Cultured cells are the starting point for a variety of investigations into differentiation and proliferation of normal cells and their pathological counterparts, in diseases ranging from cancer to diabetes, musculoskeletal and cardiovascular disorders, and in developmental stages from stem cells of human and mouse, to terminally differentiated cells from a variety of tissues.
CCMB provides training to researchers from academia and industry, and distributes a number of commonly used cells lines to research establishments throughout the country. One of the statutory functions of the HFEA is to license and monitor establishments undertaking human embryo research and this will include production of human embryonic stem cell hESC lines. Developing methods for detecting the presence of gene or chromosome abnormalities in embryos before implantation.
Although these purposes did not preclude the derivation of human ESC, the reasons for doing so would have been limited by them. The Act had been passed before human ESC had first been derived, and there had been several other scientific advances, notably SCNT or cloning, which suggested that it needed updating. Enabling any such knowledge to be applied in developing treatments for serious disease HFEA, The current version of the Act, passed in , incorporates a number of very significant amendments, which were again made to accommodate rapid advances in science as well as changes in public attitudes and clinical practice Lovell-Badge, In summary the HFEA can grant research licences for up to 3 years for individual, peer-reviewed research projects.
All new applications for a research licence must also have ethics approval see Section 2. The HFEA charge an administration fee for granting and renewing project licences, which varies depending on the nature of the research. However, as with any other cell line, their use in a clinical setting would be regulated as described below. Research involving hESCs and other human tissue-derived cell lines will involve different regulatory authorities at different stages. For example, cell-based products that involve the destruction of a human embryo in their formation are initially licensed by the HFEA.
Once an embryo has been disaggregated it is no longer subject to HFEA regulation. If the cells replicating from such a disaggregated embryo are intended for application on humans, they are then subject to the HTA up to the point of the first representative cell bank for that cell line.
These regulations are administered by the HTA. However, hESC lines derived purely for research are not subject to this regulation. Favourable opinion from a recognised research ethics committee is also required for any clinical trial of a medicinal product see Section 2. Most human cell-based medicinal products intended for cell therapy or tissue engineering purposes will be classified as ATMPs.
If opinion is favourable the MHRA will be responsible, in the UK, for authorising the clinical trial, inspecting the trial and issuing a manufacturing licence. This provides practical advice and information on best practice and current legal requirements for conducting clinical trials in the UK. There are many who might lay claim to the ownership of specimens and their derivatives, including the donor and relatives, the surgeon and pathologists, the hospital authority where the sample was taken, the scientists engaged in the research, the institution where the research work was performed, the funding body and any collaborating commercial companies.
The ultimate control of ownership, intellectual property rights and patent rights will need to be negotiated by the various interested parties. Most universities and research institutes will have a research office that deals with such negotiations, as do most of the larger funding agencies. An MTA is a legally binding contract governing the transfer of research materials between two organisations where the recipient intends to use the materials for his or her own research purposes.
Biological materials including reagents, cell lines, plasmids and vectors are the most frequently transferred materials and the MTA will define the rights of the provider and recipient with respect to the materials and any derivatives.
This should include details of ownership, intellectual property rights and patent rights. The MTA should be signed by the legal representative of both the provider and recipient before any materials are transferred.
If primary human tissue or cells are involved the MTA should include a statement confirming that ethical approval and informed consent have been obtained and the recipient should confirm that, on receipt, they will become responsible for using, storing and tracking the material in full compliance with the HT Act. The provider may also wish to state that no liability can be accepted for any problem arising from the use of the cells or tissue and that no guarantee of freedom from microbial contamination can be given.
Restrictions on the use of transferred cell lines should be minimal, but it is reasonable to insist on acknowledgement and even co-authorship where the originating laboratory has made a substantial contribution to the subsequent work. However, merely supplying a cell line would not in itself normally warrant co-authorship of any paper describing work carried out using that cell line. The MTA should also indicate that the cells must not be passed on to a third party or used for commercial exploitation.
Again most universities and research institutes will have a research office that deals with agreeing and issuing MTAs, and will usually arrange for them to be signed by a legal representative.
Scientists wishing to create cell lines from animal tissues must comply with current legislation. Further information can be found in the Guidelines for the welfare and use of animals in cancer research Workman et al, If a new cell line is successfully developed it will be important to confirm the individual and cell type of origin. This will require authentication see Section 1. Once a new cell line is established it becomes an important resource.
Its authenticity, characteristics and provenance should be recorded see Section 3. Ensure that all items of laboratory equipment cabinets, incubators, autoclaves, water filtration units, etc.
Inspect the cells under an inverted phase microscope before use. For routine culture, inspect cells daily and consult reference photographs of each cell line at different cell densities.
Get to know the cells and how they behave under different conditions. Freeze new cell lines at the lowest passage possible after clearing quarantine. If they need to be frozen before being cleared they should be treated as if they were contaminated. Detailed information is available on methodology and GCCP e. The guidelines on operator safety in cell culture presented here are meant primarily for private and academic research laboratories to be used in conjunction with local and national safety regulations and do not replace rules on safety within individual laboratories, as these vary according to local circumstances.
The advice of the local Biological Safety Officer should be sought where there is any doubt about the introduction of new materials or procedures. Employers are responsible for employee safety under the Health and Safety at Work Regulations HSE, by providing information, instruction and training and effective protection against hazard in the workplace. These regulations foster safe working practices by establishing that any proposed procedure is both justifiable and safe by requiring that a risk assessment is made before work is started.
The COSSH regulations also set out a duty for employees to collaborate fully so that employers can meet the legal obligations.
It should deal with the entire process and not just individual hazardous chemicals and biological agents. Risk assessments should not be copied from one laboratory to another since the same hazards represent different risks according to local conditions and the scale of the operation. With regard to product safety, cell culture in the commercial sector is subject to strict regulation. Possibly the greatest hazard encountered in the cell culture laboratory derives from LN 2 , which is used extensively in the freezing and long-term storage of cells and can injure by causing cold burns and frostbite or kill by asphyxiation or by the explosion of poorly designed LN 2 containers.
A worker at a laboratory in Edinburgh died from asphyxiation by LN 2 evaporation-induced oxygen depletion in BBC News, and there was a similar case in Australia in Finkel, , and a factory in Japan was destroyed by the explosion of a storage vessel in HSE, Thus it is essential that all appropriate safety measures for the handling and storage of LN 2 , as identified in the relevant risk assessment, are in place in all laboratories and that these are rigorously adhered to; LN 2 suppliers are a useful source of information and the latest regulations.
Ultimately, however, local factors may be of critical importance e. The storage area should be well ventilated and there should be an oxygen deficiency alarm and mechanical ventilation preferably activated through the oxygen monitor. A further hazard associated with LN 2 is the risk of explosion where vials are stored submerged in LN 2.
This problem was at its most acute when glass ampoules were widely used, but it still exists with poorly sealed plastic ampoules where LN 2 is drawn into the vial during storage and causes a potentially dangerous explosion when the vial is warmed at thawing. Ideally, vials should not be submerged in LN 2 , but if they are, a protective wrapping such as Cryoflex Nunc, Thermo Scientific may be considered. Appropriate personal protective equipment e.
Staff should also receive training in safe working practices for the LN 2 storage facility. Access to storage vessels should be strictly controlled. The main hazard arising from cell cultures themselves is from infectious agents carried either by the cells or from the components of the culture medium. Cells can carry viruses and at least one fatality due to a viral infection acquired from cells has been reported Hummeler et al, Sera could also contain a variety of microorganisms, including viruses and mycoplasma.
The biohazard risks associated with cell culture can be minimised by GCCP and appropriate containment and disposal protocols. Laboratory workers should also use personal protective equipment such as a lab-coat, gown or coveralls. Gloves and suitable eye protection are also recommended, depending on the task and the level of risk.
The use of blood or tissue from laboratory staff for the development of cell lines is not recommended, particularly for the generation of transformed cell lines, as the person concerned would have no immunity to the transformed cells. Testing of the donor cannot be used as proof of absence of infection of the cell line, as contamination may occur by various sources in cell culture, and tests based on a blood sample taken before the cells were donated may not reflect the actual microbiological status of the donated tissue.
Comprehensive advice on working with potentially infectious material in the laboratory is contained in guidelines from the Health and Safety Executive ACDP, Material with a high potential risk of infection should be excluded or handled appropriately.
All samples of blood, body fluids, secretions, tissues and cells are potentially infectious and must be handled at Containment Level 2 in a Class II MSC.
You may wish to consider whether vaccination, for example, hepatitis B and tetanus, should be considered for laboratory workers handling human or animal tissue. It is best to treat all cell lines as potential sources of infectious agents and handle accordingly; therefore, the above precautions should be maintained with any cell lines derived from clinical samples.
There are documented cases of serious laboratory-acquired infections e. When obtaining primary tissue from laboratory animals it is important to ensure that the animals used are free of specific pathogens SPF and suppliers should provide evidence of testing.
Alternatively, if the animals have been infected deliberately as part of an experiment, or are otherwise suspected of carrying a specific pathogen, tissues obtained from them must be handled appropriately, including the relevant level of containment.
This information should be used in risk assessments and cross-referenced in laboratory record books where the respective primary cells are used. It should be assumed that any hazards associated with primary cultures will also be present in cell lines derived from them.
In principle, the infectious hazards that may arise with stem cell lines are no different from any others in that workers should consider the likelihood of contamination with pathogens associated with the species and tissue of origin. In the case of hESCs the risk of contamination of the original donor tissue with the most serious blood-borne pathogens is very low Zou et al, However, when stem cell lines are differentiated to form tissue cell types they may provide a suitable culture substrate for the growth of pathogenic viruses such as HCV, HBV Si-Tayeb et al, and other pathogens depending on the cell types generated Bandi and Akkina, Thus, when planning experiments to provide a particular differentiated cell type, consideration should be given to the most likely contaminants that may arise in reagents, cells and any test samples that might replicate in the differentiated cell types.
Human iPSCs can be isolated from a broad range of tissues; therefore, the risk is associated with the tissue. Human and mouse feeder cell lines used to grow stem cells may also carry viruses and can present similar risks to those for continuous and finite cell lines see Section 3. In addition, where primary mouse embryo fibroblasts MEFs are used to culture stem cells a range of viruses may occur in the original colony, so a viral screen should be obtained for the MEFs and mycoplasma testing performed see Section 4.
The extensive safe use of continuous cell lines indicates that there is little risk from routine cell culture. However, as most cell lines are not fully characterised, they should be subjected to local risk assessment by the local Biological Safety Committee BSC. A tumour grew in a laboratory worker accidentally inoculated with cells of a human tumour cell line through a needle Gugel and Sanders, and cancers have been transferred between people during transplantation Stephens et al, Although the growth of tumour cells from a different person is unlikely in healthy individuals, anyone with a compromised immune system is at greater risk.
The introduction of genes can reactivate dormant infectious agents in the host cell or create new agents by recombination. Viral vectors that can infect human cells e. Recommended procedures for creation, use, storage, transportation and disposal of genetically modified organisms, including modified cell lines, are given in the Genetically Modified Organisms GMOs Contained Use Regulations, UK, and its subsequent amendments HSE, nb : these do not apply to construction of somatic cell hybrids.
These regulations describe how to make a full risk assessment, which must receive approval from the Local Genetic Modification Safety Committee and, in certain cases, specific approval from the HSE may be required. Genetically modified cells may require special conditions. For example, selective pressure may need to be maintained on transfectants to retain the genetic modification and the pressure may need to be maintained during storage.
Distribution of genetically modified cells may be subject to regulation, depending on the modification. Long-established continuous cell lines may be handled at Level 1, subject to the approval of the BSC, but in practice it may prove to be more convenient for all tissue culture facilities to be maintained at the same level, that is, Level 2.
This level of containment is also applicable to untested cell products such as monoclonal antibody-containing supernates and cell homogenates. Laminar flow devices other than MSCs should not be used for cell culture.
Horizontal flow cabinets, where the airflow is directed at the operator, are particularly hazardous and must never be used when working with cells that are known to, or may, carry pathogens, or with potentially infectious cell derivatives.
Horizontal laminar flow cabinets are still used by laboratories working with early embryos and ES cells that are known to be pathogen-free. However, the use of this type of cabinet should be strictly controlled and subject to local risk assessment and approval by the local safety committee.
Modifying MSCs and other contained cell culture hoods, for use with microscopes, may disrupt airflow so much that they are neither safe for the operator nor provide adequate protection for the cell cultures.
Such modifications should only be made following approval by the local safety committee and any modified equipment should always be re-validated before use. The spread of infection often occurs via contaminated aerosols and any process that produces aerosols from crude cell culture preparations is a potential source of infection.
Such processes e. There are special guidelines for the safe use of flow cytometers with unfixed cells Schmid et al, a , b.
Any cell samples to be submitted for specialist microscopy services e. Control of the disposal of laboratory waste should prevent exposure of staff and environment to infectious hazards and prevent contamination. In the UK those producing clinical waste including drugs, pharmaceuticals, animal and human material and any items contaminated with these materials have a duty in law to ensure its safe disposal Environmental Protection Act, All infected waste arising from work in laboratories should be made safe to handle by appropriate means e.
England and Wales, Scotland and Northern Ireland each have their own hazardous waste legislation in the form of statutory instruments and rules to implement the EU Directive on Waste, which sets out a framework within the Member States for controlling the production, transport and disposal of hazardous waste Hazardous waste, — New staff should not be allowed to work in the tissue culture facility until deemed competent.
Both practical, hands-on training and theoretical training should reinforce the need to use good aseptic technique and awareness of contamination as an important issue that can be minimised through GCCP. Practical training is best carried out on a one-to-one basis with an experienced member of staff, with extensive reference made to any relevant SOP.
As compliance with any demanding technique tends to decrease with time and familiarity, performance should continue to be monitored. Individuals experienced in cell culture starting in a new laboratory should read the protocols specific to the laboratory, such as routine handling and monitoring of cell cultures see Sections 3.
It is recommended that reagents and sera are purchased from suppliers who issue certificates of analysis or results of QC testing with each batch of product. Buy in bulk quantities suitable for the level of usage to minimise batch variation and store at the temperature recommended by the manufacturer.
Aliquot proteinaceous solutions, such as serum and trypsin, rather than repeatedly freeze and thaw large bottles. Most commercial suppliers offer a custom media service for specialised formulations. If concentrate is used, this is diluted into bottles containing sterile ultra-pure water. Sterile L-glutamine and sodium bicarbonate are then added and finally the pH is adjusted.
The advantage of this system is that it is quick and technically undemanding. However, several points should be borne in mind. Media concentrates have changes made to their basic formulations, mainly to overcome problems of solubility. Precipitate is often seen on storage. If the concentrate is aliquoted the precipitate can cause variation between bottles. Suppliers acidify the medium to improve solubility. This in turn requires significant amounts of base to neutralise the medium.
Powdered media produce more stable uniform products with longer shelf lives than concentrates. However, the process does require specialised equipment for filtration and bottling. The medium should be stirred until all the powder is dissolved. The presence of fine particulate matter may require pre-filtration or a change of supplier.
Sterilisation requires filtration to a pore size of 0. Cellulose filters are most common but polyvinylidene fluoride PVDF filters should be used when protein is present in the medium. Although a 0. However, it is still advisable to screen regularly for mycoplasma contamination rather than assume its absence from media. It is difficult to exclude viral contamination and pre-screening of natural products, such as serum, by the supplier is usually the only option.
Single-use disposable cartridges or filter flasks are recommended as the most convenient option for media filtration. A Class II MSC should be dedicated to media and supplement production horizontal laminar flow may be used provided there are no antibiotics or toxins in the medium.
If a dedicated cabinet is not possible, then it should not have been used for cell culture for at least 1 hour. All tubing should be clean and autoclaved before use and connections should be securely in place. Sterile bottles and caps should be stacked outside the cabinet and introduced one at a time to receive medium. Stacking bottles within the flow cabinet will seriously compromise the airflow and consequently sterility. During bottling, representative samples should be drawn off at regular intervals.
Some medium components such as glutamine are heat labile Ozturk and Palsson, This is best addressed by adding glutamine when the bottle is first used and by discarding bottles after a set time period e.
Stabilised forms of glutamine are available that avoid this problem e. If any sample shows contamination in repeated samples, the whole batch of medium should be discarded. Simple preliminary tests can help avoid the disastrous consequences of using media, sera or supplements that do not adequately support cell growth. Low serum concentrations e. It is important to limit carry-over of the old serum during testing, as this could mask differences between the old and new batches. Trypsinisation of adherent cells will usually remove most of the serum, but cells growing in suspension will need to be centrifuged.
The selected batch may be held on reserve for a maximum of 1 year and delivered as required. Developing a defined medium, that is, serum-free and preferably free of all impure or undefined products, for a particular cell line or cell type can be very time consuming.
There is a wide range of serum-free media available see Box 3 , although not all of these are totally defined. Any protein supplementation should be with recombinant protein. The advantages of defined media include standardisation, reproducibility, absence of microbiological contaminants and the potential for selective culture of specific cell types.
However, they are generally more expensive and will mean that one medium may not suffice for all cell lines in use. Some of these, for example, SIT Sigma are defined selenium, insulin and transferrin , but others are proprietary mixtures and undefined and should be batch-tested as for serum. Fully defined or at least serum-free medium should be used whenever possible.
These cabinets provide protection to the operator as defined in the BS EN British Standards Institute, and protect the external environment while maintaining a clean working environment, but give no protection against toxic, radioactive or corrosive materials for which specialised cabinets are required.
The effectiveness of a MSC is dependent on its position, correct use and regular testing. Cabinets should be sited away from doors, through traffic and air-conditioning inlets. Movement in the area of the MSC will disturb the airflow and therefore access to the area should be restricted to essential personnel. All MSCs should be tested annually for airflow, operator protection factor and filter integrity.
Cabinets used for general cell culture should be tested annually. Testing and servicing should be carried out by trained competent personnel. Before servicing and testing is carried out, adequate fumigation is required. This is usually performed using formaldehyde gas but some manufacturers offer vaporised hydrogen peroxide VHP sterilisation, which leaves no toxic residue see Table 2.
Advice should be sought from the local safety committee regarding whether this is acceptable in your institute.
Training is essential before either procedure is carried out. An equipment safety certificate is normally required by servicing engineers before testing can begin.
When performing cell culture work within an MSC it is important to minimise the potential for contamination of the working environment and cross-contamination between cultures. This can be greatly assisted by the following:. The inside of an MSC and items that you bring into it should be clean but are not sterile, and good aseptic technique requires that you do not touch any of the surfaces with sterile instruments, pipettes, and so on.
Manipulate fluids slowly and gently with the assistance of a pipetting aid to avoid the creation of aerosols. Organise the work area such that sterile reagents and cultures do not come in contact with each other.
Use caution when homogenising tissues or cells in an MSC. If high-energy processes such as sonication are used the particles cannot always be assumed to be contained by the cabinet airflow.
Some MSCs have an ultraviolet lamp installed to assist with disinfection of the cabinet. Although ultraviolet light can be useful, its effectiveness is limited and it should not replace other decontamination procedures.
A Bunsen or similar burner must not be used when working in a MSC, unless absolutely required for a specialised procedure as they disrupt the airflow pattern, reducing the cabinet's effectiveness, and they pose a fire risk. Incubators are used during cell culture to maintain an optimal cell growth environment by controlling the temperature, humidity and carbon dioxide concentration. The following points should be considered:.
Incubators should be chosen carefully with reference to their expected use and any desirable features that can be included within budget see Box 4. CO 2 levels should be checked monthly using a calibrated CO 2 meter marked deviations will be evident as a change in pH of the medium.
All shelves should be similarly removed and cleaned. Individual trays on which culture flasks can be easily moved in and out of the incubator should be used to reduce contamination from spillages. Incubators must only be used for cell culture and not for incubating microorganisms or biochemical samples. Gassed incubators should be attached to a suitable cylinder change over unit or protected central supply.
Please note: a few companies have been purchased by or merged with other companies , they remain on the list if their brands are maintained by the new entities, such as Life Technologies, Sigma-Aldrich. PO Box St. Louis, MO Friedrich-Ebert-Strae 68 Bergisch Gladbach. Cell Biolabs, Inc. We are scientists driven by a devotion to discovery and innovation, and we work hard every day to find new ways to streamline life science research.
Bairoch A. The Cellosaurus: a cell line knowledge resource. Available from: web. Check your cultures! A list of cross-contaminated or misidentified cell lines.
Int J Cancer. The culture of cell culture practices and authentication--Results from a Survey. Standards for Cell Line Authentication and Beyond. PLoS Biol. Available from: www. Learn how your comment data is processed. Facebook Twitter LinkedIn More. Written by Rebecca Roberts. Leave a Comment Cancel Reply You must be logged in to post a comment. Share via. Copy Link. Powered by Social Snap. Copy link. Copy Copied.
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