Primary keratinocyte isolation- cell loss - (Dec/02/2010 )

with almost 90% regularity we are experiencing 40-60% cell loss after centrifugation of recently isolated neonatal HEK cells.
Pre-counts are often in the E+6 range, after wards we are in the E+5 range.
This has happened with both the adult isolation we are doing and the neonatal isolation, adult isolation is less of a problem seeing we have so much more surface area to work with.
We are using standard 50ml Falcon tubes for the isolation, have tried aspirating slowly with serological pipettes, and decanting. There does not appear to be an increase in cellular debris (obvious lysing).
We have tried a few things:
Checking the supernatant = a few cells, but not enough to account for all the cells we have lost
Using a mild detergent (pluronic surfactant) to coat the tubes prior to adding the cell suspension
Changing to a milder digestion enzyme for the final disassociation step


I plan to:
Reduce the volumes of digestion enzymes and try to plate without spinning
Stain the tubes to see if there are cells adhered to them


I am wondering:
Could a pH change or osmolarity neutralize an enzyme without being destructive to the proliferating cells? Even if for just the first 24 hours or so. Normally we do not change the media on these cells for 48-72 hours.




Any advice would be well received, thanks!!

So you mean keratinocyte cells, right? HEK sounds like kidney to me...
How many live cells do you expect? I mean, skin is a cell layer comprising of few live and many dead cells, so it is not surprising that you have many dead ones. Do you use Trypan Blue for your cell counts?
What digestion enzyme do you use? I wouldn't recommend Trypsin, it is quite strong, and the absence of serum makes your cells apoptotic. Better use Collagenase P / Dispase treatment (3mg/ml each in complete growth medium, 1hr shaking @ 37C), works better (not so much cell lysis) and no harm to the cells.
Keratinocyte are quite sensitive to growth factors, Ca2+, cAMP and so on. I guess you have a good established protocol?

rsm