Although the relevance of three-dimensional (3-D) culture has been recognized for years and exploited at an academic level, its translation to industrial applications has been slow. The development of reliable high-throughput technologies is clearly a prerequisite for the industrial implementation of 3-D models. In this study the robustness of spherical microtissue production and drug testing in a 96-well hanging-drop multiwell plate format was assessed on a standard 96-well channel robotic platform. Microtissue models derived from six different cell lines were produced and characterized according to their growth profile and morphology displaying high-density tissue-like reformation and growth over at least 15 days. The colon cancer cell line HCT116 was chosen as a model to assess microtissue-based assay reproducibility. Within three individual production batches the size variations of the produced microtissues were below 5%. Reliability of the microtissue-based assay was tested using two reference compounds, staurosporine and chlorambucil. In four independent drug testings the calculated IC(50) values were benchmarked against 2-D multiwell testings displaying similar consistency. The technology presented here for the automated production of a variety of microtissues for efficacy testing in a standard 96-well format will aid the implementation of more organotypic models at an early time point in the drug discovery process.

Non-alcoholic steatohepatitis (NASH) is a progressive and severe liver disease, characterized by lipid accumulation, inflammation, and downstream fibrosis. Despite its increasing prevalence, there is no approved treatment yet available for patients. This has been at least partially due to the lack of predictive preclinical models for studying this complex disease. Here, we present a 3D in vitro microtissue model that uses spheroidal, scaffold free co-culture of primary human hepatocytes, Kupffer cells, liver endothelial cells and hepatic stellate cells. Upon exposure to defined and clinically relevant lipotoxic and inflammatory stimuli, these microtissues develop key pathophysiological features of NASH within 10 days, including an increase of intracellular triglyceride content and lipids, and release of pro-inflammatory cytokines. Furthermore, fibrosis was evident through release of procollagen type I, and increased deposition of extracellular collagen fibers. Whole transcriptome analysis revealed changes in the regulation of pathways associated with NASH, such as lipid metabolism, inflammation and collagen processing. Importantly, treatment with anti-NASH drug candidates (Selonsertib and Firsocostat) decreased the measured specific disease parameter, in accordance with clinical observations. These drug treatments also significantly changed the gene expression patterns of the microtissues, thus providing mechanisms of action and revealing therapeutic potential. In summary, this human NASH model represents a promising drug discovery tool for understanding the underlying complex mechanisms in NASH, evaluating efficacy of anti-NASH drug candidates and identifying new approaches for therapeutic interventions.

The epithelial cells lining the proximal tubules of the kidney mediate complex transport processes and are particularly vulnerable to drug toxicity. Drug toxicity studies are classically based on two-dimensional cultures of immortalized proximal tubular cells. Such immortalized cells are dedifferentiated, and lose transport properties (including saturable endocytic uptake) encountered in vivo. Generating differentiated, organotypic human microtissues would potentially alleviate these limitations and facilitate drug toxicity studies. Here, we describe the generation and characterization of kidney microtissues from immortalized (HK-2) and primary (HRPTEpiC) human renal proximal tubular epithelial cells under well-defined conditions. Microtissue cultures were done in hanging drop GravityPLUS™ culture plates and were characterized for morphology, proliferation and differentiation markers, and by monitoring the endocytic uptake of albumin. Kidney microtissues were successfully obtained by co-culturing HK-2 or HRPTEpiC cells with fibroblasts. The HK-2 microtissues formed highly proliferative, but dedifferentiated microtissues within 10 days of culture, while co-culture with fibroblasts yielded spherical structures already after 2 days. Low passage HRPTEpiC microtissues (mono- and co-culture) were less proliferative and expressed tissue-specific differentiation markers. Electron microscopy evidenced epithelial differentiation markers including microvilli, tight junctions, endosomes, and lysosomes in the co-cultured HRPTEpiC microtissues. The co-cultured HRPTEpiC microtissues showed specific uptake of albumin that could be inhibited by cadmium and gentamycin. In conclusion, we established a reliable hanging drop protocol to obtain functional kidney microtissues with proximal tubular epithelial cell lines. These microtissues could be used for high-throughput drug and toxicology screenings, with endocytosis as a functional readout.

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