extraction and culture of individual organoids

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Léa Lagny

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May 21, 2026, 8:04:13 AMMay 21

to Bone Marrow Organoids

Hello everyone,

We have a few questions regarding the Nature Protocol article, specifically about the extraction and culture of individual organoids (pp. 2134–2135), as we are approaching this stage.

First, after the centrifugation at 400g for 4 minutes (step 46), do you obtain two distinct phases? Does the centrifugation damage the organoids?

Then, after centrifugation, do you remove the medium from the top of the Falcon tube, remove the excess hydrogel, and add 10 mL of Phase IV medium? Is that correct? Is there no need to wash the organoids with PBS in order to completely remove the hydrogel?

Our last question concerns steps 50–52. You resuspend the pelleted organoids in 10 mL of Phase IV medium using a Pasteur pipette and transfer them into a 50 mL Falcon tube. Regarding the transfer into the 96-well plate, do you mix the suspension thoroughly with a Pasteur pipette, take the entire organoid suspension, and distribute approximately the same volume into each well? Is that correct? Do you have any tips for this step? In addition, have you ever tried placing the organoid suspension in a 6-well plate and, under the microscope, isolating a single organoid using a Pasteur pipette or a cut P1000 tip before transferring it into a well? We are concerned that we may not be able to see the organoids clearly in the Falcon tube.

Finally, how much volume should be added to each well at the end? In step 52, it is written: “The volume of medium per well will also vary slightly and it should be adjusted to 2 mL in the subsequent days.” However, at this stage, we are using a 96-well plate, so I am not sure I understand this point.