Lauren E. Barber

Multiplexed assay panel of cytotoxicity in HK-2 cells for detection of renal proximal tubule injury potential of compounds.

Proximal tubules of the kidneys are one of the most common targets of nephrotoxic drugs and chemicals. Screens to predict nephrotoxic potential of compounds with insights to mechanisms of toxicity facilitate lead optimization, guide structure-activity relationships, minimize risks of clinical nephrotoxicity and therefore are valuable in the process of drug discovery. We developed and characterized an in vitro assay multiplexed to measure several endpoints of cytotoxicity using HK-2 cells. Assays for lactate dehydrogenase, cellular caspase 3/7 activation, resazurin dye reduction and Hoechst 33342 DNA staining were multiplexed to maximize the ability to detect cell injury. Assays were performed after 5- or 24-h incubations to further enhance the sensitivity of detection of toxicity. Individual assays were optimized for cell density, assay linearity and assay performance under multiplexed conditions. Inducers of apoptosis (staurosporine) and necrosis (perhexiline) were used to validate the mechanistic aspects of cell death. Nephrotoxic compounds (5-fluorouracil, gentamicin, cisplatin, acetaminophen, para-aminophenol, potassium dichromate, ibuprofen, doxorubicin, cyclosporine, citrinin, puromycin) were used to determine the potential of this method to detect proximal tubule toxicity of compounds. Overall, this cost-effective multiplexed platform is more sensitive than a single endpoint assay, provides mechanistic cues of toxicity and is amenable for higher throughput screening.

Adjunct Automation to the Cellmate™ Cell Culture Robot

The design and implementation of an automated tissue culture flask labeling, delivery, scraping, and retrieval system centered around the CellmateTm cell culture robot is presented. Three new custom subsystems named FlaskMaster, FlaskLabeler, and FlaskScraper were created and integrated into the Cellmate robotic system. Flask-Master is a centralized multi-axis robot with a surrounding enclosure that houses racks of stacked tissue culture flasks (size T-175). The FlaskMaster system interfaces externally with the Cellmates conveyor system to deliver and retrieve flasks. FlaskLabeler is a print and apply system that labels flasks prior to transfer between FlaskMaster and the Cellmate. FlaskScraper is a scraping rod and blade assembly that was specifically designed for automated scraping of T-175 flasks. The combination of these systems allows for large-scale, unattended cell seeding and harvesting of cells from flasks. The addition of these new capabilities significantly reduces the manpower needed to operate the Cellmate. Moreover, these changes greatly improve the performance and capability of the stand-alone Cellmate system.