Animal cell cultures : Risk assessment and biosafety recommendations
Risk assessment of human/animal cell cultures is based on both the intrinsic properties of the cell culture - including subsequent properties acquired as a result of genetic modification(s) - and the possibility that the cell culture may inadvertently or deliberately be contaminated with pathogens. In addition, risk assessment should be performed according to the type of manipulation.
The following intrinsic properties of cell cultures should be considered while performing the risk assessment: source (species of origin), cell types or tissues, culture type.
Recombinant cells may have increased (or decreased) abilities to cause harm to human health and environment compared to their non-recombinant counterparts. The properties that recombinant cells acquire following genetic modification must be determined as well. This includes an evaluation of all events intervening in the process of genetic modification:
Commission Decision 2000/608/EC provides guidance notes for risk assessment of genetically modified microorganisms.
see example 1 : transfection of mouse T-lymphoma cells with human interleukin 2 using mammalian expression vector
see example 2: retroviral packaging cell lines
Cells may be deliberately infected with pathogens. In this case, the determination of potential hazards related to infected cell cultures requires an examination of the inherent properties of the infecting pathogen. This implies an assessment of a list of criteria, which are specific to the pathogen, along with aspects such as the existence of effective therapies or prophylaxis. An evalutation of these criteria have been used to classify pathogens into classes of biological risk, also called risk groups. The biological risk of infected cell cultures will depend on the biological risk of the infecting pathogen(s).
see example : culture of bovine leukocytes infected with Theileria parva
When manipulating cell cultures, the presence of adventitious contaminating agents constitutes the main hazard to humans. Contamination may occur by the source (e.g. infected animals or tissues), during cell handling (repeated passages and use in the laboratory) or by using contaminated biological reagents (e.g. media and additives derived from bovine sources are often contaminated with bovine viral diarrhoea virus, BVDV).
Bacteria and Fungi
In general bacterial or fungal contamination can be readily detected in cell cultures because of their capacity to overgrow cell cultures. Bacterial and fungal infections are relatively easy to prevent and to treat.
Compared to bacterial or fungal infections, contaminations with mycoplasma give more problems in terms of incidence, detectability, prevention and eradication. Mycoplasma infection may go undetected for many passages, causing a variety of unpredictable effects causing harm to the host cell. Mycoplasma infection may also influence the sensitivity of host cells for growth of viruses (Hargreaves et al., 1970). Beside the fact that this intracellular bacteria is one of the most common cell culture contaminants, it should be considered that some of contaminating Mycoplasma spp. belong to class of risk 2. Together with M. arginini, M.orale, M.pirum and M. fermentans, pathogenic organisms like M. gallisepticum (class of risk 3 for animals), M. hyorhinis (class of risk 2 for animals), and M. pneumoniae (class of risk 2 for humans) account for more than 96% of mycoplasma contaminants in cell cultures.
Viral contamination needs particular attention because infection may be without cytopathic effect for the cell culture or may be latent (e.g. herpesvirus) and hard to detect. Human and non-human primate cultures are more likely to harbour viruses that are highly pathogenic to humans. Of particular concern are the blood-borne viruses such as Hepatitis B Virus (HBV), Human Immunodeficiency Virus (HIV) and others such as Hepatitis C virus (HCV) and Human T-cell lymphotropic viruses (HTLV). However, non-human cell cultures are not without risks as they may contain viruses with a broader host range able to infect humans such as rodent cell culture carrying hantavirus (Lloyd G & Jones N, 1986) or primate cells harbouring Marburg virus.
see example : Lymphocytic choriomeningitis virus (LCMV) contamination of murine cell cultures
Adventitious contamination with parasites may be an issue when handling freshly prepared primary cell cultures or tissue cultures originating from a donor organism that is known or suspected to be infected with a specific parasite. Well-known intracellular protozoan parasites for which laboratory-acquired infections have been reported are Toxoplasma gondii, Trypanosoma cruzi, Leishmania sp, Cryptosporidium parvum , Plasmodium sp. etc. (reviewed by Herwaldt et al., 2001).
Though a limited number of cultured cell lines (e.g. mouse neuroblastoma cell lines Sc N2a) have been shown to promote, upon subpassaging, stable and persistent replication of PrP(Sc) as well as infectivity (Solassal J, et al., 2003), most cell lines are resistant to prion infection (Butler, et al., 1888). However, in contrast to most of the infectious agents, prions are particularly difficult to inactivate. In fact no method can guarantee total inactivation of these agents. So, one should bear these considerations in mind when using growth media of bovine origin.
In addition to the determination of biological risk of animal cell cultures, the consideration of the type of manipulation constitutes a key aspect in the completion of a thorough risk assessment. Issues regarding the type of manipulation include:
As some clinical approaches such as stem cell therapy, gene therapy, xeno- or allotransplantation involve ex vivo cell culturing, many aspects of the risk assessment – as mentioned above- should be applied. However, culturing cells for therapeutic purposes justifies more careful consideration regarding quality, efficacy, safety, ethical, social and regulatory issues that will not be addressed on this webpage. More information can be obtained on Research and development activities with GMO-medicinal products.
The examination of biological risks related to animal cell cultures and the consideration of the type of manipulation allows the determination of adequate containment level in order to protect human and environmental health. The set up and implementation of an adequate containment level include a list of general and more specific work practices and containment measures.
The flowchart hereunder offers a schematic guidance for the assignment of appropriate containment levels and is based on, but not limited to, key features of risk assessment as outlined above. This flowchart is only indicative and should be applied and /or reconsidered according to case specific conditions and risk assessments.
BSC type II = Biosafety cabinet type 2. Contrary to a biosafety cabinet of class I, which only protects the operator and the environment, the use of a biosafety cabinet of class II aims at protecting the operator, the environment and the cell culture, the latter being of importance in order to prevent contamination of cell cultures. In view of this, animal cell cultures should never be manipulated in a biosafety cabinet with horizontal laminar air flow.
The assignment of appropriate containment requirements cannot be generalised, it varies on a case-by-case basis and should rely on a thorough risk assessment, including considerations of intrinsic cell properties (including recombinant properties), potential contamination with pathogens and type of manipulation. Adventitious contaminating agents probably constitute the main hazard associated to the manipulation of cell cultures since they are often difficult to detect and therefore less verifiable.