How to Culture Mouse Intestinal Organoids: Isolating Intestinal Crypts and Establishing Organoids
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- Опубліковано 18 жов 2024
- See how to isolate mouse intestinal crypts and establish organoid cultures using IntestiCult™ Organoid Growth Medium (Mouse). Organoids can be established in less than a week and passaged indefinitely.
Learn more about IntestiCult™ Organoid Growth Media at www.IntestiCult....
Watch the video protocol for passaging organoids: • How to Culture Mouse I...
#IntestinalOrganoids #IntestinalStemCells #3Dculture
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This is an EXCELLENT protocol video. It's rare to find a protocol video where the instructions and the video are so very clear, easy to understand and easy to follow. THANK YOU!
You're very welcome! We're thrilled to hear you found this video informative.
This is wonderful work by video.
This is so much WORK!
Scientific research is hard work! But our products and expertise are available to make the work more efficient and manageable :)
good vid
Seem most work at room temperature. What's different in Sato et al 2011 Gastroenterology paper, most performed on ice such as with cold PBS?
Hi Jing-Tan, the tissue post-harvest should be kept on ice to stay cold. We recommend using cold PBS for the wash steps. Likewise, the fractions collected during crypt isolation should be kept cold (e.g. using an ice bucket). The detailed step-by-step protocol can be found here: www.stemcell.com/how-to-isolate-mouse-intestinal-crypts.html.
If you have any more questions, feel free to email us at techsupport@stemcell.com.
Hello! the same procedure is carried out for colorectal cells?
Although we don't work with diseased mice models in-house, and this protocol was tested with healthy mice intestinal sections, it should be possible to follow this protocol for crypt isolation from mice colorectal samples.
It might be worth noting that there are some differences in wash steps and processing between small intestinal and colonic samples (colorectal cancer affects the colon, hence the relevance). The differences between processing small intestine versus colon are captured in detail in this protocol (www.stemcell.com/technical-resources/educational-materials/how-to-isolate-mouse-intestinal-crypts.html). If you have any more questions, feel free to email us at techsupport@stemcell.com.
How about the other cancer? If we use the kit, we have to create especially kit for each cancer type?
We expect different tumor mice models to grow well in IntestiCult™. It is possible that there will be some variance between different cancers, and organoid growth may be variable with cancer samples depending on the type of mutation in the specific sample. If you have further questions, feel free to email us at techsupport@stemcell.com!
How about isolating mouse intestinal epithelial cells and goblet cells?
Hi Sergio, this protocol is not designed to isolate epithelial cells or goblet cells, as the goal of this protocol is to isolate crypts (containing stem cell population) from the intestinal tissue and use the isolated crypts to establish intestinal organoids. It should be possible to isolate different cell types from the intestinal tissue using cell isolation techniques by targeting specific cell surface markers (e.g. EasySep™) if that's of interest. Feel free to contact us at techsupport@stemcell.com if you have any more questions!
How about human colon tissue crypt isolation, since the biopsy samples are usually small, not like mouse intestine tissues taking whatever you want to take?
Hi Jing-Tan, the detailed protocol for human crypt isolation from human colonic biopsies can be found in Section A of the Product Information Sheet (Isolation of human colonic crypts from biopsies) cdn.stemcell.com/media/files/pis/10000003510-PIS_03.pdf.
Although the size of the biopsy often varies, we suggest using ~5mg of tissue whenever possible. If the biopsies are much smaller, please ensure that Rock inhibitor is added.
If you have any more questions, feel free to email us at techsupport@stemcell.com.
@@STEMCELLTechnologies thanks
Beautiful
Highly potential contamination at 3:16. WHY?
One possible explanation for discovering contamination at this stage is as a result of the initial washing steps. If you do not thoroughly wash the tissue during the first couple of steps, some of the residue from the intestine can carry over into the isolation. This might be why instead of seeing "clean" prep with chopped tissue pieces + D-PBS, you might be seeing floating debris carryover from the intestine.
When processing the intestine, we also recommend removing the mesentery prior to cutting the intestine. If it is not removed, it will be hard to spin it out when the intestine is cut and the segments have to be cleaned.
If you have any more questions, please feel free to email us at techsupport@stemcell.com.
@@STEMCELLTechnologies
At minute 3:16 of this video, the technician touches the pipette tip end with his hand. Even if you use antibiotics there is a high risk to spread contaminations into PBS, cell culture, media...
@@lesson3thelarch Our apologies - we misinterpreted your initial comment. At the step you note, it is essential to use sterile gloves and ensure that the pipette tips are opened in a sterile environment. If these procedures are followed, the risk of contamination is low.
@@STEMCELLTechnologies
Once you touch the wrapped serological pipette your gloves are not sterile anymore. It is better to hold the pipette tip through the wrapping bag instead of touching directly the sterile serological pipette.
@@lesson3thelarch It's just a demonstration video. And a good one :) At least they sprayed the items before putting them under the hood. I've been in a lab where they don't even do that. But they don't have contamination problems either (using P/S)