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Generation Of A New Renal Nephrotoxicity Model By Using A Microfluidic System
Author(s) -
SanchezRomero Natalia,
Arias Cristina,
Martinez Laura,
Torcal Pilar,
SanchezZalabardo Manuel,
Iñigo Pablo,
Gimenez Ignacio
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.lb731
Subject(s) - cisplatin , nephrotoxicity , cytotoxicity , chemistry , viability assay , microfluidics , cell culture , cell , microbiology and biotechnology , multiplex , in vitro , biophysics , toxicity , biochemistry , biology , nanotechnology , materials science , bioinformatics , genetics , organic chemistry , chemotherapy
Aim To generate a biomimicking cell culture system to study the toxicity caused by exposure to cisplatin and the process of reparation‐regeneration after the injury, in proximal tubule cells grown in microfluidic devices. Methods Primary cultures of human proximal tubule cells (hPTPC) are generated from healthy tissue isolated from nephrectomy specimens. The hPTPC are cultured on conventional cell culture containers or inside the channels in polysterene microfluidic devices. Morphological and biochemical characterization of the cultured cells is performed by using PCR, immunofluorescence (IF) and immunocytochemistry (ICC) to verify the expression of selected proximal tubule and epithelial markers. Confluent hPTPC monolayers are exposed to six cisplatin concentrations in the 0–300 uM range. Cisplatin citotoxicity is assessed by determining three parameters in two different optical reactions: the first assay combines PrestoBlue reagent, a cell viability indicator, with a gamma‐glutamyl transferase 1 (GGT1) substrate. GGT1 is a specific luminal border enzyme in hPTPC and is responsible for the bioactivation of cisplatin into a more potent toxin. In the second assay, cell number is estimated through nuclear DNAstaining with crystal violet. Results The phenotyping techniques have been optimized to work with the small volumes and cell numbers present in microfluidic devices. Biochemical characterization of hPTPC through multiplex analysis of phenotypic markers shows these cells retain the most relevant PT markers when cultured in microfluidic channels. Cisplatin exhibits cytotoxicity on hPTPC when grown on multiwell plates or microfluidic channels. The EC50 was found to be 30–50 μM for the three parameters determined in our assay. Maximal effects were observed in approximately 48–72h after exposure to cisplatin. Our data suggest that hPTPC cultures do not possess autoregenerative capacity. The cytotoxicity model has been successfully adapted for its use in microfluidic devices, with preliminary data showing no change in hPTPC sensitivity to cisplatin. Conclusions The phenotypic characterization of our hPTPC confirms typical characteristics of proximal tubule, and its preservation in cells cultured on microfluidic channels. The effects of cisplatin are time and concentration dependent. Simultaneous reading of assays for cell viability, enzymatic activity, and cell numbers in hPTPC allow us to perform studies of renal toxicity and repair in cells grown inside microfluidic devices. This is the first step towards a cell culture model for the study of proximal tubule physiology and physiopathology under biomimicking conditions that will include exposure to flow‐mediated shear stress.Support or Funding Information Spain's Ministerio de Economía, grant DPI2011‐28262/C02 and fellowship BEP‐2012‐059562 (to NS‐R)