The following article originally appeared on June 22, 2020 in The Cell Culture Dish here.
We concluded our Ask the Expert session on Bringing 3D models into a High Throughput Environment. 3D cell culture models offer an advanced tool for many key research areas including: understanding biological mechanisms, modeling disease states, therapeutic drug screening, cancer research, and toxicology screening. As throughput needs increase in several areas of 3D cell culture modeling, the need for new tools that support increased throughput are required.
3D cell culture is frequently grown in the presence of an extracellular matrix. For example, Corning’s Matrigel matrix, a natural extracellular matrix (ECM)-based hydrogel, is widely used and referenced in 3D cell culture, in support of organoid and spheroid formation. As throughput needs increase in several areas of 3D cell culture modeling, the need for new tools that support increased throughput are required. This includes the need for more convenient, consistent, pre-coated Matrigel matrix options.
To increase the throughput for screening with 3D models, Corning has developed 96- and 384-well microplates pre-coated with Corning Matrigel matrix specifically for 3D cell culture. These plates are easy-to-use and provide the convenience of pre-dispensed Matrigel matrix in high-throughput formats while providing the consistency required for such assays. These products enable ‘on-top/sandwich’ and ‘embedded’ workflows to generate 3D cell cultures.
For this Ask the Expert Session, we assembled a team of experts to answer questions on increasing 3D cell culture throughput to address the needs of cell culture modeling. Experts discussed imaging best practices, screening troubleshooting, and implementation of automation. In addition, vessel selection and culture tips including harvesting recommendations for small well format plates were covered.
Elizabeth Abraham, Ph.D. – Senior Product Manager, Corning Life Sciences
Elizabeth has been employed at Corning Life Sciences (CLS) since 2008 and has held multiple senior roles in various functions including R&D, Project Management and Business Operations. At CLS, she led the development of various new products including advanced extracellular matrices/surface coatings and laboratory devices for culture of mammalian primary and stem cells. She has authored 23 journal articles, technical notes and is an inventor on 11 patents. In her earlier career, she worked in Cell Therapy evaluating potential stem cell treatments for Diabetes. Currently, she is the business lead on products for organoid and 3D cell culture; translating voice of customer into new concepts and commercializing them globally.
Hilary Sherman – Senior Scientist, Corning Life Sciences
Senior Scientist in the Corning Life Sciences Applications Lab located in Kennebunk, ME. Hilary has been with Corning Incorporated since 2005 and has worked with a wide variety of cell types including mammalian, insect, primary, stem cells and organoids in a vast array of applications. Her key roles at Corning involve creating technical documents such as protocols and whitepapers as well as providing technical support and training for both the Corning sales force and customers. Hilary received her B.S. degree in Biology from the University of New Hampshire. In the last several years, Hilary has focused on 3D cell culture applications including human organoid culture
With flat bottom ULA, round bottom, Microcavity and Matrigel options for 3D how do I pick the right model for my application.
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.
What concentration Matrigel should I use for 3D screening?
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.
I see some protocols where cells are mixed with Matrigel before seeding and others where cells are added on top of Matrigel. Which is best?
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.
I see the value of 3D cell culture and its applications in high throughput screening, but what I have trouble with is imaging systems that can keep up with the demand and also how to quickly organize and evaluate all the data that is generated as a result.
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.
We are using a scaffold-free method for culturing our cardiac spheroids and are having problems getting uniform size, which is leading to inconsistent information and uneven distribution of nutrients. Do you have any suggestions?
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.
Do you recommend imaging spheroids while still embedded in Matrigel matrix or should we dissolve the Matrigel matrix before imaging?
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.
We have found background noise when imaging our cells in Matrigel matrix, do you have a recommendation for removing this?
Immunostaining has been done with all types of Matrigel matrix. However, the use of phenol red-free Matrigel matrix will reduce autofluorescence/ background noise.
Will the new Matrigel matrix-3D plates make automation easier to implement? We have had some difficulties in the past with Matrigel matrix temperature requirements for use with our liquid handling systems.
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.
Media changes in high throughout plates-possible? If so, what’s the best way to do this?
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.
If my application requires more dilute Matrigel, can I easily dilute the pre-coated plates to obtain my desired final concentration?
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.
What is the best way to harvest organoids or spheroids generated in these small well format plates?
In order to recover viable organoids or spheroids we recommend using corning cell recovery solution to depolymerize the Matrigel. Here is a general protocol.