Although hepatocytes in vivo experience intra-abdominal pressure (IAP), pressure is typically not incorporated in hepatocyte culture systems. The cuboidal cell shape and extent of intercellular contact between cultured hepatocytes are critical parameters that influence the differentiated hepatic phenotype. Using a microfluidic device, the application of pressure to artificially compact cells and forge cell–cell interactions was previously demonstrated to be effective in accelerating hepatic repolarization. In seeking to implement this approach to higher throughput culture platforms for potential drug screening applications, we specifically designed a vertical-flow compaction bioreactor array (VCBA) that compacts hepatocytes within the range of IAP and portal pressure in vivo in a multi-well setup. As a result of vertical perfusion-generated forces, hepatocytes not only exhibited accelerated repolarization, an in vivo-like cuboidal morphology, but also better maintained hepatic functions in long-term culture as compared to the same cells cultured under static conditions. As a novel engineering tool to modulate cell compaction and intercellular interactions, this platform is a promising approach to confer tight control over hepatocyte repolarization for in vitro culture.

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.

The perfusion culture of primary hepatocytes has been widely adopted to build bioreactors for various applications. As a drug testing platform, a unique vertical-flow bioreactor (VfB) array was found to create the compaction culture of hepatocytes which mimicked the mechanic microenvironment in vivo while maintaining the 3D cell morphology in a 2D culture setup and enhancing the hepatic functions for a sustained culture. Here, we report the methodology in designing and fabricating the VfB to reach ideal bioreactor requirements, optimizing the VfB as a prototype for drug testing, and to demonstrate the enhanced hepatic function so as to demonstrate the performance of the bioreactor. This device enables the modular, scalable, and manufacturable construction of a functional drug testing platform through the sustained maintenance of model cells.