We use a collagenase perfusion method to isolate hepatocytes from turkeys. We are culturing them in monolayer with the ultimate goal of using them to evaluate drug metabolism. The experiments were going really well (see July 10 picture of monolayer 20hrs after isolation, 20X), but all of a sudden the cells won't attach well (see patchy attachment and vacuolation in Sept272905 file, ~20hrs after plating, 40X) or die off quickly (see cell debris in Oct 3 file, 2hrs after plating, 40X).
- The media components have not changed. The attachment/plating media is: WEM, HEPES, chicken serum, ITS, Pen-Strep, dexamethasone (5nM), and L-glutamine (2mM).
- We're using a consistent collagenase lot. Concentration in media: 200 U/ml
- The cell yield and the viability are very high, with live cells being >90% consistently.
- There are no obvious signs of contamination (i.e. colour change in media, turbidity, etc.)
- The plating density is 0.5 million/ml
Any thoughts?
Thank you.
Subhash C. Juneja
Did you use any non-adherent dishes by mistake? Check if you order new dishes at that time or borrowed dishes from someone? That is what I can think of if you did not change in media composition.
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