Spheroid Culture Advancements – Microplates

Corning® Elplasia® round bottom plates are available in 6-, 24- and 96-well formats and feature Corning Ultra-Low Attachment (ULA) surface. Corning ULA surface is a proprietary, animal-free, covalently bonded hydrogel surface that is hydrophilic and neutrally charged. The ULA surface promotes the formation and easy harvesting of anchorage-dependent scaffold free spheroids.  Corning Elplasia round bottom plates are optimal for bulk spheroid formation, collection, and expansion.

Corning Elplasia Square bottom plates are plasma-treated for self-coating applications and come in 6-, 24-, 96- and 384-well formats. Square bottom plates feature a surface with optical qualities suited for image analysis, making them an ideal solution for clonal selection and high magnification imaging of very small clusters.

Yes, the Corning Elplasia plates will support consistent spheroid formation. If you add a homogenous mix of cells to each well, a similar number of cells will fall into each microwell resulting in spheroids of a similar size in each microwell across each well. You can learn more about the Corning Elplasia plates or review the guidelines for use on Corning’s website.

Yes. The Corning Elplasia plates feature a clear bottom and low background autofluorescence. We have successfully imaged with the Elplasia plates using a 20x objective with a confocal imager. Corning Elplasia are also compatible with staining protocols. However, you should take care when adding reagents to the well in order to not disturb the spheroids. We recommend avoiding pipetting near the bottom of the wells as well.

Some adherent cell lines do not form spheroids as well as others. The Corning scientific applications team have noticed that MCF7 cells form loose aggregates when using ultra low adhesion methods alone. One way we have been able to help cells like this successfully form spheroids is to seed cells in the presence of methylcellulose or overlay the cells with Corning® Matrigel® matrix the day after seeding in a round bottom ultra-low attachment vessel.

To dissociate cells from spheroids, we recommend incubating the spheroids with a dissociation reagent, such as Accutase, 5 mM EDTA, 1X Trypsin/EDTA, or 1X, 5X, or 10X TrypLE. The incubation time required to dissociate cells will depend on the cell type, spheroid size, and length of culture time. Once the spheroids have started to dissociate, pipette to mix a few times, which will generate a single cell suspension.

There are a few different methods that can be used to change media in Corning® spheroid microplates. A multi-channel pipette can be used to perform half media changes without disturbing the spheroids. We recommend placing the pipette tips to the side of each well while aspirating and dispensing. Another option for removing media is to use an autoclavable multi-pin aspirator hooked up to a vacuum such as a Stainless Steel Manifold, which can be purchased from Drummond, VWR, or Fisher Scientific. Washers or tubing can be placed on either end of the aspirating pin tool to set the height of the pins, which will limit how far they go into the spheroid microplates. We recommend leaving at least 20 uL/well of culture media so as not to disturb the spheroids. The final option is to use automation. For example, Biotek has an AMX module for their MultiFlow FX Multi-Mode Dispenser that is designed specifically for media changing spheroids.

There are several different methods for spheroid formation that can be used depending on your cell type and application. If you are interested in forming many spheroids of varying sizes, a flat bottom Ultra Low Attachment coated flask or a spinner flask could be used. If you would like to assay single spheroids of the same size, we would recommend Corning 96-, 384-, or 1536-well spheroid microplates. If you need to produce many spheroids of the same size, we would suggest the Corning Elplasia 24-well or 6-well multi-well plates. Corning Elplasia microplates also may be used to generate multiple spheroids per well in order to increase assay response signal or cell number, if that is what your assay requires.

Both cryo and paraffin methods have been successfully used for spheroid staining. The Corning Life Sciences applications team has a protocol available for paraffin embedding. Download the application note, “Spheroid Processing and Embedding for Histology”.

One suggestion is to use pipet tips that are specifically designed to prevent sticking, such Axygen Maxymum Recovery® tips. Another suggestion is to pre-wet the pipet or tip with culture media that contains protein.

Generally, you can make this distinction using phase contrast or brightfield microscopy. If you have a spheroid, you should not see the edges of the individual cells as you would with just a group of cells lumped together. The timing for this is cell type dependent. For example, HT29 cells typically form a spheroid within 1-2 days, but primary human hepatocytes can take up to 7 days to form a spheroid.