Organoid Cell Culture – Frequently Asked Questions

We recently finished our Ask the Expert discussion, “Going tiny is the next BIG thing: Tools and Techniques for Organoid Cultures”. During this Ask the Expert session, we covered topics related to the culture of organoids including, media requirements, co-culture with other cells, surface requirements, culture vessels, nutrient delivery and gas exchange, harvesting organoids, and key points on the cell culture process timeline. In addition, we also had questions about making organoid cell culture more high throughput to meet the needs of drug screening, the difference between spheroids and organoids, and best practices for immunostaining of organoids.

Organoids have become increasingly popular as they allow scientists to create lab-grown miniature versions of organs that more closely resemble the composition and functionality of organs.  These “mini-organs” so far include kidney, liver, brain, prostate and pancreas. Organoids support advancements in the study of organogenesis, disease modeling and subsequently the development of new therapies.

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.

This Ask the Expert session, helped to demystify some of the most frequently asked questions about organoid culture. The session was hosted by 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 attachement 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.

Below is a sneak peek of the discussion, for a full transcript, please see – Ask the Expert – Going tiny is the next BIG thing: Tools and Techniques for Organoid Cultures



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.


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:

  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.


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.

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