My experiment aims to make spheroids by sacrificial micromolding technique.
I am following the easy protocol:
"...The PDMS stamps were deposited onto a 250 μl drop of 3% liquid agarose within the wells of a 24-well plate. Agarose gelation occurred within minutes of placing the 24-well plate at room temperature..." Karen E. Samy, 2019.
The PDMS stamp has pillars with 120 um in diameter and 80 um in height according to the article.
However, the problem is, that during pull out of the PDMS stamp the bottoms or walls of agarose microwells are somehow stuck on the pillars, and they are pulled out with them. So, instead of having nice microwells, there are cones made out of pulled microwells, and the cells can't get inside and form a spheroid.
I have tried 3% agarose (according to protocol), 5% agarose, different timing, place the stamps with agarose to fridge or freezer for 10 - 20 minutes, used plasma on PDMS stamps to make them more hydrophilic and all these combinations together.
I am running out of any other ideas. Does anybody know, what could help my situation?
Unfortunately, the corresponding author hadn't answered.
The photo of the "microwells" is attached. In this picture, you see two successful microwells and poorly done the rest of them.
The elastomeric properties and hydrophobic surface of PDMS should itself facilitate the removal of the master from the stamp leaving the wells intact. Though I am not an expert in the field, I could think of two possible reasons for the issues you are experiencing. Gels made from lower concentrations of agarose will be fragile and lose their integrity of the imprinted features especially while removing the stamp. As you already tried 5% agarose, may be a bit high percentage of agarose (7.5%) can be helpful. Secondly, the thickness of the well bottom also plays a significant impact on the stability of the well. As you are placing the stamp on a 250ul drop of agarose, adding any little extra pressure can result in forming thin and fragile well bottoms.
How about pouring agarose solution in the well plate and molding on it with the second pouring?
Hello guys, thank you for your advice. However, it turns out that whole SU-8 wafer was made in the wrong way. There were missing another nanolayer of the certain chemical substance that makes it easy to remove PDMS from wells. In the first case, the residues of PDMS left in the holes. So now they have adjusted it and it is working well.
- Badges
- Science topic
- Similar topics
- Biological Science
- Immunology