Migrating cell-based assay workflows from manual to automated processing typically enhances throughput and assay robustness, but also frees scientists to perform more value-added tasks. Yet the transition is often not straightforward, as each cell line behaves differently and requires optimization. Although no prevailing parameters exist when transitioning cell-based assays into automated workflows, the considerations below may help to maintain the integrity and functionality of plated cells during optimization, validation and development.
#1 – Optimizing Automated Liquid Handling Steps
Cell-based assays are liquid handling-intensive and typically involve cell seeding, media and buffer exchanges, compound and reagent dispensing, and cell washing throughout the workflow. Automating these procedures eliminates variability from human error while reducing time spent on the overall process. When aspirating, tube height and the flow rate must be fine-tuned so that the cells adhered to the well of the microplate are not disturbed or aspirated; however, as little residual liquid as possible should be left in each well before new liquids are dispensed. When dispensing, the same procedure of care holds true. Additionally, when dispensing low volumes with narrow tubing, the prime volume may require optimization to account for the minute change in tubing diameter when the liquid is initially introduced. Lack of conditioning can result in dispensing lower volumes than expected and ultimately, in inconsistent results across the microplate.
#2 – Protecting Cell Health
The direct force of liquid impacting adhered cells during compound and reagent additions using straight tips from a microplate reader’s injectors or a reagent dispenser can cause the adherent cells to detach from tissue culture-treated microplates. Alternatively, angled injection or dispense tubes in a reader, washer or dispenser provide indirect contact and gentler flow rates to protect the integrity of the cells adhered (Figure 1). The same holds true when washing cells or performing automated media and buffer exchanges, in that low flow rates and angled dispense tubes provide non-disruptive aspiration and dispensing steps. When miniaturizing an assay, cell health may deteriorate if the cell density is too crowded or sparse. This can be countered by optimizing the proper amount of cells per well and by confirming cell health through imaging.
Figure 1. Comparison of cells before and after fluid dispensing using either straight or angled dispense tips. Wells imaged at 4x.
#3 – Maintaining Precise Environmental Control
Figure 2. Example of a gas controller used in conjunction with a microplate reader.
Live cell kinetic assays, especially those monitored over long periods of time, may be affected adversely when exposed to unregulated environments. Ultimately, the best results can be obtained where the cells experience optimal conditions in the readout system used. By integrating precise CO2, O2 (Figure 2) and temperature control directly in the microplate reader, the cell-based assay can be conducted in an optimal culture environment, even over long kinetic reads. Edge effects, where liquid in the perimeter microplate wells evaporates at a faster rate than in the central wells, may also skew results, especially in long-term kinetic studies. Humidity levels can be controlled to reduce this effect via a gas-permeable sealing tape that allows adequate gas exchange while maintaining sterility and humidity.
#4 – Instrument Care and Proper Maintenance
Protein buildup and reagent crystallization are common sources of assay failure, and can be prevented through regular and thorough instrument care. Additionally, proper disinfection and separate dispense channels help to prevent cross-contamination during dispensing steps. Ensure that the automated instruments are properly cleaned and maintained per manufacturer recommendations, at regular intervals, to extend instrument life and provide consistent, robust processes from run-to-run.
Cell-based assays are a useful, robust addition to high-throughput drug discovery screening applications. The variable nature of cell lines calls for careful consideration when adapting these assays to simple and robust automated methods. Nonetheless, establishing methods to maintain cell adherence throughout the workflow aids in final assay performance.