I would like to get organoids of the same size. We filter organoids using a 100 um filter and then pass that filtrate through a 40 um filter followed by flipping the 40 um filter. Then we wash the filter continuously to retrieve the organoids that are stuck. However, that results in a low yield of organoids/cells because I believe they are stuck in the filter and that the washing does not help. Is there a better approach than filtering cells or is there a better way to retrieve organoids that are stuck in the filter? All help is welcome!
Dear Marco, using single-cell seeding in Matrigel often results in a homogeneous population of organoids (although, sometimes there are bigger ones, but these are easily filtered out by slow centrifugation and using the supernatant for your assays containing the same-size organoids). Another way to prevent sticking of organoids, is dipping or prewashing the filter with pure FBS or DMEM-F12 (+5% FBS). Let me know if this helps :-)
Bart Hee This is amazing advice and thank you for your quick answer. I will test it out as soon as possible. How slow is slow regarding your centrifuging step?
Dear Marco Huberts , it depends mainly on the size of your organoids, if they are rather big, gravity is sufficient (place the tube on ice for a few minutes). When they are somewhat smaller, centrifugation around 100 x g is more than enough, but perhaps optimize first (make sure to use a swingout rotor and cooled centrifuge
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