polinares

Thanks Charon, do you think a 10kDa filter would work?

Hi all! I'm a PhD student in the Netherlands and I'm in my second year... hence I'm really starting to worry about my PhD project. It's about developing an in vitro testing method for a vaccine that is already commercialized and in use - only they need to test the batch-to-batch variation on mice, and they'd like to switch to cells. Long story short: the products I've been given have proved to be toxic on my cells. One product is the final vaccine, which has a very low viral protein concentration (3 ug/ml) and high alum concentration (2 g/l); the other is the non-adsorbed vaccine (no alum), with a higher protein concentration (60 ug/ml) but also high sucrose concentration (42% v/v). For one reason (too high alum) or another (too high sucrose) these products not only fail to activate my dendritic cells cultures, but they cause a high amount of debris, cell death, and the few results are hardly reproducible given the low viability. Also, the controls (excipient with alum and 42% sucrose in medium) cause the same effects, so it's obvious the problems are caused by the formulation and aren't specific of the inactivated virus (i wished...). I've tried to remove the sucrose/concentrate the proteins with dialysis cassettes, but it hasn't really worked - my first product has really really few proteins and they stick to the cassette, so it's not really concentrating, more like losing I'm trying with the second product now, but I'm quite skeptical as well. These troubles seem something that I can do little about - basically the company thought "let's just give what we use on grown up humans and that should work as well on a 100.000 cells well!", well it doesn't. It has affected my motivation a lot, since I've been trying for long (several dilutions.. timepoints.. cells) and with no good results. Does anybody have any suggestions?