3D Cell Culture: Is Your Microplate Reader Ready for an Extra Dimension?

Traditional 2D cell cultures have been used in drug discovery and cell-biology research for decades, providing quick and convenient in vitro models to study cellular pathways and the effects of small molecules. However, cells growing on a flat, rigid substrate experience an unnatural environment, resulting in markedly different behavior compared with in vivo. Morphology, proliferation, gene expression, metabolism and viability can vary significantly, and this can have a significant impact on the way cells respond to drugs, both small molecules and biopharmaceuticals. As a result, 2D cell-culture techniques are not considered sufficient for many preclinical investigations, meaning a large number of experiments still need to be performed in animal models—a process that is difficult, time consuming and expensive.

Laboratories increasingly are looking to 3D cell-culture techniques to help bridge the gap between 2D in vitro studies and animal models. Although growing cells in 3D is not a recent invention—a hanging-drop technique for growth of frog neurons was first demonstrated in 1907—the quest for greater biological relevance in cell-based assays has led to the rapid development of a number of novel 3D cell-culture techniques over the last decade.

Broadly divided into two categories—scaffold-based and scaffold-­free—these techniques are designed to offer a more in vivo-like environment for cell growth, proliferation and differentiation. Many are designed to enable complete automation of the culture-maintenance processes in a convenient microplate format, providing reproducible 3D cultures for medium-throughput, cell-based screening activities. But will your existing microplate reader let you realize the benefits of 3D cell-based assays, and how can you make sure you’re getting the most out of your instrument setup?

Reading modes

A reader with instrument optics located below the microplate is vital for all cell-based applications, as the quantity and optical properties of the medium within the wells can have a significant impact on top-down measurements, leading to poor reproducibility and loss of data. This is particularly important for 3D cultures because of the increased depth of field. Ideally, the instrument should have an adjustable Z-focus, allowing the user to optimize the height within the well at which the measurement is taken for maximum sensitivity. Some systems provide fully automated Z-focusing, with the optimal Z-position determined by pre-scanning a reference well or multiple wells.

The size of the excitation aperture is also important; ideally, the exciting light source spans the entire culture. This avoids the need to generate average results for multiple ‘spot’ measurements at different points within each well, and several 3D cell-culture technologies have been developed specifically to meet this requirement.

On the emission side, there are several considerations for 3D cell cultures. Scaffold-based 3D cell-culture approaches can suffer from high background fluorescence because of autofluorescence from the scaffold biomaterials; some form of background correction is essential to avoid masking subtle changes in cellular response. A large dynamic range is also important, as the higher concentration of cells per well for 3D cultures can easily lead to large signal variations across a sample cohort.

For some applications, multi-analyte measurement may be possible, so it is important to check that the reader optics offer precise definition of excitation and emission wavelengths for each analyte, with suitable filter sets or monochromators to avoid signal crossover.

Environmental control

Rigorous environmental control is vital to any cell-based application, with even small variations often leading to experimental bias or erroneous results. Although most readers offer some form of temperature control, it is important to understand how this is achieved, as many systems suffer from poor thermostatic control, with large fluctuations over time as the system responds too slowly to increases or decreases in temperature within the measurement chamber. The location of the heating element also can affect well-to-well performance. Uneven heating can cause temperature gradients to form across the plate, affecting both cell growth and enzymatic activity. For long-term studies, this also can lead to differing evaporation rates between wells, which can significantly affect assay performance.

One of the key objectives of 3D cultures is to deliver greater biological relevance for cell-based assays, and maintaining in vivo-like partial pressures of carbon dioxide and oxygen is critical, particularly for the eukaryotic cells generally used in drug-discovery applications. The proliferation and survival of these cell lines is strictly dependent on the culture-medium conditions; the pH of the medium typically is maintained by a bicarbonate buffer system, using precise control of atmospheric CO₂ partial pressure to stabilize the buffer system. In addition, the hypoxic conditions found in vivo can have a significant effect on drug uptake and metabolism, making precise regulation of the O₂ partial pressure equally desirable; this is particularly useful when investigating hypoxia-induced transcription factors in cancer cells.

Most currently available microplate readers lack the ability to control CO₂ and O₂ levels inside the measurement chamber, requiring regular transfer of the microplate between an incubator and the reader for measurement. Although this is possible with some robotic systems, it is not ideal, and manual plate transfer will result in the measurement series being distorted by overnight gaps. Ideally, the reader should offer precise regulation of both CO₂ and O₂ within the measurement chamber, enabling continuous measurement without biasing results.

Summary

3D cell-culture systems represent a real advancement in cell-based assays for drug development, offering greater biological relevance and potentially significant downstream savings. Implementation of 3D culture­-based assays requires a thorough understanding of your multimode reader’s software and hardware architecture to achieve optimal performance, with careful matching of instrument parameters to the requirements of both the culture system and the assay. Ideally, the reader should enable complete automation of the assay, allowing long-term, continuous measurements of cellular response within the measurement chamber and, consequently, improved data quality.

Michael Fejtl is Market Manager for Detection at Tecan Austria

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