ISCBI guidance
Final DRAFT For Open Consultation, v 18 February 2008
Consensus Guidance for Banking and Supply of
Human Embryonic Stem Cell Lines for Research Purposes -
ISCF International Cell Banking Initiative
For tables and figures relating to this document please see ISCBI Guidance Tables and Figures
For part two of this document please see Part Two
The International Stem cell Banking Initiative welcomes comments on the Guidance document below and these comments will be considered before a final document is published later in 2008. Please submit any comments you may have to the following email using the word template. You are asked to add your name and affiliation but this is purely for administrative and governance purposes and no names or affiliations will be used in any output from the ISCBI activity.
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Contents:
Part One
Part two
1. Background and Scope
In just a few years hundreds of human embryonic stem cell (hESC) lines have been established in laboratories around the world and many programmes of research initiated to investigate their properties and broad ranging potential in therapy and for other research applications such as developmental biology, toxicology and drug discovery. This work is being performed with a variety of cell lines using a variety of culture conditions; a situation that makes standardization between projects and publications very difficult and could prevent the identification of cells that have undergone permanent deleterious changes. Clearly, the consequence of using such cells would be wasted time and resources, but more seriously, the generation of erroneous data in the literature which could both confuse and delay scientific progress in this area. Thus, ensuring that cell lines used in this dynamic field have the correct identity and characteristics is critical to the delivery of effective and efficient research of acceptable quality.
Many centres now distribute hESC lines around the world but the preparation and testing of cell stocks released to other researchers is generally based on local ‘norms’ and naturally varies from centre to centre. The challenge of preparing satisfactory cells for use in research work has been recognized and guidance has been developed by international groups on good cell culture practice (Coecke et al., 2005) and cell banking (ISBER (2005), OECD (2007)). In the absence of formal coordination between the active distributing centres from different countries the International Stem Cell Forum, a group of national and international stem cell research funding bodies, has supported an initiative, the International Cell Banking Initiative (ICBI), to establish a dialogue between the distribution centres to develop a consensus on best practice for the banking, testing and distribution of hES cells. The first meeting of this group was held in at the Jackson Laboratories (Maine, USA) in October 2007 and this guidance document represents the first output from the ICBI. The document has been prepared from the perspective of hES cell culture but is broadly applicable to all human stem cell lines. There are a number of critical concepts and definitions that must be clear to enable appropriate interpretation of this guidance and these have been drafted as a glossary at the end of the formal guidance (below).
2. The Cell Banking Process
Today the conditions of consent for the use of donated cells and tissue for research purposes is rightly subjected to appropriate scrutiny and accordingly, traceability should be established between each stem cell line and fully informed consent from the original donor of cells used to derive the cell line (see section 6, ‘Ethics’).
The establishment of archive material is important for future reference and useful samples include cells from the original donor and fixed cells (e.g. “Guthrie cards”, ‘FTA’ cards (Whatman)) or DNA from early passages of the cell line to enable confirmation of genetic identity of cell banks. In addition vials of the cryopreserved cell line supplied by depositors can be held unopened in case the banked cells are later found to have altered characteristics or contamination.
Fundamental information from depositor of a cell line in the bank should include cell identity, contamination status, viral testing, passage level, mycoplasma testing and key cell antigen and molecular markers. Details of the testing recommended and cell bank specifications and release criteria are given in sections 2.2, 2.4, 3 (‘Cell Bank Release Criteria’) and 5 (‘Cell Characterisation’).
2.2 Cell banking procedures and documentation
The bank should follow applicable national laws and guidelines and formally adopt appropriate and international ’norms’ and guidance established for the banking and use of human stem cell lines. Formal ethical oversight for each bank is necessary to give confidence to users of the bank that all bank procedures have been performed according to national and local rules (see ISCF website for information sources on regulation in different countries).
Operation of a formal and documented Master Cell Bank and Working Cell Bank system (Coecke et al., 2005) is strongly recommended to enable supply of reproducible cells at the same passage level over long time periods. Cells would normally be supplied from the ‘Working Cell Bank’ which may be called the ‘Distribution Cell Bank’. In addition, the bank must establish a quarantine procedure for newly acquired cell lines in order to avoid spread of any contamination they may contain.
For all processes involving culture or manipulation of cells the risk of microbiological contamination from raw materials should be evaluated (see section 3, ‘Microbiological Testing’). The use of antibiotics in culture media may be necessary for derivation of cell lines, but is not recommended for preparing cell banks and other experimental work, with the notable exception of cell clones expressing ‘marker’ genes that are maintained under antibiotic selection. This is important to avoid the longer term problems that can be associated with suppressed contamination.
Documentation of banking procedures is vital for the bank to be able to demonstrate that it is has worked in compliance with appropriate regulatory standards and to address and ‘troubleshoot’ complaints from recipients and internal failures in quality control. Documentation should include all information provided with deposited cell lines, consistently recorded data from quality control and characterisation and Standard Operating Procedures for all key processes and protocols. Materials and procedures used to prepare each cell bank should also be traceable. All data available on a particular cell line should be collated or referenced in a ‘Cell Line Master File’ that will provide a central source of information on that cell line and the stocks established in the bank.
Frozen stocks of a particular cell line should not be stored in a single location. In order to avoid the risk of complete loss of cell lines, the bank should provide back-up storage facilities at a second site that would enable regeneration of the original stocks. Off-site stored material should be held under conditions equivalent to those at the bank site. The bank should operate an inventory management system and procedures should be in place to ensure the accuracy and reliability of labeling and location records. The loss of cell lines due to substandard maintenance of cold storage would have a significant impact on the credibility of the bank and auditing the bank storage system is an important factor in assuring secure long-term storage of material and is strongly recommended. As a minimum an internal audit process should be employed to carry out periodic checks on the reliability and robustness of maintenance procedures for stored materials. Where formal standards apply for medical use, testing or other purposes, a formal independent audit process may be required for all cell culture and testing procedures.
The past experience of other culture collections distributing cell lines has identified that an important principle of quality control (QC) for cell lines is that it enables the bank to state, subject to certain qualification, that the cells are what the bank says they are and that they are free of microbial contamination. In order to achieve this for human stem cell lines, and indeed any stored cells, it is vital to establish key criteria for the quality control and characterisation of the cells and these are addressed in detail in sections 3 (‘Release Criteria’), 4 (‘Microbiological Testing’) & 5 (‘Cell Characterisation’). Clearly qualification of cell lines, through appropriate and rigorous quality control, is critical and should be a mandatory function for a bank. It should also be remembered that QC is just one part of overall quality assurance procedures for the bank which should also cover aspects such as correct function of facilities and equipment, and staff training. In some applications of cell lines where specific quality standards apply the bank will need to understand and possibly comply with such standards examples of which include standards from the International Standards Organisation (www.iso.org/):
- ISO9001:2000 a general quality management standard for provision of services and products
- ISO17025 for laboratory testing and monitoring including the use of cell lines for ‘batch release’ testing of medical products,
- ISO13485 for diagnostic testing procedures including the use of cells or cell-derived reference materials
- ISO34 Guide for preparation of reference materials
Other international guidance is also available for general cell culture (e.g. Coecke et al., 2005), safety testing (OECD, 2004) and establishment of cell lines for the manufacture of medicinal products (ICH (1998),WHO (1998)).
2.4 The process of releasing cell banks
Historically researchers have often sourced lines from unqualified sources in their colleagues’ laboratories, and this practice unfortunately promoted the widespread use of cross-contaminated and mycoplasma infected cell lines (Chatterjee (2007), NIH (2007)). Banks should actively work towards establishing expedient procedures to promote prompt access to cell lines to minimize the need for ‘colleague-supply’ routes. As part of this process banks need to work towards low cost simple testing regimes
For each cell bank there should be an associated ‘specification’ which describes the key characteristics of each line. The ‘specification should meet the requirements for the cell to be ‘fit for purpose ‘ i.e. for use in stem cell research work. The specification must be matched by the quality control and characterisation data for each bank of cells before they can be released (for details see section 3, ‘Release Criteria’)
It is recommended that with current culture methods, banks should avoid shipment of growing cultures. Pooling multiple straws or vials from the same frozen stock may be necessary for a recipient to successfully recover a culture. However, banks should aim to provide sufficient ‘viable’ cells in a single container to enable appropriately trained staff to recover a representative culture.
Culture manuals should be available from the bank, ideally ‘on-line’, along with key Standard Operating Procedures. Release of cells to researchers should be accompanied with advice and training, recipients should either have evidence of past training or training should be provided as part of cell supply.
It is recommended that the bank should make stocks of especially critical reagents, such as feeder cells, available to recipients to get their work started efficiently. Banks should advise recipients to produce a bank of each stem cell line on supplied feeders before switching to local feeder cells.
A complaints procedure should be in place which should be responsive to avoid frustration for recipients and enhance reputation of the bank. The bank should also have a clear replacement policy for cultures which fail to thrive in the hands of recipients. All complaints should be reviewed to assess the effectiveness of the corrective actions taken and to look for opportunities to improve service. Technical support is important to users of the bank and should be provided.
3. Release Criteria for Cell Banks
Bank cultures should be representative of the originally deposited material i.e. quality control and characterisation data should show no evidence that the banking process has altered cells.
The bank should provide assurance that cell lines released have no detected microbial contamination and that as far as can be determined they are ‘monoseptic’ i.e. a single uncontaminated viable cell line. This assurance can be provided through a combination of cell-identity testing and microbiological isolation methods but should be qualified based on the types of organisms covered by the testing regime used.
Identity testing is critical to demonstrating that the cell line is unique (i.e. not switched or cross-contaminated by another cell line). Banks should share identity data to avoid spread of cross-contaminated cultures but should take care in general with dissemination of genetic information that may be donor specific.
‘Sterility’ tests from antibiotic-free cultures should be performed on cell banks to provide evidence for absence of bacterial and fungal contamination. Banks should seek to extend these tests to cover fastidious organisms, including mycoplasma and certain bacteria that may contaminate cell cultures but are not detected by standard sterility test methods for bacteria and fungi.
Mycoplasmas are a frequent cause of contamination in cell culture and can be introduced from a variety of sources including bovine serum, feeder cells, animal-derived raw materials (e.g., trypsin) or humans. Mycoplasmas are one of the greatest risks for potential contamination of mammalian cell lines with several studies estimating rates of contamination as high as 15-30% in cell lines that are shared between research labs (Bolske, 1988; Rottem and Naot, 1998; Timenetsky et al., 2006). Although over 20 different mycoplasmas have been isolated from cell lines, the vast majority of contaminations are due to eight species: Mycoplasma hyorhinis (porcine trypsin), M. arginini (bovine serum), M. fermentans (human), Acholeplasma laidlawii, M. hominus (human), M. orale (human), M. bovis (bovine) and M. pulmonis (murine). Due to their small size filtration is not always effective at removing mycoplasma from cell culture media and reagents. In addition, mycoplasma contamination of cell cultures is often difficult to detect since gross changes in culture (e.g., turbidity) are not commonly observed. Mycoplasma contamination, however, can result in significant changes in cell metabolism and growth characteristics, and can have a dramatic effect on cell phenotype and chromosome structure.
Due to the high frequency of mycoplasma contamination, good cell banking practices should include measures for early detection and prevention.
Cells should not be released if they are known to carry a viral hazard to laboratory workers or a virus that may influence research data from the culture in question. It is possible that persistently infected cells would have significant value in research but these would have to be handled separately in appropriate facilities and the infection notified to recipients appropriately.
In addition to tests for certain viral pathogens (see section 4, ‘Microbiological Testing’), banks should also consider inclusion of testing for other contaminants prevalent in the local environment, media supply and donor groups.
3.2 Technical aspects of release procedures
Each bank should establish a clear set of release criteria that should apply to each bank of cells. A recommended minimum set of criteria for release of hES cell banks is given in Table 1.
A similar test regime should be established for feeder cell banks based on a minimum requirement for sterility testing, mycoplasma testing and viability as described for hES cells (Table 1). In addition, identity testing and appropriate virological testing (see section 4. ‘Microbiological Testing’) should be carried out for human feeder cells. Mouse embryonic feeder cells should be obtained from animal colonies with an appropriate standard of husbandry compliant with national and/or international standards, including colony screening for common murine pathogens. Mouse Antibody Production tests are recommended for mouse feeder cells and it is also recommended to prepare large homogenous stocks of these cells to promote consistency and economy.
Mycoplasma tests should be performed on newly deposited cells under quarantine and all cell banks established. Several different assay methods are currently available for mycoplasma testing including culture methods, PCR-based assays, and detection methods based on the presence of mycoplasma-specific metabolic enzymes. Mycoplasma testing of distribution cells is typically conducted using either culture or PCR-based assays since these methods can provide broad coverage of mycoplasma and acholeplasma species. A brief overview of these methods is provided in Table 2. The chosen test methods should be validated for their specificity and sensitivity for testing cell lines and for testing cell banks a combination of methods used to give high sensitivity and detection of non-culturable strains.
Identity testing should be performed on samples of early stage material (early passage cell DNA preserved either frozen or on storage cards e.g. ‘Guthrie cards’, ‘FTA’ cards (Whatman)) and all cell banks established (see section 2, ‘The Cell Banking Process’). It is recommended to use systems with core common alleles represented in forensic work and examples from commercially available STR kits are given in Table 3. Publication of STR profiles of recently isolated human stem cell lines may present ethical issues in some countries. However, it is recommended that banks should share such data for the purpose of identifying cross-contaminated cell lines and preventing their release; which could have a serious long term effect on the validity of research data performed with such lines.
“Viability” is a difficult parameter to define - frozen vials or straws should enable recovery and expansion of a ‘representative’ culture within a certain time (see Table 1). The bank should formally qualify the viability method it uses. Viability test data can be used as a release test but should not be used as an absolute indicator of the ‘quality’ of a stem cell bank. This can only be demonstrated through successful completion of a range of quality control and characterisation tests.
Cell composition should be evaluated for test samples from each bank and should include percentage of cells positive for markers and the level of background differentiation (see section 5, ‘Cell Characterisation’). The functional properties of cells should also be addressed and the key property of pluripotency is dealt with in section 5 on ‘Cell Characterisation’.
The bank should remind recipients of lines that the testing performed on each cell bank may not detect microbial contamination below the level of detection or organisms not covered in the testing regime, and thus recipients should be advised to handle the cell as if potentially infectious.
4. Microbiological Testing
Microbial contamination can change the characteristics of cells without causing obvious cytopathic effects and the unwitting use of such cultures could not only expose researchers to infectious agents but could also affect the quality of research data due to influence of viral infection on the biological performance of the cells. In principle stem cell lines should be free of any microbial contamination as far as can be reasonably determined by the bank. However, cells with established persistent infection with specific agents may have useful research applications, but should be handled separately to ‘clean’ cultures with no evidence for infection.
It is vital that the bank has a rationale for the tests performed. Sterility and mycoplasma testing are microbiological tests that should be performed on all cell cultures used in the bank and these are covered in section 3.2. The bank’s drivers for safety testing will normally be to protect bank staff and users and deal with liability that may be associated with the supply of cells to research workers. There is a formal but low risk of tumor formation following subcutaneous inoculation of a tumorigenic cell line as a result of accidents in the use of ‘sharps’ such as hypodermic needles. Whilst this risk would appear to be very small, the bank should aim to minimize it and take special care with immuno-compromised bank staff. Furthermore, where human or animal cells have been subjected to transfer in animals, it should be remembered that there is potential for transfer of animal virus from the most.
The bank should be aware of donor testing results relating to individual cell lines, but these should not be used solely to demonstrate ‘safety’. Caution should be taken in the use of donor testing as the reliability of test data may vary and contamination can occur after cell isolation. In addition, ‘positive’ viral testing in donors does not necessarily exclude the use of cells for cell line derivation e.g. hCMV. Where reagents of biological origin are used there may be a risk of contamination (see Table 4 for examples) the bank should consider obtaining risk-evaluated products that have been appropriately tested for microbial contamination. The infectious risk represented by different cell growth media products can be difficult to assess but can be evaluated in the light of a number of aspects outlines below.
- Issues relating to source animals: Species and tissue of origin and the geographical location, husbandry and health/microbiological screening of source animals and raw product harvesting procedures.
- Product manufacture: Nature of the processing, purification and formulation methods, including assessment of any added materials and the capacity of different steps in this process for reducing the level of any potential contaminants.
- Nature, effectiveness and reliability of any sterilisation or disinfection steps. Many proteins, such as growth factors, will not survive methods of sterilisation such as autoclaving or destructive irradiation. However, processes such as ‘Pasteurisation’, filtration, chemical treatment and lower level irradiation can be quite effective at reducing the viable microbial load in cell culture products.
Testing performed on the product. Viral testing of animal derived products will be most appropriate when no sterilisation process can be used. Care should be taken to assess viral testing to ensure that appropriate methods were employed that have been demonstrated o give defined and acceptable levels of sensitivity and specificity and were performed by an qualified or accredited laboratory with appropriate experience in the tests performed.
It should be remembered that no current testing regime can guarantee absence of microbial contamination and therefore cells should be considered to remain potentially infectious even where comprehensive testing has been carried out.
4.2 Microbiological test procedures
Documentation of genetically modified components and pathogenic agents (e.g. recombinant cells, contaminated cell lines, control organisms) is important and may be a legal requirement (see relevant national rules and ISCF web site).
Virus testing should be performed on early archive or master stocks that will be used for establishment of any future distribution stocks.
It is recommended that banks should test human cell lines for serious blood born pathogens e.g. HepB, HepC, HIV, HTLV I/II, EBV and hCMV. This may be expanded to other viruses that may contaminate cells from the human reproductive tract (e.g. HPV, HSV, HHVs) depending on local policy, incidence of disease amongst donors and other risk factors identified for the individual cell line. The bank should have a documented procedure for dealing with positive microbiological results arising during testing.
Currently no recommendation can be made on tests for prions and agents of Transmissible Spongiform Encephalopathies (TSEs) due to lack of validated sensitive methods and lack of information specific to contamination of stem cells. However, banks should carry out periodic reviews of developments in microbiological testing to ensure that they maintain current best practice in testing regimes.
Microbio
