Different cells have diverse, sometimes highly demanding, requirements to grow in a culture. In this article, we address key challenges that cell-based assays impose in high-throughput screening.
Selecting microtiter plate surfaces for cell-based assays
Surface for adherent cell culture
The standard TC (Tissue Culture) surface has polar groups such as carboxyl and hydroxyl residues due to a physical surface treatment, which functionalizes the hydrophobic polystyrene surface. This enables improved, consistent cell attachment – ideal for the cultivation of adherent cells. [1]
Surface for suspension cell culture
Suspension microtiter plates with their strongly hydrophobic surfaces are particularly well suited for non-adherent cell cultures, hybridomas and embryonic stem cell cultures.
Cell-repellent surface
Microtiter plates with a cell-repellent surface have been specifically developed to effectively prevent the attachment of semi-adherent and adherent cell lines. As the cell-repellent surface prevents cell-surface interactions, it is an ideal substrate for 3D cell culture applications such as the formation of tumor spheroids or the cultivation of stem cell aggregates.
Figure 1: Human Bone Osteosarcoma Epithelial Cells: Blue – nuclei, red – mitochondria, green – actin fibers
Protein coatings and advanced cell culture surfaces
These surfaces are intended for primary and sensitive cells as well as for cells cultured under restricted growth conditions or stressed by transduction or transfection. Advanced cell culture surfaces are chemically modified to positively affect cellular properties and functions. Improved cell attachment and higher proliferation rates enhance and accelerate cell expansion. Protein coatings are suitable for primary cells, stem cells and applications that require surfaces coated with extracellular matrix proteins such as collagen type I, fibronectin, laminin or with synthetic proteins such as poly-D-lysine or poly-L-lysine. Biological coatings facilitate the growth of many cell types, including hepatocytes, muscle cells, epithelial/ endothelial cells, neural cells, and transfected cell lines. Many otherwise difficult-to-culture cells adhere to biological coatings, enabling successful culture. Additionally, protein coating can have a positive effect on differentiation and morphology for certain cell lines. Those surfaces are also ideal for serum-free and serum-reduced cell cultivation, promoting cell adhesion and stressful procedures such as transfection or automated washing. [1]
Evaporation
One group of experimental errors associated with microtiter plates are the so-called edge effects, which occur when wells along the edge exhibit differences in assay activity compared to wells further in-board. Perimeter wells are not surrounded on all sides by other wells and therefore have at least one side adjacent to the outer environment, resulting in different thermal conditions. Edge effects can lead to additional analytical uncertainties. One of the main causes of edge effects is evaporation along the outer edges of the microtiter plate. Evaporation is a threat to assay performance and can be minimized by using microtiter plates with excellent water and vapor barrier functions. Other strategies to reduce edge effects include ensuring adequate humidity in incubators, microtiter plates with dummy perimeter wells, and filling perimeter wells with assay buffer or media but not including them in the analysis. [2]
New complex cell models
Cell models such as 3D magnetic cell culture and stem cells are opening new research opportunities. In our blog series on 3D cell cultures, you will find detailed information and examples about their use in cutting-edge research, potentials, and practical implementation. Read the articles here.
Miniaturization
Miniaturization usually comes with technical challenges for the assay and requires instrumentation capable of accurately dispensing compounds and reagents into higher density formats. For more information please read our Assay Miniaturization articles. Overall throughput depends on a variety of technical factors, including instrumentation e.g., plate reader speed, and the characteristics of the assay itself. Well-by-well reads are slower than whole-plate reads, which can be an issue if readout stability is time-critical. The appropriate level of miniaturization also depends on the availability of reagents, e.g., valuable cells or cells that are difficult to culture.
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References
[1] Greiner Bio-One Microplate Selection Guide https://www.gbo.com/fileadmin/media/GBO-International/01_Downloads_BioScience/SALES_Brochures/English/F073048_microtiter plate _Selection_Guide_EN.pdf
[2] Auld DS Ph.D., Coassin PA B.S., Coussens NP Ph.D., et al. Microplate Selection and Recommended Practices in High-throughput Screening and Quantitative Biology. In: Markossian S, Grossman A, Brimacombe K, et al., eds. Assay Guidance Manual. Bethesda (MD): Eli Lilly & Company and the National Center for Advancing Translational Sciences; June 1, 2020. https://pubmed.ncbi.nlm.nih.gov/32520474/