We are thawing reference vials with minimal cell clumping problems; however, the magnitude of the problem will relate to the %PMN in your product. The majority of our apheresis products contain <25%PMN. We thaw the reference vials rapidly in 37C water bath and immediately transfer the thawed sample to cold PBS+1%HSA, using a dilution factor to bring the DMSO concentration to less than or equal to 2.5%. This method minimizes aggregation even on the occasional high PMN contaminated samples. If you are using ACD-A be sure that the PBS does not contain any divalent ions, or this will add to your problems! The cells are then washed and used for CFU or long term culture and flow evaluation. We know longer use DNAse, as it didn't seem to improve the thaw system we are currently using, which has been adapted to a larger scale for washing prior to infusion using CS3000. Based on our previous experience, if you use DNAse, it should be added just as the samples are beginning to thaw, as once the aggregates start, even DNAse doesn't seem to help. The amount is critical, as too high a concentration is toxic; a final concentration of 1-5ug per mL should be adequate to minimize aggregation due to DNA release. To: cyto-inbox cc: (bcc: April G. Durett/MDACC) From: peterc@petermac.unimelb.edu.au Date: 10/20/97 05:17:12 PM ZE10 Subject: CD34 selection and DNAse Dear Group, I have occasionally recovered archival PB CD34 cells from the freezer to use in some CD34 selection/expansion experiments. The usual cell clumping problems are always present and contribute to very high cell losses ( ... cest la vive). I have been thinking about including some DNAse into my thawing buffer to try and minimise this problem (I am assuming that free DNA from disintegrated cells is the major culprit in the whole process). Has anyone in the group tried this ??? what final concentration of DNAse should I aim at ?? Thanks in advance Peter Chapple Melbourne AUSTRALIA
This archive was generated by hypermail 2b29 : Wed Apr 03 2002 - 11:50:15 EST