Abstract
Microfluidic 3D tissue culture systems are attractive for in vitro drug testing applications due to the ability of these platforms to generate 3D tissue models and perform drug testing at a very small scale. However, the minute cell number and liquid volume impose significant technical challenges to perform quantitative cell viability measurements using conventional colorimetric or fluorometric assays, such as MTS or Alamar Blue. Similarly, live-dead staining approaches often utilize metabolic dyes that typically label the cytoplasm of live cells, which makes it difficult to segment and count individual cells in compact 3D tissue cultures. In this paper, we present a quantitative image-based cell viability (QuantICV) assay technique that circumvents current challenges of performing the quantitative cell viability assay in microfluidic 3D tissue cultures. A pair of cell-impermeant nuclear dyes (EthD-1 and DAPI) were used to sequentially label the nuclei of necrotic and total cell populations, respectively. Confocal microscopy and image processing algorithms were employed to visualize and quantify the cell nuclei in the 3D tissue volume. The QuantICV assay was validated and showed good concordance with the conventional bulk MTS assay in static 2D and 3D tumor cell cultures. Finally, the QuantICV assay was employed as an on-chip readout to determine the differential dose responses of parental and metastatic 3D oral squamous cell carcinoma (OSCC) to Gefitinib in a microfluidic 3D culture device. This proposed technique can be useful in microfluidic cell cultures as well as in a situation where conventional cell viability assays are not available.
Highlights
Microfluidic cell culture platforms have gathered growing attention to perform in vitro drug testing studies due to their ability to control cell microenvironments while utilizing a small amount of samples [1]
A microfluidic cell culture device consists of fluidic networks ranging from 10 to 500 μm, which serve as connections to chambers housing different cell types [2,3,4,5,6,7], with fluid volumes ranging from nanolitres to microlitres [8,9]
The practical translation of microfluidic cell culture devices for drug testing applications must be complemented with cellular assays, which can be implemented in a format compatible with microfluidic devices
Summary
Microfluidic cell culture platforms have gathered growing attention to perform in vitro drug testing studies due to their ability to control cell microenvironments while utilizing a small amount of samples [1]. As more microfluidic devices are designed to support the 3D tissue culture by incorporating either microstructures that physically pack cells at high density [12,14,15]; cell-laden hydrogels [16]; scaffolds [17]; or micropatterning [18] to improve physiological relevance [2,4,5,12,19,20], it becomes increasingly challenging to segment and quantify individual cells within a compact 3D cell mass during image processing to get quantitative readouts [14,21] This approach is mainly limited to the qualitative assessment of cell viability in microfluidic cell cultures [12], which does not provide the level of sensitivity required to discern between subtle changes in cell viabilities across different drug concentrations or between cell types with differential drug responses. We employed the QuantICV assay to determine the differential drug response of parental and metastatic 3D microfluidic oral squamous cell carcinoma (OSCC) models
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