Do you have any recommendations on which plate to use for EB/spheroid formation (from iPSCs in order to start a cerebral organoid culture)? I have previously tried ULA 96-wp from PerkinElmer. The cells don't always form just one EB er well but several small ones.
Have you used AggreWell or any other plates and noticed any benefits?
Thanks!
Before anyone starts on a quest for growing entities in culture one needs to assay all available plasticware, media, buffers, sera, etc., to determine what is best for their system. Most Tech Reps will supply a small amount of their materials for testing with the hope that they will receive continuity of orders. Usually, if someone finds something that works, they are hesitant to change to an unknown that has the potential to alter the results from experiment to experiment.
For example, we tested 96-well plates, 48-well plates, 24-well plates, 6-well plates, T-25 flasks and T-75 flasks from ten different manufacturers. Corning and Falcon, included. We had assumed that the same plastic was used for all their culture vessels for each manufacturer. We discovered that that was not the case through culturing with our cells. Falcon T-25's and T-75's worked great, and we have used them throughout our studies. However, for our cells, Falcon's 96-well plates did not work at all. However, Corning's 96-well plates worked great. But, Corning's T-25's and T-75's did not work out. So in the end, we use 96-well plates from Corning and T-25 and T-75 flasks from Falcon. And continuity and reproducibility between experiments has lasted for 20+ years.
Hello,Elina. This is Soyoung.
Actually i have the exactly same problem while i generate organoids.
I used both 96 U-bottom dish and aggrewell.
When I used 96 U-bottom culture dish, several small organoids were generated.
When I used aggrewell, one organoids but several single cells around the organoid were detected.
How did you solve the problem?
Thanks. Greeting. Soyoung
We discovered that if we coated any culture dishes with a 1% type-1 collagen solution and allow to air dry, that almost any cell type tested would attach to the dish. We then used EDTA in species’ buffer, followed by type-1 collagenase (Worthington) to release viable intact cells from the dish. EDTA removes calcium from calcium-dependent binding sites and collagenase digests type-1 collagen at RGD binding sites.
We discovered that if we coated any culture dishes with a 1% type-1 collagen solution and allow to air dry, that almost any cell type tested would attach to the dish. We then used EDTA in species’ buffer, followed by type-1 collagenase (Worthington) to release viable intact cells from the dish. EDTA removes calcium from calcium-dependent binding sites and collagenase digests type-1 collagen at RGD binding sites.
We discovered that if we coated any culture dishes with a 1% type-1 collagen solution and allow to air dry, that almost any cell type tested would attach to the dish. We then used EDTA in species’ buffer, followed by type-1 collagenase (Worthington) to release viable intact cells from the dish. EDTA removes calcium from calcium-dependent binding sites and collagenase digests type-1 collagen at RGD binding sites.
Hi! We have had sinilar situation, in our experience the quality of the cells, meaning passage number, presence of direrentiates cells, the quality of the single cell suspension. Good luck! Btw, We use corning 96 well plates low atatchment.
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