Safe sorting of GFP-transduced live cells for subsequent culture using a modified FACS vantage.
BACKGROUND
A stream-in-air cell sorter enables rapid sorting to a high purity, but it is not well suited for sorting of infectious material due to the risk of airborne spread to the surroundings.


METHODS
A FACS Vantage cell sorter was modified for safe use with potentially HIV infected cells. Safety tests with bacteriophages were performed to evaluate the potential spread of biologically active material during cell sorting. Cells transduced with a retroviral vector carrying the gene for GFP were sorted on the basis of their GFP fluorescence, and GFP expression was followed during subsequent culture.


RESULTS
The bacteriophage sorting showed that the biologically active material was confined to the sorting chamber. A failure mode simulating a nozzle blockage resulted in detectable droplets inside the sorting chamber, but no droplets could be detected when an additional air suction from the sorting chamber had been put on. The GFP transduced cells were sorted to 99% purity. Cells not expressing GFP at the time of sorting did not turn on the gene during subsequent culture. Un-sorted cells and cells sorted to be positive for GFP showed a decrease in the fraction of GFP positive cells during culture.


CONCLUSIONS
Sorting of live infected cells can be performed safely and with no deleterious effects on vector expression using the modified FACS Vantage instrument.
Sørensen
T U
TU
Department of Infectious Diseases 144, H:S Hvidovre Hospital, Hvidovre, Denmark.
Gram
G J
GJ
Nielsen
S D
SD
Hansen
J E
JE
D001435:Bacteriophages; D001709:Biotechnology / Q000592:standards*; D002469:Cell Separation / Q000295:instrumentation / Q000379:methods; D002470:Cell Survival; D002478:Cells, Cultured; D005434:Flow Cytometry / Q000295:instrumentation* / Q000379:methods*; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D006801:Humans; D007202:Indicators and Reagents / Q000032:analysis*; D008164:Luminescent Proteins / Q000032:analysis*; D017056:Medical Laboratory Personnel; D009784:Occupational Diseases / Q000517:prevention & control; D012190:Retroviridae / Q000235:genetics; D012192:Retroviridae Infections / Q000517:prevention & control; D012449:Safety; D014161:Transduction, Genetic
D007202:Indicators and Reagents; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins