Review the top 9 technical questions on HUB organoid development from our recent webinar, answered by Dr Sylvia Boj, Scientific Director of Hubrecht Organoid Technology (HUB).
Can you Explain How HUB Organoids are Derived?
HUB Organoid establishment from different tissues is explained in multiple publications from our academic group, the Clevers lab, and is also covered in an earlier blog post:
- Human intestinal organoids - Sato et al. Gastroenterology 2011;(141):1762-72; van de Wetering et al. Cell 2015;(161):933-945.
- Pancreas - Boj et al. Cell 2015;(160):324-38.
- Breast - Sachs et al. Cell 2018;(172):373-86.
- Airways - Sachs et al. EMBO J 2019;(38).
- Ovary - Kopper et al. Nat Med 2019;(25):838-49.
- Oral mucosa - Driehuis et al. Cancer Discov 2019;(7):852-71.
Have you Isolated Adult Stem Cells Other than Intestinal Ones?
We’ve identified Lgr5+ cells with the capacity to generate new cells in liver and pancreas after inducing injury in these tissues.
Other stem cell markers have also been identified, such as:
Can HUB Organoids be Generated from Frozen Tissue?
Yes, organoids can be generated from cryopreserved tissue. The efficiency is lower than using fresh tissue, however, so we only recommend doing this from resected tissue rather than biopsies.
What is a Good Method for Counting Organoids?
We are evaluating several cell counters on the market for organoid counting. Currently, we make a dilution step of organoid suspensions, spreading 10µl of organoid solution to make a long line (rather than a drop) and then count the organoids.
How are HUB Organoids Expanded?
Organoids are expanded by disrupting them into several pieces. This disruption can be:
- Mechanical - by pipetting them with either low retention tips or glass pipets (though this last technique should only be used when the amount of organoids for disrupting are plated in 200µl of ECM).
- Tryp-shearing - a mechanical disruption that includes the presence of TrypLE (between 25 and 50% volume).
This last method is recommended for compact organoids that cannot be disrupted just by shearing. Importantly, we do not trypsinize organoids in the traditional way (100% TryPLE and incubate in a water bath). This is because, for most organoid models, if they reach single cell level only a few cells will grow and generate new organoids.
After disrupting the organoids, they are centrifuged and resuspended in new ECM.
Regarding Expansion - Have You Evaluated the Genetic Profile of Organoids between Passages? What's the Highest Number of Passages You Recommend?
For normal models generated from non-cancerous tissue, organoids have been expanded for a year, being passaged weekly. The accumulation of mutations was comparable to the accumulation of mutations that our somatic cells experience (Huch et al. Cell 2015;(160):299-312; Blokzijl et al. Nature 2016;(7624):260-264).
For cancer models, the intrinsic nature of the tumor is to accumulate more mutations or acquire genomic instability. When generating a new model from isolation or after receiving a cryovial, we always recommended to generate a master cell bank (MCB) and after that a WCB, and thaw the WCB for the experiments.
Dependent on the organoid model, for organoids that are expanded weekly (e.g. intestinal, pancreatic, or liver models) 2-3 months is the average time they can be expanded. For models that are expanded every 15-18 days (breast or lung models) they can be in culture up to 4-5 months.
How do HUB Organoids Change over Passage?
Organoids are susceptible to the quality of growth factors and organoid density in the ECM drop. If media is not refreshed frequently, organoids are plated too crowded (meaning there is not enough signaling to maintain proliferation of all cells) or too diluted, organoid cells can differentiate. This loses proliferative cells and the possibility to expand the organoids.
Can Organoids be Cryo-Preserved after Formation? And then Thawed Later Ready for Immediate Use?
Yes, HUB organoids can be cryopreserved. After thawing, we always recommend at least one passage to let them recover. But if the experiment requires it, the organoids can be used right after thawing. In this case, it’s important to use a good freeze-thaw protocol for organoids to ensure more than 85% viability after thawing.
Can Organoids be used for Organ on a Chip Technologies and Studies?
Yes, HUB is collaborating with some companies to include organoids rather than cell lines in microfluidic plates. So far, we have obtained interesting results with intestinal and kidney organoids.
