Going tiny is the next BIG thing: Tools and Techniques for Organoid Cultures

A spheroid can be a mere aggregate of one or more cell types where cells do not attach to a surface other than to other cells to form a spheroid. Ultra low attachment spheroid microplates are an ideal tool to form and assay spheroids in the same microplate. Organoids, on the other hand, differentiate and self-organize to form 3D structures that resemble and at least partially function like the organ they resemble. They require attachment to extracellular matrix like Corning® Matrigel® matrix or Collagen-I and specific developmental signals to organize into organoids.

Corning® Matrigel matrix is one of the most successful animal based extracellular matrix that has been used to culture organoids. There are multiple reports on engineering a synthetic matrix to grow organoids with the studies being compared to their morphology/functionality in Matrigel matrix. Researchers have succeeded in generating them for a specific organoid or application and there is a lot of ongoing work. Example can be found in references listed below.

http://www.nature.com/nature/journal/v539/n7630/abs/nature20168.html

http://www.pnas.org/content/113/44/E6831

High-throughput screening with organoids is an emerging technology and scientists are looking for efficient ways to address it. Corning® Matrigel® matrix is the matrix of choice for organoid workflows and lends itself well to the growth of various types of organoids.  There are two commonly used methods to dispense organoids in HTS formats; a “sandwich method” wherein the matrix is dispensed into the plate-format of choice and allowed to polymerize. Next, the organoid cell suspension is mixed with a dilution of matrix and dispensed on top of the polymerized layer and allowed to incubate for an additional duration (Cell 2015 161, 933–945). The other option is to mix the organoid cells with Matrigel® matrix and dispense it into HTS-format of interest using a robotic platform while the plates are kept on a cool rack (Journal of Biomolecular Screening 2016 Vol. 21(9) 931–941). They were then pre-cultured for a few days before challenged with drug compounds. We have also had success with utilizing Corning 96-well spheroid microplates to differentiate iPSC spheroids and then overlay with Corning Matrigel matrix. This method allows for a single organoid to form in each well. An imaging-compatible plate format is ideal if the end-point is microscopy; Corning offers a variety of imaging-compatible plate formats that are either round-bottomed, flat bottom or spheroid bottom. Often, researchers also use viability assays also to assess drug toxicity in these 3D cultures.

Organoids can either be recovered from the hydrogel/extracellular matrix or they can be stained in the matrix itself depending on what matrix and concentration is used. For recovering organoids from Corning Matrigel matrix, the culture can be treated with Corning Cell Recovery Solution (not Dispase as it will make single cell suspension, unless this is the desired result) to release organoids and then proceed with fixing, permeabilizing and staining the organoids. Another option is to stain the organoids directly in Matrigel matrix. This method can give higher background, so blocking/washing will need to be optimized for optimum signal: noise ratio. The use of phenol red-free Matrigel is recommended to reduce background during immunostaining. Often, permeabilization and staining concentrations will need to be higher and incubation steps will be longer in order to penetrate through the entire organoid. If embedding organoids prior to processing, Corning has a protocol available that guides you through the spheroid processing and embedding for histology guidelines (CLS-AN-431). Visikol, is a clearing agent that allows better visualization of 3D structures that have been immuno-labelled. Requirement for antigen retrieval and subsequent staining protocols for embedded organized will have to be optimized for target antigen. Please see some references below:

Nguyen-Ngoc, Kim-Vy, et al. "3D culture assays of murine mammary branching morphogenesis and epithelial invasion." Tissue Morphogenesis: Methods and Protocols (2015): 135-162.

Lee, Genee Y., et al. "Three-dimensional culture models of normal and malignant breast epithelial cells." Nature methods 4.4 (2007): 359-365.

Organoids need a continuous feed of fresh nutrients and waste removal as the culture expands in size and since they are not vascularized, as suggested in your question. There are 3 recommendations that have been used for this purpose:

1. Researchers have been successfully culturing and maintaining organoids embedded in Corning Matrigel matrix droplets which are then suspended in media in Corning Spinner Flasks. Lancaster and Knoblich maintained cerebral organoids of 4 mm in size for up to 15 months. Please refer to the protocol in this paper.
2. We have successfully cultured intestinal organoids by frequently (2 to 3 times a week) changing the media for 2 mm organoids and maintained them for up to 6 weeks. McCracken et. al., passaged these organoids for up to 140 days using this protocol.
3. Perfusion is an option that can continuously replenish the nutrients and remove wastes and is more increasingly utilized in organ-on-chip cultures to maintain all the nutrients, growth factors, metabolites in a constant equilibrium once optimized so cultures can be maintained much longer than static cultures.

Developmental progression of organoids and the timing of different steps in organoid culture are dependent on origin of cells, protocol being used (e.g. media formulations) and the desired organoid type. In some protocols, organoid-like structures or buds can be observed as early as 24 hours after organoid culture process is started. For example, during the generation of cerebral organoids from human pluripotent stem cells, neuroepithelial buds were seen within 1-3 days after seeding of EBs into Matrigel matrix (Lancaster and Knoblich, Nat Protoc, 2014). The long-term differentiation/maturation process might be continued for several weeks or months.

Below are a few review articles that may be of interest on this topic:

• Picollet-D'hahan, et al., Trends Biotech, 2017
• Akkerman and Defize, Bioessays, 2017

The type of culture vessels used for organoid culture is dependent on the protocol being used for organoids. Different culture vessels are used at the various stages of generation, culture and characterization of organoids. Tissue culture-treated vessels (multi-well plates or dishes) can be used together with a natural (Corning Matrigel matrix, collagen, laminin/entactin) or a synthetic hydrogel, to initiate the organoid formation and culture in static culture. Long-term organoids cultures have also been performed in an agitated culture system like an orbital shaker or spinner flasks (Lancaster and Knoblich, Nat Protoc, 2014). Corning spheroid microplates can also be used to generate and culture organoids.  Organoids have also been cultured in drops of a culture medium, either hanging from a plate sustained by gravity and surface tension or in Corning low attachment plates. (Hohwieler M, et al. Gut, 2017, Shamir and Ewald, Nat Rev Mol Cell Biol, 2014).

In terms of imaging, Corning provides a number of microplates in 96 & 384-well formats that are compatible with high-throughput imaging technologies.

We recommend Corning® Cell Recovery Solution, 100mL (Product #354253) for harvesting organoids from Corning Matrigel matrix. Below is a general guideline, using an example of six week old organoids in 96-well spheroid microplates. Incubation time may be optimized for best results.

1. Wash wells twice with 150 µL cold PBS (organoids can be settled by gravity or centrifugation)
2. Add 150 µl of cold Corning® Cell Recovery Solution per well of 96-well spheroid microplate
3. Incubate at 4°C for one hour
4. Wash organoids 1x with 150 µL cold PBS
5. Re-suspend organoids in fresh medium or desired buffer for subsequent analysis

At this point in vivo organoids do not have their own vasculature but endothelial cells have been shown to form rudimentary vascular structures in some organoids like liver buds. When they are implanted into immune-deficient mice, the host vasculature fuses with these structures to form functional vascular network and eventually a functional liver bud in these mice. Please refer to this journal article for further details. There is a significant amount of research where endothelial cells and MSCs with iPSCs are being mixed or where vasculature is being bioprinted in 3D structures. In other cases, endothelial cells were included in co-culture spheroids to enhance the functions, e.g. hepatocyte spheroids as described in this article.

With regards to the second questions, once media is optimized for organoid cultures, adequate nutrition and waste removal is achieved by using spinner flasks, perfusion or frequent media changes. Organoids need a continuous feed of fresh nutrients and waste removal as the culture expands in size and since they are not vascularized. There are 3 recommendations that have been used for this purpose:

1. Researchers have been successfully culturing and maintaining organoids embedded in Corning Matrigel matrix droplets which are then suspended in media in Corning Spinner Flasks. Lancaster and Knoblich maintained cerebral organoids of 4 mm in size for up to 15 months. Please refer to the protocol in this paper.
2. We have successfully cultured intestinal organoids by frequently (2 to 3 times a week) changing the media for 2 mm organoids and maintained them for up to 6 weeks. McCracken et. al., passaged these organoids for up to 140 days using this protocol.
3. Perfusion is an option that can continuously replenish the nutrients and remove wastes and is more increasingly utilized in organ-on-chip cultures to maintain all the nutrients, growth factors, metabolites in a constant equilibrium once optimized so cultures can be maintained much longer than static cultures.

The size and compactness of your organoids do play a role in nutrients and oxygen distribution. There is a limitation of relying on passive diffusion to supply nutrients or oxygen within the organoid mass.  As a result, the metabolic status of cells within a spheroid mass could be very different. Take an example of tumoroid studies, necrosis occurs and signs of hypoxic responses inside those larger spheroids. So factors of organoid type, medium and other culture conditions all need to be considered for nutrient and oxygen supply.

Spheroids and organoids are both 3D structures made of many cells. Although this terminology has been interchangeably used there are distinct differences between them. An organoid is a “collection of organ-specific cell types that develops from stem cells or organ progenitors and self-organizes through cell sorting and spatially restricted lineage commitment in a manner similar to in vivo” (Science 2014. 345:124). On the other hand, multicellular tumor spheroid model was first described in the early 70s and obtained by culture of cancer cell lines under non-adherent conditions (J. Natl. Cancer Inst. 1971. 46:113). Tumorospheres, is a model of cancer stem cell expansion; tissue-derived tumor spheres and organotypic multicellular spheroids are typically obtained by tumor tissue mechanical dissociation and cutting (Neoplasia (2015) 17, 1–15). Generally, there is a higher order self-assembly in organoids as opposed to spheroid cultures and the former is more dependent on a matrix for its generation.

Organoids have recently become of great interest as a model, primarily as may serve as a better in vitro model as compared to 2D or even 3D co-culture systems. Common areas of interest for organoid research include organ development, drug screening, disease modeling, and toxicity testing. The hope is that organoids will bring researchers one step closer to in vitro models. Here are some reviews that discuss recent organoid publications:

Ranga, Adrian, Nikolche Gjorevski, and Matthias P. Lutolf. "Drug discovery through stem cell-based organoid models." Advanced drug delivery reviews 69 (2014): 19-28.

Yin, Xiaolei, et al. "Engineering stem cell organoids." Cell stem cell 18.1 (2016): 25-38.

It depends on the organoid of interest as described by a recent commentary by Sato and Clevers. Researchers have been developing and optimizing their favorite organoid medium for kidneys (Xia Y. et al. (2014) Nature Protocols. 9:2693) (Freedman B. et al. Nat Commun. 2015; 6: 8715), brain (Lancaster M.A. and J. A. Knoblich (2014) Nat. Protocols. 9:2329) or lung organoids (Dye B.R. et al. (2015) eLIFE. 4:e05098) (Nikolick and Rawlins, Curr Pathobiol Rep. 2017; 5(2): 223–231). Usually, a good starting point would be the media used for 2D monolayer differentiation with modifications and optimizations.

There is growing interest in using organoids in drug screening and the pharmaceutical industry is now looking to use the platform as well. However, there are still many challenges to overcome to make this technology routine for drug-screening. The first successes were documented in a subset of cystic fibrosis patients who carry a mutation in the cystic fibrosis transmembrane conductance regulator (CFTR)-gene. Vries team created organoids from rectal epithelium of patients with a host of CFTR mutations and measured organoid swelling in reaction to two drugs, ivacaftor and lumacaftor made by Vertex Pharmaceuticals. They found organoids from 2 patients carrying rare CFTR mutations responded to the drugs. Based on the in vitro success, these drugs were administered to the patients who responded favorably. This was the first success where events in the organoid-response corelated to physiological responses in the patient (Science Transl. Med. 2016. Vol 8, Issue 344:344ra84; Nature Rev. Drug Discov. 2017. 16: 6-7).