Cell lines are often immortalised by artificial expression of proteins which specifically cause the knockout of important cancer suppression genes or the activation of proto-oncogenes.

Cellular immortality happens upon impairment of cell-cycle checkpoint pathways (p53/p16/pRb), reactivation or up-regulation of telomerase enzyme, or upregulation of some oncogenes or oncoproteins leading to a higher rate of cell division.

However, this would appear to fall under the purview of Biosafety Level 3 since the protein expression vectors are capable of causing cancer in the researcher when they infect the researcher. Cancer can certainly be considered a serious or lethal disease, and aerosolisation of the vectors, while rare, is not impossible (for example during a spill).

According to the biosafety definitions of Columbia University,

Agents: serious or lethal diseases transmissible via aerosols, e.g., M. tuberculosis, SARS. Recombinant DNA activities using genetic material from BSL-3 organisms or such organisms as host cells.

Why then, does the CDC classify cell line work, including work with viral production cells, as a BSL-2 activity requiring only BSL-2 containment levels, when the act of immortalising the cells would involve working with viral agents that can cause cancer?

Cells immortalized with viral agents such as SV-40, EBV adenovirus or HPV, as well as cells carrying viral genomic material also present potential hazards to laboratory workers. Tumorigenic human cells also are potential hazards as a result of self-inoculation.[...]

Human and other primate cells should be handled using BSL-2 practices and containment. All work should be performed in a BSC, and all material decontaminated by autoclaving or disinfection before discarding. BSL-2 recommendations for personnel protective equipment such as laboratory coats, gloves and eye protection should be rigorously followed.

Are the transformation cells (eg the 293T viral cells containing the transformant viruses) considered Level 3 biosafety risk, and if not, why not?


2 Answers 2


Work with vectors expressing oncogenes or knocking down tumor suppressor genes (particularly if vectors are based on lentivirus) is often done under "BSL-2+" conditions. This essentially means that personal protective measures required for BSL-3 are used, but the specialized ventilation systems and so forth of BSL-3 physical laboratory space are not required. That would seem to address the investigator-safety issues raised in this question. See the complete Columbia policy as an example of the details and the risk-assessment process.

As noted already, once cultured cells have been infected with these vectors the risks to personnel are substantially diminished if not eliminated, so less stringent protections are then required.


Maybe, cells should be contained under BSL-2 and agents (viral particles etc) under stricter BSL-3? That is, after immortalization you can keep cells under lower security.

Biosafety Level 2 builds upon BSL-1. BSL-2 is suitable for work involving agents that pose moderate hazards to personnel and the environment.

Biosafety Level 3 is applicable to clinical, diagnostic, teaching, research, or production facilities where work is performed with indigenous or exotic agents that may cause serious or potentially lethal disease through the inhalation route of exposure

CDC Biosafety in Microbiological and Biomedical Laboratories, Laboratory Biosafety Level Criteria

As of HEK 293 cells, check this out:

Biosafety level 2 practices and containment facilities for all activities involving HEK-293 cell lines.

HEK 293 Cell Line Risk Assessment - IBC

  • $\begingroup$ +1 for the possible interpretation of the definitions. However, do you have any examples of such viral transformants being actually kept in Biosafety Level 3 environments by an academic research lab? $\endgroup$
    – March Ho
    Jul 12, 2015 at 13:09
  • $\begingroup$ my guess is that any research group will try to keep as low safety profile as possible (maybe, except NIH labs) to save money, or time, or stress. Low enough for NIH grants and publishing approval. $\endgroup$ Jul 12, 2015 at 13:15
  • $\begingroup$ @aaaaaa : such work is regulated by institutional biosafety committees, which are in the best position to gauge the relative risks of particular procedures and the level of protection required. That required regulation is independent of funding source. Individual labs that do not get permission for the work or do not follow procedures can be shut down. There is more stress in working with such vectors without protection than is generated in the process of applying for permission or for carrying out the protective procedures. $\endgroup$
    – EdM
    Jul 12, 2015 at 16:25

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