#3475 NOVEL OPTIMISED PROTOCOL FOR ISOLATION OF PTEC AND SYSTEMATIC CHARACTERISATION FROM HUMAN KIDNEY BIOPSY

Abstract Background and Aims Human kidneys have a role in water homeostasis, acid-base control, reabsorption of compounds, secretion xenobiotics endogenous metabolites, exposing them to substances that could cause harm. This results an alarming number acute kidney injuries (AKI) worldwide, estimated at 13%. Furthermore, one-quarter hospitalised cases are due drug-induced AKI [1]. Current methods for nephrotoxicity assays based on animal testing and/or the use simple human cell lines. Meta-analyses show we can correctly predict drug responses only 10–50% [2]. Our work aimed develop novel optimised protocol isolating proximal tubular epithelial cells (PTEC) from biopsy aid future research regarding studies. Method Isolation cultivation primary adult PTEC obtained with during regular diagnostic procedure was performed. We used consisting micro-dissection tissue sample get ∼1 mm3 fragments, enzymatic dissociation 0.2% collagenase type 1, selective culture media (Advanced DMEM/F12 added insulin, transferrin, selenite (all three together termed ITS), epidermal growth factor (EGF), hydrocortisone). Light microscopy morphologic characterisation. Some were cultured Transwell inserts, transepithelial electric resistance (TEER) measured mature formed confluent culture. For phenotypic characterisation, several markers characteristic chosen [3], immunocytochemical staining performed using fluorescent microscope evaluate phenotype. Results Following described resulted first colonies after 24 h. Using light microscopy, exhibited cobblestone appearance, reached confluence eight days, showed dome (hemicysts) formation 13 days. TEER 169 Ω/cm2 14 The isolated marked positive immunocytochemistry sodium-glucose cotransporter 2 (SGLT2), multidrug-resistant protein 4 (MRP4), organic anionic transporter 1 3 (OAT1 OAT3), cationic (OCT2), p-glycoprotein (p-gp), multidrug toxin extrusion (MATE1), N-cadherin. Conclusion In this study, developed cultivating samples. To best our knowledge, most extensive systematic characterisation following isolation reported date.