Alternatives 2007 Workshop Ankara 12 13 November 2007
Alternatives 2007 –Workshop, Ankara, 12 -13 November 2007 Good Cell Culture Practices (GCCP) and in vitro toxicology Agnieszka Kinsner and Sandra Coecke 1
2 Alternatives 2007 –Workshop, Ankara, 12 -13 November 2007 The maintenance of high standards is fundamental to ALL good scientific practice, and is essential for maximising reproducibility, reliability, credibility and acceptance of ANY results produced. Validation of alternative tests is an example of quality assurance in biomedical research How to apply Good Laboratory Practice in vitro? Good Cell Culture Practice
Alternatives 2007 –Workshop, Ankara, 12 -13 November 2007 Cell Culture: History of “Issues” • He. La cell contamination of “new” cell lines - Hep. G 2, INT 407, Chang Liver, etc…. . • Mycoplasma and other fastidious micro-organisms – silent infections which cause irreversible genetic and phenotypic changes • Cell lines are inherently unstable and some prone to change (degree of instability varies) • Cells cannot read SOPs! – careful attention to stock cultures and preparation of cells for assays 3
Alternatives 2007 –Workshop, Ankara, 12 -13 November 2007 4 Standards in Cell Culture • Need for standardisation in cell culture – why? – Inherent variation of in vitro cell-based systems – Increasingly complex in vitro cell culture systems – Lack of well trained cell culture staff • Many standards for control testing and production - but little generic guidance for the routine preparation of cells for use • Unrealistic to apply a GLP-like system in academia – Dynamic environment – Need maximum flexibility and minimum cost • Need for generic good practice for any cell culture work
5 Alternatives 2007 –Workshop, Ankara, 12 -13 November 2007 Start of GCCP • 1999 General Assembly of the Third World Congress on Alternatives and Animal Use in the Life Sciences stated: “The participants … call on the scientific community to develop guidelines defining minimum standards in cell and tissue culture, culture to be called Good Cell Culture Practice … should facilitate the interlaboratory comparison of in vitro results … encourage journals in the life sciences to adopt these guidelines. . . ”
6 Alternatives 2007 –Workshop, Ankara, 12 -13 November 2007 Good Cell Culture Practice GLP OECD Draft Consensus Document ECVAM Workshop Report 1999 . . . . World Conference Workshop on GCCP 2002 2003 ECVAM Task Force Report OECD Task Force ECVAM ICCVAM 2004 2005 ECVAM GCCP Guidance Document Coecke et al. (2005) ECVAM Good Cell Culture Practice Task Force Report 2, ATLA
Alternatives 2007 –Workshop, Ankara, 12 -13 November 2007 7 GCCP Guidance Document AIM • Reduce ‘uncertainty’ in data from cell culture systems • Support best practice in all aspects of the use of cells and tissues in vitro • Complement, but not to replace, any existing guidelines and regulations (Eu. Pharmacopoeia, OECD, etc. ) • Consensus guidance to enable greater international harmonisation and standardisation
Alternatives 2007 –Workshop, Ankara, 12 -13 November 2007 GCCP Guidance Document • Sets the minimum standards for any work involving cells and tissues of human and animal origin • Discusses issues related to: 1. Characterisation and maintenance of essential features of the in vitro system 2. Quality control of the systems 3. Recording and reporting (in-house and in scientific journals) 4. Safety 5. Ethics 6. Education and training 8
Alternatives 2007 –Workshop, Ankara, 12 -13 November 2007 9 Six principles of GCCP 1. Establishment and maintenance of a sufficient understanding of the in vitro system and of the relevant factors which can affect it 2. Assurance of the quality of all materials and methods, and of their use and application, in order to maintain the integrity, validity, and reproducibility of the work 3. Documentation of the information necessary to track the materials and methods used, to permit the repetition of the work, and to enable the target audience to understand evaluate the work 4. Establishment and maintenance of adequate measures to protect individuals and the environment from any potential hazards 5. Compliance with relevant laws and regulations and with ethical principles 6. Provision of relevant and adequate education and training of all personnel, to promote high quality work and safety
10 Alternatives 2007 –Workshop, Ankara, 12 -13 November 2007 Six principles of GCCP Principle 1 Establish and maintain a sufficient understanding of the in vitro system and of the relevant factors which can affect it
Alternatives 2007 –Workshop, Ankara, 12 -13 November 2007 11 GCCP – principle 1 The essential elements for assuring reliable and accurate work when using cell and tissue-based systems are: • Authenticity, including identity of the system, e. g. morphological evaluation, provenance and confirmation of genotypic and/or phenotypic characteristics • Stability and functional integrity of the system in relation to its intended use • Purity, e. g. freedom from biological contamination
12 Alternatives 2007 –Workshop, Ankara, 12 -13 November 2007 GCCP – principle 1 Authenticity He. La cell contaminatio n of “other” cell lines (Stacey, Dev. Biols. , 2000) 24 Kb 5 Kb Southern Blot Multilocus Fingerprints probe 33. 15
13 Alternatives 2007 –Workshop, Ankara, 12 -13 November 2007 GCCP – principle 1 Microbial contamination Mycoplasma: Vampires of Cell Culture! • Cell transformation • Chromosome damage • Physiological changes • Changes in expression of surface markers • Etc….
14 Alternatives 2007 –Workshop, Ankara, 12 -13 November 2007 GCCP – principle 1 Microbial contamination Endogenous viruses
15 Alternatives 2007 –Workshop, Ankara, 12 -13 November 2007 GCCP – principle 1 Stability • Prepare and quality control master and working cell stocks • Control within qualified passage ranges • Check whether cell performance changes in new environments [e. g. atmosphere, nutrition (e. g. serum batches, medium replenishment), culture surface] PC 12 cell Primary neuronal Reconstituted skin
Alternatives 2007 –Workshop, Ankara, 12 -13 November 2007 Six principles of GCCP Principle 2 Assure the quality of all materials and methods, and of their use and application, in order to maintain the integrity, validity, and reproducibility of the work 16
17 Alternatives 2007 –Workshop, Ankara, 12 -13 November 2007 GCCP – principle 2 Quality control Cells and tissues: authenticate and monitor Culture conditions, cell handling and maintenance: • Specification and monitoring of critical reagents/culture-ware (direct contact with cells) • Specification, calibration and monitoring of critical equipment (influence on cell growth/product – e. g. temp. , p. H, CO 2)
18 Alternatives 2007 –Workshop, Ankara, 12 -13 November 2007 GCCP – principle 2 In vitro culture conditions Changes of batches of cell and tissue culture materials, test systems and other supplies should be monitored with regard to their influence on in vitro growth conditions and principal endpoints in the study NGF (ng/ml) 0 mg/ml tetracycline 0 10 50 2 mg/ml tetracycline 0 10 50 53 k. Da PC 12 engineered with p 53 TET on/off
Alternatives 2007 –Workshop, Ankara, 12 -13 November 2007 Six principles of GCCP Principle 3 a Document the information necessary to track the materials and methods used and to permit the replication of the work 19
20 Alternatives 2007 –Workshop, Ankara, 12 -13 November 2007 GCCP – principle 3 a Origins of cells and tissues: • Ethical or other relevant committee approval, if appropriate • Health status of animal • Species/strain of donor animal • Method of isolation employed • Tissue origin • Sex of donor animal • Age of foetuses or animals and numbers used • Any special treatment of the animal • Cell type(s) isolated • Date of isolation • In case of cell line: passage nr used • Name of user Records on handling, maintenance and storage Standard Operating Procedures / optimised protocols
21 Alternatives 2007 –Workshop, Ankara, 12 -13 November 2007 Six principles of GCCP Principle 3 b Report the information required to enable the target audience to understand evaluate the work
Alternatives 2007 –Workshop, Ankara, 12 -13 November 2007 22 GCCP – principle 3 b To produce a high-quality scientific report, various components have to be incorporated: generation of ideas, planning and experimental design, execution of the study, data collection and analysis, and discussion and conclusions. The final report will depend on the requirements of the audience : • scientific research community • a client • in-house personnel • a regulatory body • a grant reviewer etc.
23 Alternatives 2007 –Workshop, Ankara, 12 -13 November 2007 Six principles of GCCP Principle 4 Establish and maintain adequate measures to protect individuals and the environment
24 Alternatives 2007 –Workshop, Ankara, 12 -13 November 2007 GCCP – principle 4 Biological: Proper use of laminar-flow cabinets Physical: Proper handling of liquid nitrogen during cryopreservation of cells and tissues and retrieval of vials from frozen storage
Alternatives 2007 –Workshop, Ankara, 12 -13 November 2007 Six principles of GCCP Principle 5 Provide relevant and adequate education and training of all personnel to promote high quality work and safety 25
Alternatives 2007 –Workshop, Ankara, 12 -13 November 2007 26 GCCP – principle 5 Provide adequate training in: • Principles of sterile technique and aseptic manipulation (including principles of disinfection and sterilization) • Principles of in vitro cell culture (use of culture media, subculture methods, viability testing, cryopreservation, quality control including authenticity, mycoplasma and sterility testing) • Microscopy techniques • Centrifugation techniques • Laboratory design and safety (including liquid nitrogen storage activities) • Risk assessment and risk management of in vitro work • Quality assurance of in vitro work
27 Alternatives 2007 –Workshop, Ankara, 12 -13 November 2007 Six principles of GCCP Principle 6 Ensure that all work is performed in an ethical, responsible and accountable manner, and in compliance with relevant laws and regulations
Alternatives 2007 –Workshop, Ankara, 12 -13 November 2007 28 Application of GCCP – concluding remarks GCCP sets the minimum standards for any work involving cell and tissue cultures, however its detailed implementation depends on the nature of the work Ø Basic research involved: Ø Testing procedures in diagnostics, pharmacology, regulatory toxicology Ø Manufacture of products and therapeutics preparation of cells and tissues (vaccines, antibodies, hormones, tissue engineering, gene therapies)
29 Alternatives 2007 –Workshop, Ankara, 12 -13 November 2007 More information can be found on the ECVAM web site: http: //ecvam. jrc. it Workshop reports Task force reports Mailing list
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