Organoid culture has been a game-changer for scientific research, enabling investigators to model complex tissues in vitro. During a typical application, organoids are mixed with an extracellular matrix (ECM), such as Corning® Matrigel® matrix, and droplets are applied to a cell culture surface. Here, the droplets are allowed to polymerize into domes before being overlaid with pre-warmed cell culture medium. Although seemingly straightforward, this process can result in poor growth, detached or dissolved domes, and/or unwanted differentiation if not properly implemented. This article shares tips to improve organoid culture in dome applications when using Corning Matrigel Matrix.
Dome size can have a significant impact on organoid culture since it influences the diffusion of oxygen, nutrients, and other essential factors into the ECM. While mouse organoids are often cultured in a single 50 µL dome per well of a 24-well plate, human organoids frequently require smaller domes to ensure adequate penetration of growth factors. If differences in organoid morphology are observed between the edge and center of a dome, as shown in Figure 1, it is recommended that the dome size be reduced. For experiments involving microscopy-based analysis, the use of smaller domes simplifies imaging due to the organoids being in a more uniform focal plane.
Figure 1. Impact of Corning Matrigel matrix dome size. Representative human intestinal organoids cultured in large (left) vs. small (right) Matrigel matrix domes. Organoids in the center of the larger dome appear more spherical and less complex than organoids on the edges of the dome or in the smaller dome. Scale bar is 800 µm.
Creating robust domes is critically important to maintain organoids for the duration of culture, including media exchanges. A key way of ensuring dome integrity is to dilute Matrigel matrix by protein concentration—not percentage—to account for any lot-to-lot differences. Additionally, repeated freeze/thaw cycles of the Matrigel matrix should be avoided. Researchers are also advised to check that domes are fully polymerized before adding cell culture medium, keeping in mind that larger domes will take longer to polymerize than smaller ones.
Because contact between organoids and the cell culture surface can lead to unwanted differentiation, most applications require that tall domes of Matrigel matrix are formed. These are commonly produced using tissue culture-treated plasticware, which ensures the domes remain attached to the surface throughout the course of the experiment. Alternatively, untreated plates result in less spread domes that are taller but can more easily become detached during frequent medium exchanges. Factors to consider for surface selection include the type of organoid you are working with and the length of the culture period. To increase dome height and speed up polymerization, plates can be placed in a cell culture incubator for ≥24 hours prior to use. Polymerization can also be accelerated by placing a heat block beneath the plate when dispensing Matrigel matrix. However, because this approach may not allow the organoids to settle into a uniform focal plane prior to polymerization, it may not be ideal for imaging assays.
While it is possible to stain organoids without removing them from Matrigel matrix, experimental conditions must be carefully optimized to ensure that reagents both reach their targets and have ample time to bind. The type of organoid, dome size, and Matrigel matrix concentration should all be considered when identifying optimal incubation times and reagent concentrations, and it is suggested that researchers refer to the literature for guidance with their specific model system. In Figure 2, the effect of incubation time on staining with Calcein AM is shown, clearly demonstrating that longer incubation times are needed to stain cells at the center of the Matrigel matrix dome structure.
Figure 2. Effect of incubation time when staining structures embedded in Corning Matrigel matrix. Representative images of 5 µL Matrigel matrix domes with cells stained with 0.04 µg/mL Calcein AM for various amounts of time. Scale bar is 100 µm.
Organoid culture presents unique challenges for optimization, with dome size, Matrigel matrix concentration, and plate type all having a role to play when it comes to producing consistent and meaningful results. Corning® Life Sciences is a leading provider of products for organoid culture, including Corning Matrigel matrix and various tissue culture-treated surfaces, and has developed a suite of resources that can be used to improve organoid culture outcomes. To learn more, visit 3D Cell Culture Solutions.