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EVOS FL Auto 2 and Organoid Research

2,066 views · Published 19 July 2018 · 3:31 · Indexed 4 November 2026

Channel: Thermo Fisher Scientific · 2018 · Science & Technology

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https://thermofisher.com/us/en/home/life-science/cell-analysis/cellular-imaging/cell-imaging-systems/evos-fl-auto.html?cid=BID_R01_PJT3354_BID88888_VI_YUT_OD_KT_615
Isolation, culture, and imaging of mouse small intestine organoids as taught at the CamBioScience workshop, Cambridge, UK.
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At the CamBioScience Discovering Organoids Course instructed by world leading specialists including Dr Bon-Kyoung Koo, the Invitrogen EVOS FL Auto 2 Cell imaging System from Thermo Fisher Scientific has proved an ideal system for the study of mouse small intestinal organoids.

Within the mouse small intestine, villi are protrusions that consist predominantly of mature cells that live only for a few days
Crypts are invaginations of the epithelium around the villi, and are lined largely with younger epithelial cells which are involved primarily in secretion. Toward the base of the crypts are stem cells, which continually divide.

The first step to preparing the crypts is to remove excess fat.
The intestine is flushed with pre-chilled buffer,  carefully cut open and spread with tweezers, before being scraped with a coverslip to remove the villi.

The prepared tissue is cut into small pieces and transferred to a tube containing pre-chilled Gibco Magnesium and calcium-free PBS buffer.  The tube is shaken vigorously to help remove remaining villi, before transferring the tissue pieces to another tube and repeating the process.

Once the supernatant is mostly clear, the sample is transferred into dissociation reagent and mixed continuously for 15 minutes.
A small aliquot is removed and checked under the microscope for the presence of crypts.

The supernatant is spun and the pellet resuspended in pre-chilled Gibco Magnesium and calcium free PBS.  A sample is examined under the microscope to allow counting of the number of crypts.
Once counted, a volume of supernatant estimated to contain 50 –100 crypts is transferred into a 1.5 ml tube and centrifuged to pellet the crypts.

The resuspended  crypts are seeded into a tissue culture plate containing a 3D matrix and a Gibco media before being incubated at 37 degrees Celsius.
 
The media will be changed every few days and Crypts will typically start budding after 2-3 days.

The number of orgaoids per well can be measured using images taken with EVOS FL Auto2 and analysed with our Invitrogen Celleste data analysis software. The whole well can be scanned, background removed and the no. of organoids counted.
Time-lapse live-cell imaging means we can follow the outbudding process over several days, as highlighted by the arrow.
Alternatively, slices can be taken through the entire depth of the organoid to produce a static z-stack image.

Organoids can be easily fixed and stained with fluorescent reagents to look at structural components.

In this example you can see an organoid stained with two different primary antibodies and the corresponding Invitrogen AlexaFluor secondary antibodies.

Find out more about the new EVOS FL Auto 2 and how it can help you make your next big scientific breakthrough.
https://thermofisher.com/us/en/home/life-science/cell-analysis/cellular-imaging/cell-imaging-systems/evos-fl-auto.html?cid=BID_R01_PJT3354_BID88888_VI_YUT_OD_KT_615

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