactness. Contrary to monolayer (2D) cell culture, they display cell–cell and cell–matrix interactions.2 Tumor cell lines are often able to spontaneously form these 3D constructs when cultured in hanging droplets, low-attachment plates or passivated microfluidic chips.
The microfluidics community has put considerable effort in the past ten years to develop chip-based platforms capable of forming and/or testing MCTS.1,5–13 Some of them can be used to synthesize thousands of spheroids in one step.14,15 Others can be used to form spheroids of different sizes utilizing a single cell suspension.16 They can also hold (or trap) spheroids in place during medium changes or while adding/ removing reagents without the risk of pipetting them out, an issue often encountered when manipulating spheroids with micropipettes in 96-well plates.
Still, microfluidic chips are not yet fully adopted by research biologists, one of the main reasons being the complexity of use. Many microfluidic chips are designed to perform experiments efficiently but need complex pumping systems or handling.20 Also, while microfluidic chips are able to easily produce large amounts of spheroids, very few commercial applications exist to analyze this massive amount of data. Typical techniques used by researchers consist of confocal, two-photon, and light sheet microscopy, and flow and imaging cytometry. Confocal microscopy is often used in conjunction with live/ dead fluorescent markers to count the numbers of viable cells compared to dead cells in a spheroid.10,21–23 Mohapatra et al. acquired spheroid fluorescence emission spectra for whole optical sections during confocal imaging.24 While this technique offers high resolution imaging and spatial information on spheroid viability, it is limited to the first few cell layers (50–100 µm) due to limited light penetration in the 3D culture. Imaging larger fields of view also requires multiple acquisitions and image stitching.25–27 Loss of signal-to-noise with imaging depth can prevent accurate measurement of the spheroid center response to a treatment, where necrotic, senescent, or slowly proliferating cells are present.
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