Bringing 3D models into a High Throughput Environment

The right product really depends on the model you are trying to create. The ULA products are great if you are trying to create 3D structures that don’t require polarity like spheroids. Flat ULA products will create lots of spheroids but they will be of different shapes and sizes. If uniform spheroids are needed round bottom ULA plates like the Spheroid microplate are a good option. Microcavity ULA plates offer the best of both flat and round ULA products by forming multiple uniform spheroids per well. If polarity of the model is required like with Organoids than Matrigel or some other ECM will likely be required.

We recommend using at least 150-200ug/cm2 of Matrigel for 3D applications.  The concentration may need to be optimized for your specific applications. Alternatively, you could try out Matrigel Matrix-3D plate which has been optimized for 3D culture and is already coated with unpolymerized Matrigel.

It really depends on the application. If the goal is to make lots of 3D structures to extract for protein analysis or a homogenous end point assay embedded protocols might be a good option as they are quick and straightforward. If imaging is required embedded protocols are not ideal as the 3D objects are mixed throughout which can cause long scan times from having to focus on so many different planes. Sandwich protocols involve laying a bed of polymerized Matrigel down before adding cells. This allows cells to settle in a more narrow focal plane making for easier and faster imaging.

The points you bring up can certainly be a limitation. One solution to consider is to limit the amount of data being analyzed. For some applications, analyzing a max projection of a 3D object might be enough to answer the questions being asked. The max projection is the representation of all the confocal images of an object stacked into one image. This requires a lot less time to analyze compared to looking at all the individual z-stacks.  Unfortunately, the technology is not quite there yet, but it is getting better. 3D image quality is improving and getting faster, with features that allow for pre-scanning at lower magnifications to identify objects of interest first before moving to more time-consuming higher magnification imaging. Limiting high resolution images to an “as needed” basis is another way you can reduce data collection.

Scaffold-free products like Corning® spheroid microplates and Corning Elplasia® plates have specialized geometries that are designed to create uniform spheroids. The unique well geometry forces cells to evenly settle into the cavities to form consistent spheroids. Flat bottom scaffold-free products do not feature specialized well geometries, which typically results in many non-uniform 3D structures.

Spheroids and organoids can be imaged while they are still embedded in Matrigel matrix depending on the magnification and what is being imaged. In fact this may be necessary for live/dead staining and organoid swelling assays.  We have found that the sandwich/overlay workflow method, wherein cells are seeded on top of a thick layer of Matrigel matrix are easier and faster for imaging as the spheroids are mostly on a single focal plane. Alternatively, small Matrigel matrix droplets are also thin enough for fast imaging at low magnification. For marker staining of larger organoids at higher magnifications or when organoids are embedded in a thick gel it might be helpful to remove structures prior to staining. If you do need to remove cells from Matrigel matrix, we recommend Corning cell recovery solution for cells/spheroids cultured in Matrigel matrix. This solution will allow non-enzymatic retrieval of spheroids and organoids. It can de-polymerize a thick Matrigel matrix layer at 4°C and facilitate cell retrieval. Here is a protocol. It should also be noted that certain fixatives such as paraformaldehyde can depolymerize Matrigel matrix. If it is desired for the Matrigel matrix to remain intact adding up to 1% glutaraldehyde to fixative can help.

Immunostaining has been done with all types of Matrigel matrix. However, the use of phenol red-free Matrigel matrix will reduce autofluorescence/ background noise.

Yes, new Corning® Matrigel® matrix-3D plates are automation compatible and provide the convenience and consistency required for high-throughput assays. The plates eliminate the need to handle ECM dispensation. This off-the shelf pre-coated option reduces inaccuracies that can occur during manual operations. Corning® Matrigel® matrix-3D plates are available in 384-well and 96-well formats. To learn more about using Corning® Matrigel® matrix-3D plates, read the Guidelines for Use.  You also may find this application note that cover using the new plates in high throughout assays helpful.

It is possible as long as aspiration is not too close to Matrigel layer. For example, I add 50 uL of cell suspension to the 96 well plates and try to remove 40uL or so before adding another fresh 50uL. Alternatively, I have also had success simply adding more fresh medium or compounds without aspirating any medium.

While the format may allow you to do so, the watch out is that it can impact the ability to form 3D structures; we recommend to follow our user guideline for best success.

In order to recover viable organoids or spheroids we recommend using corning cell recovery solution to depolymerize the Matrigel. Here is a general protocol https://www.corning.com/catalog/cls/documents/protocols/CLS-AN-528.pdf