ISCBI Guidance2
Final DRAFT For Open Consultation, v 18 February 2008
Part Two
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 one of this document please see: Part One
Contents
Part One
5. Cell Characterisation
Banks are considered to have a primary international role to play in meticulous characterization of stem cell lines available for research. They must also confirm consistency for each cell line between deposited cells, Master stocks and cells for distribution (section 2, ‘Banking Procedures’).
A critical feature regarding the pluripotency of hESC lines is that they should form ‘teratomas’ in immunocompromised mice. The definition for nomenclature of these tumors has been a matter of recent debate but the conclusion recently adopted by Nature Biotechnology (Editorial, 2007), following expert consultation, is as follows:
‘…. Nature Biotechnology will adopt the term ‘teratocarcinoma’ to describe malignant tumors comprising both somatic tissues and undifferentiated malignant stem cells, identifiable as EC cells. .... We will apply the term ‘teratoma’ only to tumors composed of normal, ‘benign’ somatic tissue and their immature (fetal) precursors derived from more than one of the three embryonic germ layers (ectoderm, mesoderm and endoderm). Teratomas comprising nonproliferating somatic tissue may be further labeled as ‘benign’, ‘mature’ or ‘fully differentiated’. Teratomas composed of immature, proliferating fetal-like tissues may be labeled ‘immature’.
It is strongly recommended that distributors of stem cell lines should adopt this terminology.
Morphology continues to be an important criterion in stem cell research and banks should provide representative images of undifferentiated and differentiated cells (NB under defined culture conditions and post seed/subculture time) for users to compare with their own cultures. Ideally the bank should also make images available of typical immuno-cytochemistry and examples of undesirable features such as differentiation.
There are certain key cell markers for the characterisation of stem cell lines that should be used by the bank, and these are described in section 5.2.1 below.
5.2 Methods and measurements
A standardised international study (ISCI, 2007) of hESC phenotypic markers confirmed for a large group of hES cell lines that a typical surface marker profile for these cells is SSEA-1 (negative or very low), SSEA-3 ‘positive’, SSEA-4 ‘positive’, TRA160 ‘positive’ and TRA 181 ‘positive’ (NB SSEA-3 & -4 have the potential to reveal different patterns for a very small number of individual donors i.e. 1%). In the same study microfluidic arrays for Q-RT PCR revealed that expression of six genes could be recommended for confirmation of the hESC phenotype: Nanog, Oct 4, DNMT 3B, TDGF, GABRB3, GDF3. These were found to be strongly correlated with each other in stem cells. It is important to note that other cell types may express these markers but it is the pattern of reactivity that is key; not expression of individual markers.
Flow cytometry (FC) is the central methodology for canonical markers of stem cells identified above. Immuno-cytochemistry is also valuable to provide additional data on morphology and localization of antigen. Multiple fluorochromes are useful for flow cytometry to evaluate coexpression etc. Measurement of expression by FC should be recorded and available to bank users along with the method of FC data collection and the FC instrument used. Care should be taken in how data is collected and expressed and this should be clear to bank users. In particular the bank should carefully and unambiguously report the meaning of data referring to “% positive cells”. Banks should use appropriate controls in FC analysis (fixed cell preparations are under development in the International Stem Cell Initiative stemcellforum.org.uk). Bank SOPs for such phenotypic analyses should be made available to users.
RNA expression profiling gives desirable additional supporting data on associated gene groups but for critical interpretation such data should be confirmed at the protein level.
In all analytical work on feeder cell dependent stem cells, care should be taken to exclude the feeder cells from cell samples to avoid interference in data on stem cell lines. In addition it is useful to use a preparation of pure feeder cells as a control.
Each genotyping technique gives a different type of data and it is important that the bank understands and reports the advantages and limitations of the methods it uses.
Karyology by Geimsa-banding should be performed as a routine genotyping technique but it should be recognized that some diploid cells may carry undetected genetic alterations. Other methods can provide useful information and techniques such as “spectral karyotyping” (SKY), comparative genome hybridization (CGH) microarray and multiple single nucleotide polymorphism (SNP) analysis could provide useful supplementary data. However, comparative data from different methods will need to be collated from different centres over time. NB the number of cells analysed can be critical for sensitivity of detecting abnormal clones and the bank should make this information available to users.
G-banding analysis can detect the appearance of chromosomally abnormal clones however, the number of metaphase spreads analysed is critical to the sensitivity of their detection. Where karyologically abnormal cells are found repeat testing is recommended to confirm the findings (see below). Abnormal sub-clones of stem cell lines should be renamed and information and cells made available as they may be useful in genetic research or for high throughput screening methods.
The following guidance indicates the minimum work necessary for the cytogenetic analysis of human embryonic stem cell cultures. Standard G-band Analysis of prepared cell metaphase ‘spreads’ should include a chromosome count for 20 metaphases and banding patterns analysed in a further 10 metaphases. It is to be expected that occasional abnormal karyotypes will be observed in hESC line analyses and these may appear to be present in all cells (i.e. clonal) or in a minority of cells (non-clonal). Chromosome abnormalities that appear to be of clonal origin should be confirmed in a second later passage culture, to allow further interpretation of their significance. Abnormalities seen in single cells in a set of cell analyses (e.g. aneuploid chromosomes, structural rearrangements) will require further investigation in some cases to exclude mosaicism, depending on the chromosome involved. Standard terms and methodology for G-banding analysis of hESCs adopted in this guidance are given in Table 5. These were developed as a consensus between the standards used in the USA and the Association of Clinical Cytogeneticists General Best Practice Guidelines (2007) available on the website (see website list)
The bank should provide some form of evidence for the potential of each stem cell line to produce cells representative of the three germ layers that ultimately give rise to all cells of the body. The ‘gold standard’for this pluripotency is considered to be the ability of stem cells to form teratomas in SCID mice. This should be performed on each cell line at least once in its history, and the bank should make all efforts to ensure that this characteristic is not lost by in vitro manipulation in the bank.
Assays of cell features deemed to be subject to variation in culture should be carried out by the bank. If certain hESC lines are found that do not to form teratomas they may also have value for research into pluripotency. However, it is considered important that teratoma formation tests should be applied to all cells that may be used for clinical purposes.
Alternative methods are available for the determination of pluripotency including formation and characterisation of embryoid bodies and in vitro induced differentiation. Standardisation of any pluripotency assay will prove challenging. This is an area which is under development and banks should keep up to date on current technical progress. In the future it will be extremely helpful if the methods used are sufficiently accessible that they can be reproduced in user laboratories.
5.4 Cell line stability and epigenetics
In order to avoid any subtle changes in cell characteristics, it is recommended that for each cell line banks use the depositor’s methodology at least up to the first cryopreserved stock of cells or Master Cell Bank. Banks should monitor cultures for adaptation to in vitro conditions e.g. changes in growth rate, cloning efficiency) and attempt to minimise the risk of changes in stem cell cultures by:
• Minimizing culture passages (a Master- and Working cell bank system is key to this, see section 2, ‘Banking Procedures’) and being aware of culture methods that could induce change
• Publishing subculturing methods for recipients to use
• Passaging cells beyond typical levels used by recipients of cell lines and requalify cells at these time points
• Making recommendations to recipient not to use cells beyond a specified passage level from a characterised cell bank.
It is important to record actual passage no. post derivation and passage of cells provided to recipients. Population doublings may be difficult to estimate with current protocols, however, this is considered a more accurate and useful measure of replicative ‘age’ of a cell culture and it would be highly desirable to have such data available from the bank. Banks should keep up to date with current technical developments in this area to ensure that bank procedures reflect current best practice.
Investigation of epigenetic variation occurring in stem cell lines is at an early stage and more data is needed before routine tests can be recommended for stem cell banks. However, banks should keep up to date on current scientific and technical developments in this area.
6. Ethics
Banks must comply with their own national laws and regulations (see ISCF website) and should also check consistency with international ethics guidance established by the International Society for Stem Cell Research (ISSCR) and other relevant bodies.
For each cell line a process of obtaining fully informed consent from the donor should have been completed which includes a document signed by the donor which demonstrates what was explained to the donor about the use of their tissues and what constraints, if any, were applied by the donor on the use of their tissue. Ideally this should also enable traceability to the original donor but this should be coded, or otherwise anonymised, such that the bank cannot access details of donors directly but through a formal mechanism via another independent party or organisation. A review of international ethical safeguards and further information by the ISCF Ethics Working Party and others and information can be found at stemcellforum.org/ .
6.2 Procedures to assist ethical operation
Each bank should have an independent and transparent governance structure which reviews and authorises the ethical status of cell lines accepted by the bank, and ethical approval should be traceable for each cell line. Depositors of cells should demonstrate, through the governance mechanism used by the bank, that they have met all legal and ethical requirements in the countries associated with procurement of tissue and derivation of the cell lines. The information provided by the depositor of the cell line should also provide information that enables the banks to determine that these conditions are consistent ‘broadly’ with the bank’s national regulation. It is also important for the bank to have in place some mechanism to maintain awareness of changes in regulation.
In principle only scientifically justified and ethically approved projects with named principal investigators at bone fide institutions should receive cells from the bank and these requirements may be dealt with in an MTA (see below). To avoid distribution to ungoverned uses the users should be prohibited from third party distribution at least without prior permission from the bank. The bank should consider ethical responsibility in case of discovery of infectious disease which may be indicated in national guidance (see ISCF website for sources of information).
7. Shipment of Cells and Information for Users
“Instructions For Use”(IFU) and/or Standard Operational Procedures for culture and preservation should be provided to bank users. The IFU should typically contain information prescribing general culture and preservation methods and what procedures the cells have been qualified or consented for (e.g. “in vitro research only”, “not for generation of gametes”, “not for reproductive cloning”).
Lot numbers for the cells shipped should be provided to users that are traceable to the ‘lot’ or cell bank. It is also recommended that data on Master cell bank is made available by the bank. Banks should supply, with the cells, test results obtained from that specific stock of cells (i.e. a Certificate of Analysis for each bank listing data including quality control and characterisation). It is also desirable for these to be available from the bank website. A statement or Materials Safety Data Sheet on hazards associated with the cells should be supplied with cell shipments. Terms and Conditions or a warrantee should be provided which qualifies cell potential and characteristics based on testing performed by the bank.
7.2 Information available to users of the bank
Information should include: Standard Operating Procedures (culture, preservation etc.) and characterisation data from the depositor for each cell line in the bank. Each bank should provide a statement on policy for the quality and sourcing of certain raw materials subject to national or international restrictions (e.g. serum). It would also be desirable for data generated on cell lines supplied by each bank to be made available to assist future users of the bank.
Ethical issues may be particularly important for bank users and each bank should provide information to facilitate efficient selection of suitable lines. Relevant information would include date of preservation of tissue, date of attempted ‘derivation’ (for hES cells usually considered to be the date the inner cell mass was isolated or plated in vitro), whether fresh or frozen embryos used, whether payment was made for the donation of embryo/tissue, whether the embryo created for research, the existence of fully informed consent obtained from the donor, for use of the original tissue for research, and any associated constraints on the use of the derived line. Information should also be available on the derivation procedure, seminal and key publication for each cell line.
7.3 Materials transfer agreements (MTAs)
Banks should work within national guidelines and laws on ownership and patenting of biological materials (see the World Intellectual Property Organisation website www.wipo.int/ for general information and for information on patent office contacts in the 184 WIPO member states see www.wipo.int/members/en/ ).
A common MTA would not be practicable for all banks, however, biological resource organisations have identified key generic elements that should be included (e.g. European Culture Collection Organisation www.eccosite.org/) and there are other National examples that could be considered as templates (e.g. National Cancer Institute – see also links to banks on the ISCF web site for template MTAs from suppliers of cells).
7.4.1 Technical issues
Vials and straws shipped should be from a homogenous distribution bank of cells (see section 2, ‘Banking Procedures’) and contain sufficient cells to readily recover a culture (see section 4, ‘Release Criteria’). The method of transport should be consistent with the method of preservation and validated by the bank. Typically cryopreserved vials (i.e. preserved by a slow cooling method) can be shipped on “dry-ice” (solid carbon dioxide) and ‘vitrified’ materials should be shipped in the vapour phase of liquid nitrogen (i.e. in ‘dry-shippers’). In the international distribution of mouse embryos; test vials (or ‘dummy’ straws) have proven helpful for recipients to test their thawing process.
Preservation methods are developing and improved methods are needed to assist in stable storage and shipment and it is important that banks maintain awareness of current developments in preservation science and technology.
7.4.2 Administrative procedures
Banks should have a planned and documented shipment process to identify recipient contacts (i.e. a detailed local contact name/tel. no. to receive cells as well as the shipment address), shipment, delivery dates and shipment tracking information from the courier.
For each shipment the bank should check all local import regulations with the recipient. Helpful information is available from the World Federation of Culture Collections on import regulations http://www.wfcc.org - see also the ISCF webpages for links to different national import/export regulations). Before dispatching cells the bank should also check the necessary international packing and labeling requirements for national and international shipment (see www.iata.com).
Preference should be given to use of shipping agents that can refresh ‘dry-ice’ or liquid nitrogen and the chosen method of shipment (insulated box of ‘dry-ice’ or nitrogen ‘dry-shipper’) and required storage temperature on receipt should be communicated to the recipient laboratory.
Websites
www.cytogenetics.org.uk/prof_standards/acc_general_bp_mar2007_1.01.pdf
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