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Assessment of Chemosensitivity | Formation of Co-Culture Spheroids | Assay Performance

Excellent Assay Performance in Perfecta3D™ Hanging Drop Plates

Perfecta3D™ Hanging Drop Plates offer simplified liquid handling procedures and compatibility with high-throughput screening (HTS) instruments, which are essential to wider usage of 3D cultures in research and pharmaceutical development. One distinguishing feature of Perfecta3D™ Hanging Drop Plates is the ability to perform assays and obtain assay readouts directly on the 3D culture plates. This is a key differentiator over traditional hanging drop culture methods and competing 3D cell culture technologies.

In drug discovery, the ability to identify true active compounds, or hits, depends greatly on the quality of assays and proper analysis of data. Therefore, high-throughput 3D cell culture needs reliable assays for endpoint analysis to obtain accurate readouts. This means to identify hits, a high degree of accuracy and sensitivity in the assay is essential, and a high quality assay must be able to identify the few compounds with desired biological activity with high confidence.

To demonstrate the performance of HTS assays in Perfecta3D™ Hanging Drop Plates, the quality of HTS assays performed on the Perfecta3D™ Hanging Drop Plates were characterized. Z-factor calculations were used as assay performance measures to validate the robustness of fluorescence- and colorimetric-based assays. Z-factor is a widely accepted standard to evaluate the quality of a HTS assay. The range of Z-­factor is from negative infinity to 1, with > 0.5 as a very good assay,> 0 an acceptable assay, and < 0 an unacceptable assay.

To calculate the Z-factor of Perfecta3D™ Hanging Drop Plates for fluorescence and colorimetric-based assays, hanging drops from various concentrations of fluorescein and yellow food color, respectively, were formed according to the concentration maps shown below. The placement setups were designed to remove any positional effects contributing from each column and row and thus normalize the entire plate.

The prepared Perfecta3D™ Hanging Drop Plates were read on a microplate reader from the bottom of the plate to obtain fluorescence intensity or absorbance readouts, from which the Z-factor at each concentration was then calculated. The same fluorescein and yellow food color solutions were also prepared in standard 384-well microplates and the readouts were similarly obtained to calculate the Z-factors.

Overall, the data shows that Z-­factors calculated for the Perfecta3D™ Hanging Drop Plates are comparable to those values obtained from conventional clear 384‐well microplates. For fluorescence-based assays, Z-factors are all well-above 0.5 at all the fluorescein concentrations tested in the Perfecta3D™ Hanging Drop Plate. This indicates that the fluorescence-based assays performed in the Perfecta3D™ Hanging Drop Plate are excellent within the range of concentrations tested. For colorimetric-based assays, with the exception of the lowest two yellow food color percentages, all the other percentages have Z-factors above 0.5 in the Perfecta3D™ Hanging Drop Plate. This indicates that the colorimetric-based assays performed in the Perfecta3D™ Hanging Drop Plate are excellent within the 30% to 100% yellow food color solutions tested.

Images adapted from:
Tung et al. High-throughput 3D spheroid culture and drug testing using 384 hanging drop array. Analyst, 2011, 136, 473-478. > Read Paper
Hsiao et al. 384 Hanging Drop Arrays Give Excellent Z-factors and Allow Versatile Formation of Co-culture Spheroids. Biotechnology and Bioengineering, 2012. > Read Paper