I am a green hand in 3D cell culture. I used U-shape bottom 96-well plate with a hydrophobic coating for 3D cancer cell culture, and I found it very difficult to remove or change media without aspirating the spheroid. Are there any tips for that? Thanks!
I wonder what is your goal in completely removing the media. However, if you only wish to renew/refresh the old medium, I suggest you to do it by half the total volume, e.g., 100 uL removal from a total of 200 uL. Aspirate the said amount slowly without touching the U bottom. You can tilt the plate to the side, so your pipette tip is aiming to the wall. Do the medium renewal more often if you worry about not providing optimal condition for your spheroid.
Win Sen Heng Thank you for the suggestion! My goal is to remove the media and embed my spheroid in collagen. What I did was remove part of the media and then add collagen. However, it seems like the media and collagen were not mixing well since I saw some cloudy gels. That's why I would like to remove all of the media. If it is not likely to do that, maybe I should pipette more after adding the collagen.
I think it is technically challenging to entirely remove the medium. Now, I am curious as to why you would like to add collagen. Is it not expected to have cloudy gel when collagen gets solidified? I think solidified gel can get cloudy when solidified depending on the amount and concentration.
Win Sen Heng I would like to add collagen because I want to see the branched morphology (here is an exampleArticle Hypoxia Induces a HIF-1-Dependent Transition from Collective...
). The collagen looked cloudy with naked eyes but rather clear under the microscope. And they are kind of floating in the media...
From that paper, I understood that you need to achieve a certain concentration from your concentrated form of collagen. Hence, you do not really need to completely remove the medium from your spheroid culture. Make it so that the concentration can allow good polymerization of the collagen. Cloudiness may not be a problem since based on your observation, microscopic view is not hindered by the fact that it is cloudy when viewed by naked eyes. The fact that they are floating in the media may be due to inappropriate concentration and polymerization protocol. You may need to check how to use your collagen from its provider.