A Dusty Miller, Fred Hutchinson Cancer Research Center
29 September 2009
Speaking as an investigator who does much work with retroviruses, and as the creator of the pAMS hybrid retrovirus mentioned in this report as having spread promiscuously in multiple cell lines, I find it incredible to see such widespread contamination by retroviruses in the authors' laboratories. Provided the PCR data in Table 3 are correct and do not represent amplification of endogenous retroviral sequences, and because I work with many of the listed cells lines and know them to be free of replication-competent retroviruses, I conclude that most of the spread has occurred in the authors' laboratories. I also know that it is relatively easy to prevent spread of retroviruses in cultured cells by using good cell culture techniques. Presumably one or a few infected cell lines were imported into the lab and provided the source of the subsequent widespread contamination.
First, I would ask whether many of the authors' cell lines are also contaminated with mycoplasma. This would be another sign of poor cell culture technique where organisms secreted by one cell type are transferred to another, probably by transfer of culture medium containing the organisms. This can occur by using the same pipette to feed multiple cell types, or by feeding a contaminated cell type and then using that pipette to get more medium from a bottle that is later used to feed other cells.
Beyond the use of poor cell culture practices, the other worry is transfer of viruses during storage of cells in liquid nitrogen. Liquid nitrogen can enter and leave standard freezing vials, and I have always wondered if it would be possible that frozen virus might be transmitted between cell lines by this route, although I have never detected this. Storage of cells in the vapor phase above the liquid nitrogen should prevent this provided the liquid nitrogen level never rises above the stored vials. Which type of frozen cell storage was used in this case? In my lab we usually freeze virus-producing cells in sealed glass vials to prevent any possibility of virus transmission, especially when dealing with pathogenic retroviruses. I believe many of the central cell line repositories store cell lines in glass vials as well.
I'd be interested in the authors' thoughts.
Competing interests
None.
Response to “How did these retroviruses spread so widely?”
Thomas Grunwald, Department of Molecular and Medical Virology, Ruhr-University Bochum, Germany
7 October 2009
As mentioned in our paper “Unintended spread of a biosafety level 2 recombinant retrovirus” contamination of cell lines with replication competent retroviruses has been repeatedly observed, even in laboratories of experienced retrovirologists. Systematic investigations on the frequency of such contaminations are, to the best of our knowledge, missing. In response to a recommendation of the German Central Commission for Biosafety (ZKBS), that laboratories working with gene-modified organisms should test their cell lines for the presence of squirrel monkey retrovirus (SMRV), 128 of 4279 tested aliquots of cell lines were reported to be positive. Approximately 70 different cell lines from at least 13 different laboratories throughout Germany were positive for SMRV. Since not all aliquots of the same cell line were positive, this indicates that contaminations do occur under commonly employed cell culture techniques. We agree with Dr. Miller´s assumption, that we have imported one or a few infected cell lines and that the contaminants subsequently spread in our laboratory. However, we reject his insinuation that this has been due to poor cell culture techniques. We are using sterile, disposable tissue culture plastic ware and receive media, fetal calf serum and all other cell culture solutions from common suppliers. Cell cultures are regularly checked for mycoplasma and in the rare case of positivity the cell lines are either disposed off or treated with antibiotics until mycoplasma can no longer be detected. All lab members working with tissue cultures are of course trained not to use the same pipette to split different cell lines or to re-fill a pipette that has been previously used to feed cell lines. At least since 2001, all cell lines are stored in the vapor phase above the liquid nitrogen in plastic cryo tubes with a screw cap. Virus stocks are stored at – 80°C, and not as virus infected cultures in liquid nitrogen tanks. Thus, it remains unclear, as in most previous reports on retroviral contaminants, how these contaminants spread. Formation of aerosols during the splitting of a contaminated culture and subsequent contamination of cultures splitted in the same tissue culture hood via aerosols should also be considered. Even if this is a rare event, the virus might accumulate over time in several cell lines, since different cell lines are maintained simultaneously in the same laboratory for years, particularly if a contamination is not suspected due to lack of cytopathic effects. While one can only speculate, how the retroviral contaminants spread so widely, we do know that by regular testing for the contaminants and systematic elimination of all contaminated cultures we were able to maintain our cell cultures free of SMRV and the hybrid retrovirus without other major changes in cell culture techniques and storage.
How did these retroviruses spread so widely?
29 September 2009
Speaking as an investigator who does much work with retroviruses, and as the creator of the pAMS hybrid retrovirus mentioned in this report as having spread promiscuously in multiple cell lines, I find it incredible to see such widespread contamination by retroviruses in the authors' laboratories. Provided the PCR data in Table 3 are correct and do not represent amplification of endogenous retroviral sequences, and because I work with many of the listed cells lines and know them to be free of replication-competent retroviruses, I conclude that most of the spread has occurred in the authors' laboratories. I also know that it is relatively easy to prevent spread of retroviruses in cultured cells by using good cell culture techniques. Presumably one or a few infected cell lines were imported into the lab and provided the source of the subsequent widespread contamination.
First, I would ask whether many of the authors' cell lines are also contaminated with mycoplasma. This would be another sign of poor cell culture technique where organisms secreted by one cell type are transferred to another, probably by transfer of culture medium containing the organisms. This can occur by using the same pipette to feed multiple cell types, or by feeding a contaminated cell type and then using that pipette to get more medium from a bottle that is later used to feed other cells.
Beyond the use of poor cell culture practices, the other worry is transfer of viruses during storage of cells in liquid nitrogen. Liquid nitrogen can enter and leave standard freezing vials, and I have always wondered if it would be possible that frozen virus might be transmitted between cell lines by this route, although I have never detected this. Storage of cells in the vapor phase above the liquid nitrogen should prevent this provided the liquid nitrogen level never rises above the stored vials. Which type of frozen cell storage was used in this case? In my lab we usually freeze virus-producing cells in sealed glass vials to prevent any possibility of virus transmission, especially when dealing with pathogenic retroviruses. I believe many of the central cell line repositories store cell lines in glass vials as well.
I'd be interested in the authors' thoughts.
Competing interests
None.
Response to “How did these retroviruses spread so widely?”
7 October 2009
As mentioned in our paper “Unintended spread of a biosafety level 2 recombinant retrovirus” contamination of cell lines with replication competent retroviruses has been repeatedly observed, even in laboratories of experienced retrovirologists. Systematic investigations on the frequency of such contaminations are, to the best of our knowledge, missing. In response to a recommendation of the German Central Commission for Biosafety (ZKBS), that laboratories working with gene-modified organisms should test their cell lines for the presence of squirrel monkey retrovirus (SMRV), 128 of 4279 tested aliquots of cell lines were reported to be positive. Approximately 70 different cell lines from at least 13 different laboratories throughout Germany were positive for SMRV. Since not all aliquots of the same cell line were positive, this indicates that contaminations do occur under commonly employed cell culture techniques.
We agree with Dr. Miller´s assumption, that we have imported one or a few infected cell lines and that the contaminants subsequently spread in our laboratory. However, we reject his insinuation that this has been due to poor cell culture techniques. We are using sterile, disposable tissue culture plastic ware and receive media, fetal calf serum and all other cell culture solutions from common suppliers. Cell cultures are regularly checked for mycoplasma and in the rare case of positivity the cell lines are either disposed off or treated with antibiotics until mycoplasma can no longer be detected. All lab members working with tissue cultures are of course trained not to use the same pipette to split different cell lines or to re-fill a pipette that has been previously used to feed cell lines. At least since 2001, all cell lines are stored in the vapor phase above the liquid nitrogen in plastic cryo tubes with a screw cap. Virus stocks are stored at – 80°C, and not as virus infected cultures in liquid nitrogen tanks.
Thus, it remains unclear, as in most previous reports on retroviral contaminants, how these contaminants spread. Formation of aerosols during the splitting of a contaminated culture and subsequent contamination of cultures splitted in the same tissue culture hood via aerosols should also be considered. Even if this is a rare event, the virus might accumulate over time in several cell lines, since different cell lines are maintained simultaneously in the same laboratory for years, particularly if a contamination is not suspected due to lack of cytopathic effects.
While one can only speculate, how the retroviral contaminants spread so widely, we do know that by regular testing for the contaminants and systematic elimination of all contaminated cultures we were able to maintain our cell cultures free of SMRV and the hybrid retrovirus without other major changes in cell culture techniques and storage.
Competing interests
None.