Abstract: SA-PO013
Case Series of Novel PTEC Isolations from Human Kidney Biopsy: Optimized Protocols and Systematic Characterisation
Session Information
- Bioengineering: Modeling, Diagnosis, Therapy
November 04, 2023 | Location: Exhibit Hall, Pennsylvania Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Bioengineering
- 400 Bioengineering
Authors
- Bevc, Sebastjan, Department of Nephrology, University Medical Centre Maribor, Maribor, Slovenia
- Petreski, Tadej, Department of Nephrology, University Medical Centre Maribor, Maribor, Maribor, Slovenia
- Varda, Luka, Department of Dialysis, University Medical Centre Maribor, Maribor, Maribor, Slovenia
- Gradisnik, Lidija, Faculty of Medicine, University of Maribor, Maribor, Slovenia
- Maver, Uros, Faculty of Medicine, University of Maribor, Maribor, Slovenia
- Piko, Nejc, Department of Dialysis, University Medical Centre Maribor, Maribor, Maribor, Slovenia
- Ekart, Robert, Department of Dialysis, University Medical Centre Maribor, Maribor, Slovenia
- Hojs, Radovan, Department of Nephrology, University Medical Centre Maribor, Maribor, Slovenia
Background
Human kidneys provide water, electrolyte, and acid-base homeostasis while enabling endo- and exogenous compound metabolism and excretion. Various causes, such as medications (especially in polypharmacy), can quickly disrupt this delicate balance, and currently used methods for nephrotoxicity assays limit the prediction of human response. Our work aimed to develop novel and optimised protocols for isolating proximal tubular epithelial cells (PTEC) from human kidney biopsy.
Methods
We performed nine diagnostic kidney biopsies (informed consent was obtained from the patients) with parts of tissue used to isolate and characterise PTEC. We used two protocols, using five and four biopsy specimens, respectively. The first employed enzymatic dissociation with 0.25% trypsin/EDTA and culture with Advanced DMEM supplemented with 5% fetal bovine serum. In comparison, the second used 0.2% collagenase type 1 and was cultured in selective serum-free culture media (Advanced DMEM/F12 with added insulin, transferrin, selenite, epidermal growth factor, and hydrocortisone). Light microscopy was used for morphologic characterisation, while several markers characteristic of PTEC were chosen for phenotypic characterisation.
Results
Following the protocols resulted in isolating cells that formed first colonies after seven days (range 1-13 days) and three days (range 1-5 days), respectively. Based on light microscopy, the cells exhibited a cobblestone appearance and reached confluence after approximately three weeks following the first protocol and ten days following the second protocol. Population doubling time (PDT) for the best isolate in the first and the second protocol was 29.7 hours and 23.6 hours, respectively. The isolated cells were positive among others for sodium-glucose cotransporter 2 (SGLT2), multidrug-resistant protein 4 (MRP4), organic anionic transporter 1 and 3 (OAT1 and OAT3), organic cationic transporter 2 (OCT2), p-glycoprotein (p-gp), multidrug and toxin extrusion protein 1 (MATE1), and N-cadherin.
Conclusion
In this study, we developed two protocols for isolating and cultivating primary human PTEC from biopsy samples. To the best of our knowledge, we have performed the most extensive systematic characterisation following the isolation of PTEC from the diagnostic kidney biopsy reported.