I am growing mouse liver orgnoids. I usually change the medium every 2 to 3 days. But after 2 passages, I can see the organoid growth is deteriorating. Even when I culture the new stock I cannot see the growth. As we dont have liquid nitrogen gas in our lab, I store the stocks at -80°C. If temperature is the problem, then why continues passsaging organoids are also not growing well?
I follow this protocol:
Broutier, L., Andersson-Rolf, A., Hindley, C. et al. Culture and establishment of self-renewing human and mouse adult liver and pancreas 3D organoids and their genetic manipulation. Nat Protoc 11, 1724–1743 (2016). https://doi.org/10.1038/nprot.2016.097
I attached the images of the organoids at day 5 of expected growth and current growth
Poor growth of mouse liver organoid after each passage could be attributed to a variety of factors. Organoid culture can be sensitive and complex, and any changes in growth patterns should be carefully investigated. Here are some potential reasons for the observed poor growth:
1. Genetic Drift: over time and passages, genetic mutations or changes may accumulate in the organoids, affecting their growth and function. This can lead to reduced viability and growth rate. It’s important to periodically verify the genetic integrity of your organoids using techniques such as genetic typing and DNA sequencing.
2. Contamination: contamination with bacteria, fungi, or other microorganisms can significantly impact the growth of the organoids. Make sure your culture equipments and reagents are properly sterilized and periodically tested for contaminants. Contamination can also occur during passaging, so maintaining aseptic techniques is crucial.
3. Cell Quality: the initial quality of cells used to establish the organoid culture can impact long term growth. Poor quality or low viability cells can lead to poor organoid growth overtime. Ensure that your starting cell population is healthy and viable.
4. Media Composition: the composition of the culture media can greatly influence organoid growth. Changes in media formulations, growth factors and supplements can affect the organoids’ ability to proliferate and differentiate. Double – check and be sure that the media is consistent in each passage and stock.
5. Passaging Techniques: improper passaging techniques can damage organoids, impacting their growth. Be gentle during dissociation and ensure that the enzymatic digestion time is optimized to avoid excessive cell stress.
6. Substrate and Matrix: the substrate or extracellular matrix used to grow the organoid can impact their growth and differentiation. Make sure you are using the suitable substrate that provides the necessary cues for proper organoid development.
7. pH and Osmolarity: fluctuations in pH or osmolarity of the culture media can affect the health of the organoids. Regular monitor and maintain the appropriate pH and osmolarity levels.
8. Freezing and Thawing: if you’re using a frozen stock of organoids, improper freezing and thawing techniques can damage cells and lead to poor growth upon revival. Use proper cryopreservation techniques and test the viability of the thawed cells before proceeding.
9. Adaptation to New Conditions: when establishing a new stock or transferring organoids to a different laboratory, they might need time to adapt to the new environment. Gradually acclimatize the organoids to any changes in the culture conditions.
10. Cell Lineage Instability: some organoid cultures can exhibit lineage instability, where cells start to deviate from their original tissue types. This can lead to changes in growth behavior and differentiation potentials.
To troubleshoot this issue, you should carefully review and document your entire culture protocol, including media preparation, passaging technique, and any changes that have been introduced. Systematically address and test each potential factor to identify the root cause of the poor growth. Collaboration with colleague or experts in the field can also provide valuable insights.
