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September 15, 2009 (Vol. 29, No. 16)

Reproducible Dispensing of Live Cells

Automation Helps Remove Pipetting Bottleneck and Avoids Mechanical Stress

  • High-throughput cell-based assays are an integral part of the drug-screening process. With the ability to automate and subsequently accelerate the entire process, high-throughput screening (HTS) allows researchers to conduct biochemical assays to rapidly identify active compounds, antibodies, or genes of interest. The results of such screening experiments provide the starting point for drug design as well as an understanding of various cellular interactions.

    With the ability to screen a wide range of static assays against a library of candidate compounds, HTS provides analysis for cellular events such as kinase activation/inhibition, up/down regulation of signal transduction pathways, and apoptosis, to provide a detailed understanding of potential drug effects. The assays that are incorporated into any one screening campaign are dependent on the therapeutic targets, which can include cancer, cardiovascular disease, and inflammation. However, for any assay, an accurate method of dispensing live cells is essential and must be suitable for all assay types.

    Successfully dispensing cells while maintaining viability is essential to the entire procedure in order to ensure that accurate resulting data is obtained. Traditionally, researchers have used conventional hand-held pipettes to enable gentle dispensing that does not cause any damage, ensuring that cell viability is effectively maintained.

    Although this is effective, pipetting is often viewed as a bottleneck due to its time-consuming, labor-intensive process. Automated reagent dispensers can be used to achieve a higher throughput within screening laboratories. Dispensing in this manner must also be gentle so as not to cause any damage to the cells.

    In this article, we investigate the process of dispensing live HeLa S3 cells into 96- and 384-well microplates using the Thermo Scientific Multidrop Combi reagent dispenser from Thermo Fisher Scientific. The results are subsequently compared with cell viability data achieved using hand-held pipettes. The peristaltic pump technology and optimized dispensing cassettes of the Multidrop® Combi provide the capability to quickly dispense cells into any multiwelled plate type for all varieties of cell-based assay.

  • Culturing the Cells

    In this investigation, HeLa cells were used since they are a well-known and commonly used cell line with an innate ability to proliferate at an extremely rapid rate. As such, these cells are often used in a number of different high-throughput cell-based assays. The HeLa S3 cells were grown in adherent culture (Kaighn’s media with fetal bovine serum and penicillin/streptomycin) using standard culture flasks in a CO2 incubator at 37°C with 5% CO2. The cells were maintained at 0.2–0.6 x 106 cells/mL by passaging 1:5–1:8 every three days.

  • Detaching and Dispensing

    Click Image To Enlarge +
    Figure 1. (A) HeLa S3 cells attached to the bottom of the cell culture flask, (B) in suspension immediately after being dispensed, and (C) 24 hours after adherence to the 96-well plate.

    Enzyme-induced dissociation with trypsin was used to remove and collect the cells from the flask. Commonly used in cell culture labs, trypsins are often added to cleave the peptide chain linking the cells to the surface of the culture flask, dish, or plate. The number of cells collected was determined, and the Multidrop Combi was used to dispense both the cell suspension and reagents into cell culture plates. The tubing was prewet with phosphate buffered saline and primed with the cell suspension, before dispensing the samples. When pipetting into a number of different plates, the cassette was primed before each plate.

    The standard tube-dispensing cassette was used to dispense 100 µL of the cell suspension into 96-well plates, and the small tube plastic tip dispensing cassette to dispense 30 µL into 384-well optical bottom plates. As a result, concentrations of 5,000 cells per well and 2,000 cells per well were observed in the 96- and 384-well plates, respectively.

    As a control measure, an equal number of cells were hand pipetted into the second to last column of the plates, and growth media was dispensed as a blank control. To ensure accuracy and repeatability, cells were dispensed in four replicate plates for both plate types. 

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