3D Spheroids of Human Primary Urine-Derived Stem Cells in the Assessment of Drug-Induced Mitochondrial Toxicity

Abstract

Mitochondrial toxicity (Mito-Tox) risk has increased due to the administration of several classes of drugs, particularly some life-long antiretroviral drugs for HIV+ individuals. However, no suitable in vitro assays are available to test long-term Mito-Tox (≥4 weeks). The goal of this study is to develop a 3D spheroid system of human primary urine-derived stem cells (USC) for the prediction of drug-induced delayed Mito-Tox. The cytotoxicity and Mito-Tox were assessed in 3D USC spheroids 4 weeks after treatment with antiretroviral drugs: zalcitabine (ddC; 0.1, 1 and 10 µM), tenofovir (TFV; 3, 30 and 300 µM) or Raltegravir (RAL; 2, 20 and 200 µM). Rotenone (RTNN, 10 µM) and 0.1% DMSO served as positive and negative controls. Despite only mild cytotoxicity, ddC significantly inhibited the expression of oxidative phosphorylation enzyme Complexes I, III, and IV; and RAL transiently reduced the level of Complex IV. A significant increase in caspase 3 and ROS/RNS level but a decrease in total ATP were observed in USC treated with ddC, TFV, RAL, and RTNN. Levels of mtDNA content and mitochondrial mass were decreased in ddC but minimally or not in TFV- and RAL-treated spheroids. Thus, 3D USC spheroid using antiretroviral drugs as a model offers an alternative platform to assess drug-induced late Mito-Tox.

Precision nephrotoxicity testing using 3D in vitro models

“…It provides a large number of cells and has gained attention for its potential in research, particularly human urinary exfoliated cells (USCs), which have shown promise as a source of cells for drug-induced nephrotoxicity testing in 3D models. This method offers the advantage of being non-invasive, making it more acceptable for routine research and potentially reducing costs compared to invasive procedures [6][7][8][9].…”

“…Precision nephrotoxicity testing aims to overcome these limitations by leveraging cutting-edge technologies such as genomics, proteomics, metabolomics and imaging to develop more accurate and sensitive biomarkers. In this review, we discuss precision nephrotoxicity testing using primary human renal cells in 3D in vitro models as a method for predicting druginduced kidney injury [6][7][8][9].…”

“…3D models more closely mirror cellular architecture [9,11], gene expression patterns [12], oxygen and nutrient diffusion dynamics [13], than 2D culture models. Drugs known to affect microarchitecture, mitochondrial function, and alter gene expression, (i.e., oxygen consumption rate/extracellular acidification rate [OCR/ECAR], mitochondrial membrane potential [MMP], adenosine triphosphate [ATP] production, complex I-V expression and activity, mitochondrial morphology, DNA content-Mitochondrial [mtDNA] content, apoptosis, and reactive oxygen species [ROS]) demonstrate better physiological toxicity in 3D vs 2D models [8,9]. These provide a more realistic representation of in vivo physiology and better predict drug toxicity compared to 2D cultures.…”

“…USCs form functional tubular structures in 3D cultures. These properties make USCs a valuable cell source for the development of 3D in vitro models for evaluating drug-induced nephrotoxicity [6,7] and renal mitochondrial toxicity [8,9]. This review specifically focuses on precision nephrotoxicity testing using human renal cells, particularly USCs, in 3D in vitro models.…”

“…Spheroids: Spheroids are 3D aggregates of single renal cells, such as human renal tubular epithelial cells, or undifferentiated renal stem cells (e.g. USCs [9]). They can be generated using different techniques, such as hangingdrop, centrifugation, or non-adherent surfaces [22].…”