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Two-Dimensional (2D) and Three-Dimensional (3D) Cell Culturing in Drug Discovery
Tubular fluid flows down the loop of Henle from the proximal tubule, where water is reabsorbed further increasing the tubular concentration of drug to potentially toxic levels. Tubular cells in the collecting duct and loop of Henle are at further risk for nephrotoxicity as they are highly metabolically active due to the presence of cytochrome Ps and other enzyme systems, therefore nephrotoxicity may be mechanistically linked to reactive oxygen species ROS as well as direct effects of drug metabolites 9.
To recapitulate this complex structure and function of the kidney in vitro is challenging. Current methods to detect potential DIN using in vitro high-throughput cytotoxicity screens have primarily relied upon using 2D monolayers of either primary kidney cells or kidney cell lines derived from proximal tubules, such as HK Interference of drugs with transporters is another effect of nephrotoxicants and the uptake of proteins such as albumin can be inhibited. The use of primary kidney cells is preferred over immortilised cell lines as the latter lack many of the characteristics of their primary analogues often required for toxicity prediction with any clinical relevance.
In addition, the lack of longevity seen with 2D in vitro models limits nephrotoxicity determination and is driving the desire to develop models that are more stable in culture. In contrast to liver and cardiac 3D in vitro modelling which has been heavily influenced by microtissue formation, 3D kidney models began utilising transwell membrane culture systems and only recently expanded into microtissue development.
Proximal tubular cells grown on transwell membrane culture systems allow the formation of epithelial barriers as they occur in vivo and recapitulate the apical and basolateral uptake of compounds in vitro. DesRocher et al 11 used immortilised human renal cortical epithelial cells in a transwell dish. This model permitted long-term culture of humanderived kidney cells with in vivo-like epithelial barriers. The authors concluded that 3D culturing in a transwell format improved in vitro to in vivo correlation due to enhanced sensitivity compared with conventional 2D kidney cell culture.
Wilmes et al 12 determined the nephrotoxicity of cisplatin is associated with transporter-mediated accumulation of cisplatin and formation and accumulation of cisplatin metabolites in a human renal proximal tubule cell line which ultimately affected several cellular pathways eg Nrf2, p53 signalling. This study was conducted using a day repeat dosing regime. Prange et al 13 demonstrated the successful formation and characterisation of two kidney microtissues utilising either immortilised human kidney cell line HK-2 or primary human renal proximal tubular epithelial cells HRPTEpiC in combination with fibroblasts.
21st Century Cell Culture for 21st Century Toxicology | Chemical Research in Toxicology
The human primary kidney microtissues displayed enhanced expression of epithelial differentiation markers AQP1, megalin and cubilin. Microtissues were also found to functionally uptake albumin and respond with increased sensitivity to known nephrotoxins, gentamicin and cadmium. Cyprotex recently developed multicellular kidney microtissues alongside HEK immortilised human embryonic kidney cells spheroids. Both models were sensitive to a panel of nephrotoxins including diclofenac which elicited induced oxidative stress as detected using confocal high content screening Figure 3. Primary human kidney 3D cell models permit the in vitro replication of clinical repeat exposure strategies in a model with improved in vivo relevance and yet reduced cell usage costs.
Recent and continued developments in 3D culturing of various cell types in co-culture are resulting in a 3D microtissue portfolio with a variety of options for drug safety assessment. Microtissues and spheroids are not only characteristically more in vivo relevant models but they also minimise cell usage allowing traditionally costly primary or stem cell-derived models to migrate to earlier in the safety assessment pipeline. Typically, a well plate of microtissues would require x less cells than required in a 2D format, a dramatic cost reduction when using costly primary cells or iPSCs.
Custom HCS Cell Based Assays
These cost-effective organotypic models represent early in vitro screening tools capable of improving in vitro to in vivo translation. In the pipeline over the coming months and years it can be expected that various other organ types will be the target of microtissue development including, but not limited to, brain, bone and lung.
Organs- On-Chip are composed of a clear, flexible polymer about the size of a computer memory stick which contain hollow microfluidic channels.
The ultimate aim of this technology is to link each human 3D organ model within one system to allow complete in vitro human toxicity profiling of a novel agent. With the expansion of 3D analysis techniques such as the improvements in high content confocal imaging alongside the continual refinement of existing microtissue models and the addition of other organ types and technologies, this field is predicted to become a key aspect of the safety assessment pipeline. Her main responsibilities involve the development of new assays with particular expertise in microtissue models and high content screening.
Stephanie obtained her PhD from the University of Liverpool in collaboration with AstraZeneca and was recently shortlisted for BioNow technologist of the year Her dissertation focused on the validation of several in vitro test methods to predict the skin sensitising potential of cosmetic ingredients and chemicals. Caroline joined Cyprotex in and has been involved in the establishment and validation of in vitro screening methods. Dr Paul Walker is the Head of Toxicology at Cyprotex where he is responsible for the development of new assays and management of client work performed within the Toxicology Group.
Paul further developed his understanding of molecular biology and toxicology during his post-doctoral years at the University of Manchester with a keen interest in the application of high content screening within this field. Paul joined Cyprotex in with his research interests focused on the role of drug metabolism in drug toxicity and in vitro assays to predict toxicity in early drug discovery.
Multicellular tumor spheroids from human gliomas maintained in organ culture. J Neurosurg. Toxicology Sciences. Cyprotex in-house data.
Multi-cell type human liver microtissues for hepatotoxicity testing. Arch Toxicol. A 3D in vitro model of differentiated HepG2 cell spheroids with improved liverlike properties for repeated dose high-throughput toxicity studies. Cardiac endothelial-myocardial signaling: Its role in cardiac growth, contractile performance, and rhythmicity. Phys Revs 83; Tissue Engineering Part C: Methods.
Phenotypic profiling of structural cardiotoxins in vitro reveals dependency on multiple mechanisms of toxicity. Tox Sci 2 ; Renal vulnerability to drug toxicity.
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