The use of three-dimensional (3D) cell culture models has grown in recent years due to the advantages that these systems can offer over traditional 2D cell cultures. However, 3D cell cultures present unique challenges for optimization. This article shares tips for generating and handling spheroids—the most common 3D cultures—and suggests factors to consider when performing spheroid assays.
Spheroids are 3D aggregates, consisting of one or more cell types. They can be established from stem cells or immortalized cell lines, as well as from both normal and malignant patient tissues. Compared to traditional 2D cultures, spheroids better mimic conditions in vivo to provide more physiologically relevant insights. Spheroids can be cultured using low-adherence plates, such as those with a Corning® Ultra-Low Attachment (ULA) surface. Alternatively, they may be formed using a biological extracellular matrix (ECM) coating, such as Corning® Matrigel® matrix. Spheroids are used for applications including drug screening, disease modeling, and studying the tumor microenvironment.
Spheroid culture can be divided into four main steps: cell seeding, aggregation, maintenance, and screening. Prior to undertaking any of these, you should identify a suitable cell type for your intended application. It is also important to establish whether the cell type in question requires a physical scaffold or biological cues from an ECM in order to thrive, or whether a low attachment product is sufficient.
As a general rule, lower seeding densities and shorter culture times result in smaller spheroids, which offer the advantage of being easier to image and are thus well-suited to microscopy-based applications. Larger spheroids are more often preferred for homogenous assays, such as viability assays for enumerating total ATP, since they produce a greater signal. However, larger spheroids may develop a hypoxic core due to limited penetration of nutrients and oxygen, which can be detrimental to certain assays.
Media formulation is another key factor to consider. Different formulations should be tested with each cell type and, for applications using low-adherence plates, you may wish to think about using supplements (e.g., methylcellulose) to increase the viscosity of the medium if the spheroids are struggling to form. When treating spheroids with drugs or other reagents, longer incubation times or stronger concentrations may be required for larger spheroids.
Lastly, you will want to consider how many spheroids you need and if they must be uniform. Corning® Spheroid Microplates and Corning® Elplasia® round bottom plates both feature round well geometry and a ULA surface to ensure consistent spheroid formation. However, while spheroid microplates enable the formation of a single spheroid per well, Elplasia plates enable the formation of multiple spheroids per well, which can benefit applications requiring greater assay signal or more spheroids.
When performing media and buffer exchanges, it is crucial that the spheroids are not disturbed. This is best achieved using an automated liquid handler, but can also be accomplished by leaving at least 10 to 20 μL residual volume during manual exchanges.
For applications in which the spheroids need to be removed from ULA microplates, the use of 1–200 μL wide bore tips or a 5 mL serological pipet is recommended. Alternatively, if the spheroids are embedded in Corning Matrigel matrix, researchers can either reduce the temperature of the microplate (to liquefy the Matrigel) or add a cold buffer to the wells, depending on the Matrigel concentration. Another option is to use Corning® Cell Recovery Solution, which depolymerizes Matrigel matrix.
Where assays require single cell suspensions, spheroids can be dissociated by incubating with reagents such as Accutase®, 5 mM EDTA, 1X Trypsin/EDTA, or 1X, 5X, or 10X TrypLE™.
Many different types of assays have been developed for studying spheroids. Approaches based on fluorescent imaging are among the most common, although researchers should note that staining a 3D structure may require protocol optimization compared to the 2D equivalent. Broadly speaking, the larger and tighter the spheroid, the longer and more complex it will be for complete staining to occur.
Spheroid assays may also involve co-culturing multiple cell types, such as to investigate cell-cell interactions, create more in vivo like models, or add structure to a cell line that does not easily form a tight spheroid. Critically, whichever type of assay will be performed, rigorous optimization is essential using appropriate positive and negative controls.
Corning® Life Sciences offers an extensive selection of products for 3D spheroid culture, including Corning® Matrigel® Matrix, Transwell® Permeable Supports, Corning® Spheroid Microplates, and Corning® Elplasia® Plates. To learn more, visit 3D Cell Culture Solutions.