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Office of Research Assurances Institutional Biosafety Committee

HEK 293 cell lines

Risk summary and guidelines for risk management

The human embryonic kidney or HEK 293 cell line was derived from human embryonic kidney cells grown in tissue culture. The cell line was initiated by the transformation and culturing of normal HEK cells with sheared adenovirus 5 DNA. A portion of human adenovirus 5, nucleotides (nts) 1-4344, is integrated into chromosome 19 of the HEK cells. Human adenovirus 5 causes a range of respiratory illnesses.

HEK 293 cell lines are commonly used in cancer research.

Risk summary

Biosafety Level 2 (BSL-2) protections apply to work with pathogens or infectious organisms associated with human diseases. These organisms pose a moderate health hazard. Centers for Disease Control and Prevention (CDC) guidelines (pdf) require BSL-2 containment for work with HEK 293 cell lines.
Risk factorRisk group
Overall risk group2
RecombinantsRequires a separate risk assessment
Infectious dose3
Environmental stability2
Economic considerations2
Availability of prophylactic and therapeutic treatments3
Mode of transmission2
Host range2

Risk management guidelines

Agent informationFamily Adenoviridae—Genus Mastadenovirus— Human adenovirus Group C—Human adenovirus 5 (HadV-5). Non-enveloped double stranded DNA virus (1). Adenovirus has been completely sequenced and it includes a total of 35,937 nts (15 pp2359). The 1-4344 nts of HadV-5 represents approximately 12% of the full genome. This agent is unusually stable to chemical, physical and adverse pH conditions (2).
Proteins transcribed from this left portion of the Adenovirus include proteins that are involved in oncogenic transformation and in positive regulation of transcription of the early genes of host infection (3), as well as cell cycle genes (4). However, Group C viruses are not known to be tumorigenic (15 pp.2379). It is well established that E1A proteins have mitogenic activity (4). In mammalian cells, the human adenovirus type 5 early region 1A (E1A) oncoprotein functions as a thyroid hormone (TH) dependent activator of the thyroid hormone receptor (TR) (5). Transcription of viral early genes leads to synthesis of some 17 early proteins, many of which perturb host cell or host physiology (4). The Adenovirus DNA genome provides it with the advantage that its DNA can persist in host cells as either a circular extra chromosome (plasmid) or by integration into the host DNA after the complete viral replication has stopped. It has been recently noted that adenoviral DNA from the E1A gene is correlated with COPD (6). The DNA segment which induces transformation is located between 1 and 6% from the left end of the HadV-5 DNA molecule (7). A 2002 study indicates that HEK-293 cells exhibit a pattern of intermediate filament expression similar to that seen in early differentiating neurons as opposed to cells derived from typical kidney epithelial or mesenchymal cells. This fact could have significant implications for experiments that use these cells as kidney cell controls or as non-neuronal control cells (8).
Primary hazardsIngestion via the fecal oral route (9)
Droplet exposure of the mucous membranes (9)
Inhalation (through respiratory droplets) (10)
Contact/hand-to-eye transfer (10)
Special hazard notesGeneralized infections can occur in immunocompromised individuals. Exposure to this agent can cause serious infection in the congenitally immunocompromised, in patients undergoing immunosuppressive treatment for organ and tissue transplants and for cancers, and in human immunodeficiency virus-infected patients. Adenovirus infections in these patients tend to become disseminated and severe. In all immunocompromised patients, generalized illness involving the central nervous system, respiratory system, hepatitis, and gastroenteritis usually have a fulminate course and result in death. Treatments for adenovirus infections are of little proven value (11).
People who smoke may be at higher risk from exposure to HadV-5 (6).
Using Adenoviral vectors in conjunction with these cell lines will result in replication-competent viruses as the portion of HadV-5 incorporated into chromosome 19 contains both the E1 and E3 regions that are generally deleted in commonly used Adenoviral vectors (12).
Lab workers exposed to HEK-Cells may present an in vivo recombination opportunity between the 12% Adenovirus and latent adenoviruses in the human lab worker (host). Alternatively, poor work technique can cause propagation of adenovirus in the HEK cell lines by introduction of adenovirus from the lab worker (13).
Containment requirementsBiosafety level 2 practices and containment facilities for all activities involving HEK-293 cell lines (9).
Required PPEProcedures that may generate droplets (aerosols, splashes, sprays) with HEK-293 cell lines and their derivative should be handled in a Class II BSC with BSL-2 PPE (lab coat and gloves) (9).
When open work with HEK-293 cell lines is performed outside the BSC, recommend wearing a full face shield to protect mucous membranes from potential exposure.
Spills outside BSCAllow aerosols to settle. Wearing protective clothing, gently cover the spill with absorbent paper towel and apply 1% sodium hypochlorite starting at the perimeter and working towards the center; allow sufficient contact time (30 minutes) before clean up (9).
Effective decontaminationSusceptible to 1% sodium hypochlorite, 2% glutaraldehyde, 0.25% sodium dodecyl sulfate (14).
Post exposureThis cell line is a transformed cell line and as such is classified as a Genetically Modified Organism (GMO). The Human Embryonic Kidney cell lines have been transformed with Human Adenovirus 5. Exposure to these cell lines constitutes a reportable event to the Office of Science Policy, part of the National Institutes of Health (NIH) (15).
Report all occupational exposures to the WSU biosafety manager.

Complete an incident report if exposure has occurred.
Medical surveillanceIncubation period: 1-10 days (9)
There is a live vaccine against adenovirus that is administered to the military but is not available for general use in part due to the concern with the live vaccine’s oncogenic potential (16).
Varies in clinical manifestation and severity; symptoms include fever, rhinitis, pharyngitis, tonsillitis, cough and conjunctivitis (9). Infections in immunocompetent individuals are generally localized (16, 17).
WSU oversight1. Submit a Biosafety Approval Form (BAF) to the Institutional Biosafety Committee (IBC).
2. Obtain approval from the IBC before starting work.
3. Develop a current BSL-2 Biosafety Manual that includes an exposure control plan for established cell lines. Submit the manual to the WSU biosafety manager by the submission deadline. When developing your manual, use the BSL-2 biosafety manual template.
4. Environmental Health and Safety will contact you to schedule a facility review.

You must complete the Biosafety Manual and the facility review prior to the IBC meeting. Otherwise, the BAF will likely be deferred until these reviews have been satisfactorily completed.
References:1., Double-Stranded DNA Viruses: Adenoviruses
2. “Quantitative PCR Detection and Characterisation of Human Adenovirus, Rotavirus and Hepatitis A Virus in Discharged Effluents of Two Wastewater Treatment Facilities in the Eastern Cape, South Africa” Martins Ajibade Adefisoye, Uchechukwu U. Nwodo, Ezekiel Green, and Anthony Ifeanyin Okoh; Food and Environmental Virology, 2016; 8(4): 262–274. May 28, 2016.
3. Harvard Catalyst, Harvard Catalyst Profiles, Adenovirus E1A Proteins
4. “Adenovirus type 5 exerts genome-wide control over cellular programs governing proliferation, quiescence, and survival,” by Daniel L. Miller, Chad L. Myers, Brenden Rickards, Hilary A. Coller, and S. Jane Flint, BioMed Central, Genome Biology, April 12, 2007
5. “Cellular Context of Coregulator and Adaptor Proteins Regulates Human Adenovirus 5 Early Region 1A-Dependent Gene Activation by the Thyroid Hormone Receptor,” Xianwang Meng, Yong-Fan Yang, Xiemin Cao, Manjapra V. Govindan, Michael Shuen, Anthony N. Hollenberg, Joe S. Mymryk, Paul G. Walfish; Molecular Enocrinology, June 1, 2003
6. “Latent Adenoviral Infection in the Pathogenesis of Emphysema”, James C. Hogg and Shizu Hayashi, University of British Columbia Pulmonary Research Laboratory, St. Paul’s Hospital Vancouver, British Columbia, Canada V6Z 1Y6.
7. “Size and location of the transforming region in human adenovirus type 5 DNA,“ F. L. Graham*†, A. J. Van Der Eb† & H. L. Heijneker; Nature, October 25, 1974; 251, 687–691
8. “Preferential transformation of human neuronal cells by human adenoviruses and the origin of HEK 293 cells,” Gerry Shaw, Silas Morse, Miguel Ararat, and Frank L. Graham; The FASEB Journal, June 2002, vol. 16 no. 8 869-871
9. Public Health Agency of Canada, Adenovirus types 1,2,3,4,5 and 7, Pathogen Safety Data Sheet
10. “Adenovirus Infections,” Gale Encyclopedia of Medicine, 3rd ed., Copyright 2006, Thomson Gale
11. “Adenoviruses in the immunocompromised host,” JC Hierholzer; Clinical Microbiology Reviews, 1992 Jul; 5(3):262-74
12. “Adenoviral Producer Cells,” Imre Kovesdi and Susan J. Hedley, Viruses, 2010 Aug; 2(8): 1681–1703
13. “Using Viral Vectors in Animal Research” (pdf) presented by Dr. Bruce Crise and Dr. Steve Hughes on October 14th, 2006 at the American Biosafety Association National Conference
14. Free Safety Data Sheet Index, Adenovirus types 1, 2 ,3 ,4, 5 and 7; MSDS online
15. NIH Guidelines, National Institutes of Health Office of Science Policy
16. “The Adenovirus Family,” Sreyashi Jhumki Basu, Stanford University
17. “Adenoviruses,” Joanne M. Langley, MD, MSc, Pediatrics in Review, July 2005, Volume 26, Issue 7