I have been making mouse embryoid bodies (EBs) using the hanging drop method in bacteriological petri dishes for quite some time.
However, recently I have been having this issue where instead of a hanging drop having only one large EB there are 3-4 EBs of smaller size. Is there a possible explanation for that?
It does not look as a physical problem with the shape of the drop (as gravitation still works OK) but the problem with cell attachement to each other, especially that it is the general problem. Check the medium or make it fresh, or exchange cells for the fresher ones.
After 2 years of troubleshooting, I have finally come up with a suitable answer to this problem. The answer is "SERUM" quality.
In the past 2 years I have tested, at two separate occasions, about 5 different FBS lots from various manufacturer. Out of the total 10, only two of the serum lots helped me form single large EB in a hanging drop. Interestingly, both the serum, from different manufacturer, were HEAT INACTIVATED.
This was a little contrary to the idea propagated by various books and protocols on the topic of mouse embryonic stem cell culture. Usage of heat inactivated (needless to say tested) FBS is recommended for mESC culture but for making EBs one can use the regular (NON heat inactivated FBS).
Since it was only heat inactivated serum that gave me the required results I was looking for, I dug a little deeper into literature and realized that heat inactivation not only inactivates complement but it affects the various physico-chemical properties of the serum (http://www.nature.com/nature/journal/v134/n3390/abs/134628c0.html). One of them being VISCOSITY. Heat inactivation increases the viscosity of the serum and it seems that is what is making the single cells come together and form a single EB rather than 3-4 smaller ones.
The changes in the physic-chemical properties of serum on heat inactivation was studied in detail by Dr. P. LECOMTE DU NOÜY in the late 1920s.
The link mentioned below is for his paper published in 1929 where he showed how temperature affects the serum viscosity.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2323722/
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