extraction and culture of individual organoids
Léa Lagny
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.
Thank you in advance for your reply.
Best regards,
Lea
Abdullah Khan
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?
Yep that's correct.
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.
Yes also correct - it is tricky. Your idea of using something like a stereo microscope is a good one - we've done this before by putting them in a large petri dish in the hood and picking them using a microscope this way. It's just very time consuming - you can use a pateur pipette or a wide bore P1000.
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.
That's likely my mistake - we aim for a final volume of 150uL - but it can be a bit tricky to nail when transferring. I'd suggest looking at the comBO paper for the granular method which is much easier.
Hope that helps, thanks for tooting !
BW
Abs