title: Human proximal tubule cells form functional microtissues.
abstract: 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.
authors.affiliation: Institute of Physiology, Zurich Center for Integrative Human Physiology (ZIHP), University of Zurich, Zurich, Switzerland.
authors.forename: Jenny A
authors.initials: JA
authors.lastname: Prange
authors.affiliation: Institute of Physiology, Zurich Center for Integrative Human Physiology (ZIHP), University of Zurich, Zurich, Switzerland.
authors.forename: Manuela
authors.initials: M
authors.lastname: Bieri
authors.affiliation: Division of Nephrology, UniversitätsSpital, Zurich, Switzerland.
authors.forename: Stephan
authors.initials: S
authors.lastname: Segerer
authors.affiliation: Institute of Anatomy, University of Zurich, Zurich, Switzerland.
authors.forename: Charlotte
authors.initials: C
authors.lastname: Burger
authors.affiliation: Center for Microscopy and Image Analysis, University of Zurich, Zurich, Switzerland.
authors.forename: Andres
authors.initials: A
authors.lastname: Kaech
authors.affiliation: InSphero AG, Zurich, Switzerland.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Moritz
authors.affiliation: Institute of Physiology, Zurich Center for Integrative Human Physiology (ZIHP), University of Zurich, Zurich, Switzerland. olivier.devuyst@uzh.ch.
authors.forename: Olivier
authors.initials: O
authors.lastname: Devuyst
mesh_terms: D002454:Cell Differentiation; D002460:Cell Line; D049109:Cell Proliferation; D002478:Cells, Cultured; D018920:Coculture Techniques / Q000379:methods; D017077:Culture Media, Conditioned / Q000494:pharmacology; D004847:Epithelial Cells / Q000166:cytology* / Q000187:drug effects / Q000502:physiology; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D007687:Kidney Tubules, Proximal / Q000166:cytology*; D061251:Primary Cell Culture / Q000379:methods*; D046509:Tissue Culture Techniques / Q000379:methods
chemical_list: D017077:Culture Media, Conditioned
keywords: Albumin; Drug toxicity; Endocytosis; Epithelial cells; Primary culture