(For a context to these annexes, see the main page)
1. Primary cell cultures are obtained directly from tissues and are often the most appropriate in vitro tool for reproducing typical cellular responses observed in vivo. However, as typical cell characteristics are lost during passaging, these cultures must be obtained from fresh tissue that may contain or may become inadvertently contaminated with pathogens. Consequently, primary cell cultures have increasing risks for potential contaminating pathogens compared to continuous, established cell lines.
2. Continuous cell lines may survive almost infinite serial passages. These immortalized cells are obtained by either isolating cells from tumours (neoplastic origin), treatment of primary cells with mutagens, viruses or recombinant DNA or by cell fusioning of primary cells with a continuous cell line. As a consequence, the class of risk of cell lines is often correlated to the class of risk of primary cells of whom they are derived. Due to the immortality of the continuous cell lines, their potential ability to induce tumours has also to be considered.
3. Example: Transfection of mouse T-lymphoma cells with human interleukin-2 using a mammalian expression vector.
- Host organism properties: mouse T-lymphoma cells (derived from BW 5147 lymphosarcoma line) that only exhibit tumorgenicity property in syngeneic mice following subcutaneous inoculation. The cells belong to class of risk 1.
- Vector: mammalian expression vector containing
- mammalian and bacterial selectable marker gene(s) (f.e. beta-lactamase coding region, neomycin phosphotransferase gene)
- commonly used viral sequences to promote expression of cloned inserts and selectable markers (f.e. human cytomegalovirus (CMV) immediate-early enhancer/promoter region, Simian virus 40 (SV40) enhancer and early promoter)
- commonly used viral sequences to provide transient, episomal replication in cells expressing SV40 large T antigen such as COS-1 or COS7 cells.
Although originating from pathogens of class of risk 2, these viral sequences do not burden host cells with a phenotype that could cause illness to humans or could infer damage to the environment.
- Localisation of inserted material: integration into the genome
- Insert: human interleukin-2 (IL-2). IL-2 is a central regulator of immune responses. It acts mainly on T lymphocytes, natural killer cells, activated B cells and monocytes by stimulating their proliferation, maturation and activation. It can induce the production of 'secondary' cytokines that can be important for antitumor efficacity. IL-2 plays also a role in anti-inflammatory reactions, in hematopoiesis and in tumor surveillance.
- Biosafety recommendations: Biosafety containment level 1
4. Example: Retroviral stable packaging cell lines are established cell lines which are deliberately and stably transfected with 'helper constructs' to ensure the production of retroviral vectors. The expression of 'helper construct genes' allows high-level constitutive production of viral proteins (f.e. gag, pol and env proteins) which are missing in the genome of the viral vector but are crucial for viral replication.
One of two most important biosafety issues related to the use of packaging cell lines is the fact that replication competent retroviruses (RCR) may be generated as a result of (homologous) recombination events after transfection with retroviral vectors. Therefore the engineering of safer generations of retroviral packaging cell lines consists in minimizing the likehood of generating RCR by increasing the number of recombination events necessary to generate RCR (Dull et al., 1998)
The second biosafety concern is related to the insert of the retroviral vector itself. Clearly, the class of risk of transfected packaging cell line will depend on the class of risk of the retroviral vector itself.
5. List of criteria: Risk assessment of the infecting pathogen should consider:
- Pathogen characteristics (description of the microorganism)
- Host range
- Pathogenicity
- Mode of transmission
- Infectious dose (if known)
- Communicability
- Epidemiology (the assignment of appropriate risk group may depend on the geographic localisation)
- Reservoir
- Vectors
- Zoonosis
- Drug susceptibility
- Susceptibility to desinfectans
- Survival outside host
- Physical inactivation
along with aspects related to the disease caused by the pathogen, such as:
- Treatment
- Immunization
- Prophylaxis
- Laboratory-acquired infection
6. Example: Culture of bovine leukocytes infected with Theileria parva.
- Host cells: primary bovine leukocytes (T-cells). Class of risk 1.
- Infecting pathogen: Theileria parva, a tick-transmitted, intracellular protozoan of veterinary importance and the causative agent of East Coast fever among domestic livestock, is an animal pathogen of class of risk 3 (not pathogenic to humans). The sporozoite form (= infective form) invades bovine lymphocytes where it develops into a non-infective form (shizonts). At this stage host cell transformation and clonal expansion is induced.
- Class of risk of T. parva infected bovine leukocytes: class of risk 2.
- Biosafety recommendations:
- Biosafety level 2 during infection of bovine lymphocytes with T. parva sporozoites.
- Biosafety level 1 for manipulation of T.parva tranformed leukocytes carrying the shizont form of the parasite
7. Example: Lymphocytic choriomeningitis virus (LCMV) contamination of murine cell cultures
Lymphocytic choriomeningitis virus (LCMV) is an arenavirus that establishes a silent, chronical infection in mice but causes aseptic meningitis, encephalitis or meningoencephalitis to humans. LCMV is a potential contaminant of cell cultures. The significance of LCMV contamination has been reinforced by the description of cases of laboratory-acquired LCMV infections arising from contaminated murine tumor cell lines (Mahy, et al. 1991). Laboratory workers that manipulate infected rodents or cell cultures originating from infected animals expose themselves at risk by directly exposing cuts, open wounds or mucus membranes with infected body fluids or by inhaling infectious aerosolized particles of rodent urine, feces or saliva. The risk can be minimized by utilizing animals or cell cultures from sources that are regularly tested for the virus.
Manipulation of LCMV infected material or material with an increased likelihood of LCMV contamination necessitates the implementation of a biosafety containment level 2 (L2). However, it should be kept in mind that the handling of teh neurotropic LCMV itself requires a biosafety containment level 3 (L3).
- respect good microbiological practices, especially those that are aimed at avoiding accidental contamination
- treat each new culture that is manipulated for the first time in the laboratory facility as potentially infectious. The use of a biosafety cabinet of class type II is strongly recommended until the cells have been shown negative in sterility tests for bacteria (including mycoplasma) and fungi. Cell cultures from ill-defined sources should be handled under biosafety level 2 (L2) conditions. If there is a reasonable likelihood of adventitious agents of higher risk class, the cell line should be handled under the appropriate containment level until tests have proven safety;
- clean up any culture fluid spills immediately;
- work with one cell line at a time, disinfect the work surfaces between cell lines handling, aliquot growth medium so that the same vessel is not used for more than one cell line;
- avoid pouring actions that are a potential source of cross-contamination;
- if necessary, carry out a quality control of cells demonstrating the absence of likely contaminating pathogens (e.g. PCR, reverse transcriptase detection, electron microscopy studies for observation of retrovirus-like particles, infectivity assays with sensitive cell cultures or indicator cell cultures).
9. Conditions to be fulfilled in order to consider cells free of adventitious contaminating pathogens:
- the use of well-characterized cell lines or controlled cell sources for primary cells such as specified-pathogen- free (SPF) animals. If no well-characterized cell lines or SPF are available, tests for detection of likely contaminating agens should be negative;
- the use of media sources free from contamination;
- the use of appropriate containment measures to reduce contaminations during sample take or subsequent manipulation of cells (refeeding, washing steps).