The following article originally appeared on October 30, 2017 in The Cell Culture Dish here.
In the last decade, organoid cultures have quickly become a popular way to create “mini-organs” to support the advancements in the study of organogenesis, disease modeling and subsequently the development of new therapies. Scientists are creating lab-grown miniature versions of organs that so far include kidney, liver, brain, prostate and pancreas, that more closely resemble the composition and functionality of organs.
There are many protocols, tools and techniques that can be used for organoid cultures – ranging from microplates, extracellular matrices, hydrogels, and bioprinting to microfluidics. Depending on your cell type, research area and ultimate goals, the options can seem overwhelming.
So how does one know where to start? Learning from the work that has already been done is a great place to start. We’ve assembled a team of Corning Life Science experts, who are ready to answer your organoid-related questions. Corning has over 30 years of experience in 3D cell culture and offers some of the original and most widely used 3D tools, such as Corning® Matrigel® matrix, Transwell® permeable supports and the Corning spheroid microplate.
Corning experts include Feng Li, Senior Scientist Development, Hilary Sherman, Applications Scientist, Himabindu Nandivada, Senior Development Scientist and Nitin Kulkarni, Sr. Scientific Support Specialist. Dr. Feng Li has been working on 3D hepatic model systems for liver toxicity and disease modeling. His recent work includes establishment hepatic 3D spheroid culture procedures, testing primary human hepatocytes (PHHs) for 3D spheroid culture, and assay development for chronic liver toxicity testing and repeated-dosing with PHH spheroids in Corning ultra-low attachment spheroid microplates. Hilary Sherman is an Applications Scientist with Corning Life Sciences. She has worked with a wide variety of cell types including mammalian, insect, primary and stem cells in a vast array of applications, including 3D cultures. Dr. Nandivada has more than 10 years of experience in human pluripotent stem cell culture and material science. Dr. Kulkarni has worked in Scientific Support group for several years supporting 3D cell culture and recently presented talks and a webinar on surfaces used for organoid culturing.
What is the difference in surface requirement between spheroid and organoid culturing?
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.
Are there any animal-free/synthetic surfaces that are suited for growing 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.
Designer matrices for intestinal stem cell and organoid culture
Neural tube morphogenesis in synthetic 3D microenvironments
Is there an efficient way to make organoids compatible with a high throughput drug screening environment?
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.
I would like to hear about your best practices advice for immunostaining of organoids.
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:
Lee, Genee Y., et al. "Three-dimensional culture models of normal and malignant breast epithelial cells." Nature methods 4.4 (2007): 359-365.
I feel one of the biggest challenges in organoid culture is in delivering nutrients and gas exchange especially as the organoids grow. Thoughts or recommendations?
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:
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.
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.
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.
What is the timeline for organoid culture? When should I expect to begin to see the beginning of formation, timing for passaging, media changes?
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:
What are your recommendations for culture vessels that work best with organoids? Something that works well with imaging too.
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.
I am growing intestinal organoid from iPSC cells in Corning Matrigel matrix. I plan to collect the organoids from the matrix for subsequent tests and analysis. Can you please recommend a protocol for harvesting?
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.
- Wash wells twice with 150 µL cold PBS (organoids can be settled by gravity or centrifugation)
- Add 150 µl of cold Corning Cell Recovery Solution per well of 96-well spheroid microplate
- Incubate at 4°C for one hour
- Wash organoids 1x with 150 µL cold PBS
- Re-suspend organoids in fresh medium or desired buffer for subsequent analysis
What do you think about the research that discusses organoid co-culture with endothelial cells? Do you find that the organoids can get adequate nutrients with good culture media?
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:
- 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.
- 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.
- 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.
What is the difference between spheroids and organoids and what they can be used for?
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.
Are there any special media requirements for organoid culture or is it sufficient to just address the specific needs of that cell type?
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.
Is there any evidence of successfully using organoids as working as mini organs for drug testing? I know the thought is there but is anyone using this successfully?
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).
For more information, please see Organoid Models.