Organoids are 3D collections of cells that model the composition and function of an organ. Scientists have made organoids from a wide range of tissues, including brain, breast, intestine, kidney, liver, lung, pancreas, and prostate.1 To start an organoid, a scientist can use pluripotent stem cells or cells taken from a specific tissue. Scientists use organoids in many ways, such as studying how organs develop, modeling diseases, and developing new therapies, including personalized treatments created from work with an organoid started from a patient’s tumor.
In discussing the key challenges of working with organoids, Hilary Sherman, a senior applications scientist at Corning, says, “A main one is just the difficulty in culturing cells as organoids, because they are very sensitive and need advanced techniques to get them into culture and keep them healthy.” Organoids require unique culturing conditions and media, and the stickiness of an organoid can make working with it complicated.
There are several ways of forming organoids, such as growing them inside a dome of Corning® Matrigel® matrix for organoid culture, which will be much of the focus here, or sandwiching them between layers composed of extracellular matrix (ECM). The following tips can help scientists succeed in a variety of organoid-based applications.
In a dome-based method, organoids grow inside a drop of ECM—such as Matrigel matrix for organoid culture—attached to a culture plate. If the gel spreads too much, though, the organoids won’t be encapsulated in enough ECM, and they might attach to the plate rather than being embedded in the Matrigel matrix, which can lead to differentiation. You need to get the appropriate dome for the application, and the hydrophobicity or hydrophilicity of the surface can impact the dome. To create uniform domes, a scientist should use tissue culture–treated plates, such as Corning’s multi-well plates, and pre-incubate them at 37° Celsius overnight before using them. This step will help prevent domes from spreading out too much when plating.
Use of a dry bath in the hood can help to stabilize the temperature of the plate or dish while making domes. This aids in dome polymerization and really helps out with large domes that need to be carried from the hood to an incubator. For the best results, be sure to gel the domes on the dry bath before taking them to an incubator.
With heavier organoids, more care needs to be taken to prevent the cells from attaching to a plate’s surface. One way to prevent that is by inverting the plate after the domes are dispensed. With it upside down, the organoids stay embedded in the dome, instead of lying at the interface with the plate, which, as noted above, can lead to differentiation.
Depending on the kind of organoids, a scientist creates a dome from a 5–50 microliter droplet. The right size of a dome, though, depends on the application. Some organoids grow better in larger domes, and others prefer multiple smaller domes. Most domes include 20–40 organoids.
Organoid loss during handling can be a big problem, because organoids tend to be very sticky. To solve this, use low-attachment tips, such as Axygen® Maxymum Recovery® tips from Corning.
To harvest or passage organoids, they can be broken up with many methods. For example, enzymes or shearing forces applied by tips or needles can be used, but the structure of the organoid will determine how much force is needed. For cystic organoids that are not budded, less sticky pipette tips or fire-polished glass pipettes can be used. Budded organoids and more cell-dense ones require harsher methods, such as smaller bore tips, syringe-needle dispersion or enzymatic methods.
From start to finish, the tools and techniques determine the odds of success or likelihood of failure when creating and using organoids. In many cases, preparation—such as selecting the right plates and preparing them as needed—makes a crucial difference. Furthermore, the right tools must be used even after organoid formation, such as handling them with low-attachment pipette tips. These complex and fragile structures can only expand tomorrow’s knowledge and unveil new disease treatments when scientists can keep organoids healthy and functioning as naturally as possible.
1. Lancaster, MA; Huch, M. Disease modelling in human organoids. Dis. Model Mech. 2019. 12(7): dmm039347.