1. Alternatives 2007 –Workshop, Ankara, 12-13 November 2007 1 Agnieszka Kinsner and Sandra Coecke NOTES 1. PLACE, DATE AND EVENT NAME 1.1. Access the slide-set place, date and event name text box beneath the JRC logo from the Slide Master. 1.2. Do not change the size nor the position of that text box. 1.3. Replace the mock-up texts for the place (“Place”), the date (“dd Month YYYY”) and the event name (“Event Name”) with your own texts. 1.4. Set it in MetaPlus Book Roman, if you own the typeface. Otherwise, keep the original typeface – Arial. 1.5. Keep the original flush-left justification. 1.6. Keep the original font colour (white). 1.7. Keep the original font body size (7 pt) and the text on one single line. 2. SLIDE NUMBER 2.1. The slide number on the banner’s lower right-hand side is automatically generated. 3. SLIDES 3.1. Duplicate the first slide as needed. 3.2. Do not change the size nor the position of the slide’s text box. 3.3. Try not to place more text on each slide than will fit in the given text box. 3.4. Replace the mock-up heading text (“Joint Research Centre (JRC)”) with your own text heading. 3.5. Set it in Eurostile Bold Extended Two or in Helvetica Rounded Bold Condensed, if you own one of these typefaces. Otherwise, keep the original typeface – Arial. 3.6. Keep the original flush-left justification. 3.7. Keep the original font colour (100c 80m 0y 0k). 3.8. Keep the original font body size (28 pt) and the heading on one single line whenever possible. Reduce the font body size if needed. 3.9. Replace the mock-up text (“The European Commission’s Research-Based Policy Support Organisation)”) with your own text. 3.10. Set it in MetaPlus Book Roman, if you own the typeface. Otherwise, keep the original typeface – Arial. 3.11. Keep the original flush-left justification. 3.12. Keep the original font colour (100c 80m 0y 0k). Use black if you need a second colour. 3.13. Keep the original font body size (22 pt) or reduce it if unavoidable. 3.14. Replace the EU-27 map mock-up illustration with your own illustration(s). 3.13. Try to keep your illustration(s) right- and top- or bottom-aligned with the main text box whenever possible. NOTES 1. PLACE, DATE AND EVENT NAME 1.1. Access the slide-set place, date and event name text box beneath the JRC logo from the Slide Master. 1.2. Do not change the size nor the position of that text box. 1.3. Replace the mock-up texts for the place (“Place”), the date (“dd Month YYYY”) and the event name (“Event Name”) with your own texts. 1.4. Set it in MetaPlus Book Roman, if you own the typeface. Otherwise, keep the original typeface – Arial. 1.5. Keep the original flush-left justification. 1.6. Keep the original font colour (white). 1.7. Keep the original font body size (7 pt) and the text on one single line. 2. SLIDE NUMBER 2.1. The slide number on the banner’s lower right-hand side is automatically generated. 3. SLIDES 3.1. Duplicate the first slide as needed. 3.2. Do not change the size nor the position of the slide’s text box. 3.3. Try not to place more text on each slide than will fit in the given text box. 3.4. Replace the mock-up heading text (“Joint Research Centre (JRC)”) with your own text heading. 3.5. Set it in Eurostile Bold Extended Two or in Helvetica Rounded Bold Condensed, if you own one of these typefaces. Otherwise, keep the original typeface – Arial. 3.6. Keep the original flush-left justification. 3.7. Keep the original font colour (100c 80m 0y 0k). 3.8. Keep the original font body size (28 pt) and the heading on one single line whenever possible. Reduce the font body size if needed. 3.9. Replace the mock-up text (“The European Commission’s Research-Based Policy Support Organisation)”) with your own text. 3.10. Set it in MetaPlus Book Roman, if you own the typeface. Otherwise, keep the original typeface – Arial. 3.11. Keep the original flush-left justification. 3.12. Keep the original font colour (100c 80m 0y 0k). Use black if you need a second colour. 3.13. Keep the original font body size (22 pt) or reduce it if unavoidable. 3.14. Replace the EU-27 map mock-up illustration with your own illustration(s). 3.13. Try to keep your illustration(s) right- and top- or bottom-aligned with the main text box whenever possible. Good Cell Culture Practices (GCCP) and in vitro toxicology

2. Alternatives 2007 –Workshop, Ankara, 12-13 November 2007 2 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

3. Alternatives 2007 –Workshop, Ankara, 12-13 November 2007 3 Cell Culture: History of “Issues” HeLa cell contamination of “new” cell lines - HepG2, INT407, Chang Liver, etc….. HeLa cell contamination of “new” cell lines - HepG2, INT407, Chang Liver, etc….. Mycoplasma and other fastidious micro-organisms – Mycoplasma and other fastidious micro-organisms – silent infections which cause irreversible genetic and phenotypic changes silent infections which cause irreversible genetic and phenotypic changes Cell lines are inherently unstable and some prone to change (degree of instability varies) 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 Cells cannot read SOPs! – careful attention to stock cultures and preparation of cells for assays

4. Alternatives 2007 –Workshop, Ankara, 12-13 November 2007 4 Standards in Cell Culture Need for standardisation in cell culture – why? 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 5 Start of GCCP 1999 General Assembly of the Third World Congress on Alternatives and Animal Use in the Life Sciences stated: guidelines defining minimum standards in cell and tissue culture Good Cell Culture Practice facilitate the inter-laboratory comparison encourage journals “The participants … call on the scientific community to develop guidelines defining minimum standards in cell and tissue culture, to be called Good Cell Culture Practice … should facilitate the inter-laboratory comparison of in vitro results … encourage journals in the life sciences to adopt these guidelines...”

6. Alternatives 2007 –Workshop, Ankara, 12-13 November 2007 6 Good Cell Culture Practice GLP 1999..................20022003 ECVAM Workshop Report World Conference Workshop on GCCP 2004 OECD Draft Consensus Document OECD Task Force ECVAM ICCVAM ECVAM Task Force Report ECVAM GCCP Guidance Document 2005 Coecke et al. (2005) ECVAM Good Cell Culture Practice Task Force Report 2, ATLA 33, 261-287

7. Alternatives 2007 –Workshop, Ankara, 12-13 November 2007 7 GCCP Guidance Document AIM Reduce ‘uncertainty’ in data from cell culture systemsReduce ‘uncertainty’ in data from cell culture systems Support best practice in all aspects of the use of cells and tissues in vitroSupport 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.)Complement, but not to replace, any existing guidelines and regulations (Eu. Pharmacopoeia, OECD, etc.) Consensus guidance to enable greater international harmonisation and standardisationConsensus guidance to enable greater international harmonisation and standardisation

8. Alternatives 2007 –Workshop, Ankara, 12-13 November 2007 8 Sets the minimum standards for any work involving cells and tissues of human and animal origin Sets the minimum standards for any work involving cells and tissues of human and animal origin Discusses issues related to : 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 GCCP Guidance Document

9. 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 and 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 10 Establish and maintain a sufficient understanding of the in vitro system and of the relevant factors which can affect it Principle 1 Six principles of GCCP

11. Alternatives 2007 –Workshop, Ankara, 12-13 November 2007 11 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 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 Stability and functional integrity of the system in relation to its intended use Purity, e.g. freedom from biological contamination Purity, e.g. freedom from biological contamination GCCP – principle 1

12. Alternatives 2007 –Workshop, Ankara, 12-13 November 2007 12 HeLa cell contamination of “other” cell lines (Stacey, Dev. Biols., 2000) Southern Blot MultilocusFingerprints probe 33.15 24Kb 5Kb GCCP – principle 1 Authenticity

13. Alternatives 2007 –Workshop, Ankara, 12-13 November 2007 13 Mycoplasma: Vampires of Cell Culture! Cell transformation Cell transformation Chromosome damage Chromosome damage Physiological changes Physiological changes Changes in expression of surface Changes in expression of surface markers markers Etc…. Etc…. GCCP – principle 1 Microbial contamination

14. Alternatives 2007 –Workshop, Ankara, 12-13 November 2007 14 Endogenous viruses GCCP – principle 1 Microbial contamination

15. Alternatives 2007 –Workshop, Ankara, 12-13 November 2007 15 Prepare and quality control master and working cell stocks Prepare and quality control master and working cell stocks Control within qualified passage ranges Control within qualified passage ranges Check whether cell performance changes in new environments [e.g. Check whether cell performance changes in new environments [e.g. atmosphere, nutrition (e.g. serum batches, medium replenishment), atmosphere, nutrition (e.g. serum batches, medium replenishment), culture surface] culture surface] GCCP – principle 1 Stability Primary neuronal culture PC12 cell line Reconstituted skin

16. Alternatives 2007 –Workshop, Ankara, 12-13 November 2007 16 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 Six principles of GCCP Principle 2

17. Alternatives 2007 –Workshop, Ankara, 12-13 November 2007 17 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 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., pH, CO 2 ) Specification, calibration and monitoring of critical equipment (influence on cell growth/product – e.g. temp., pH, CO 2 ) Formal cell culture QA role in lab GCCP – principle 2 Quality control

18. Alternatives 2007 –Workshop, Ankara, 12-13 November 2007 18 0 10 50 0  g/ml tetracycline 2  g/ml tetracycline NGF (ng/ml) 53kDa 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 PC12 engineered with p53 TET on/off In vitro culture conditions GCCP – principle 2

19. Alternatives 2007 –Workshop, Ankara, 12-13 November 2007 19 Document the information necessary to track the materials and methods used and to permit the replication of the work Six principles of GCCP Principle 3a

20. Alternatives 2007 –Workshop, Ankara, 12-13 November 2007 20 Ethical or other relevant committee approval, if appropriate Ethical or other relevant committee approval, if appropriate Species/strain of donor animal Species/strain of donor animal Tissue origin Tissue origin Sex of donor animal Sex of donor animal Age of foetuses or animals and numbers used Age of foetuses or animals and numbers used GCCP – principle 3a Health status of animal Health status of animal Any special treatment of the animal Any special treatment of the animal Method of isolation employed Method of isolation employed Cell type(s) isolated Cell type(s) isolated Date of isolation Date of isolation In case of cell line: passage nr used In case of cell line: passage nr used Name of user Name of user Origins of cells and tissues: Records on handling, maintenance and storage Standard Operating Procedures / optimised protocols

21. Alternatives 2007 –Workshop, Ankara, 12-13 November 2007 21 Report the information required to enable the target audience to understand and evaluate the work Six principles of GCCP Principle 3b

22. Alternatives 2007 –Workshop, Ankara, 12-13 November 2007 22 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. GCCP – principle 3b

23. Alternatives 2007 –Workshop, Ankara, 12-13 November 2007 23 Establish and maintain adequate measures to protect individuals and the environment Six principles of GCCP Principle 4

24. Alternatives 2007 –Workshop, Ankara, 12-13 November 2007 24 Physical: Proper handling of liquid nitrogen during cryopreservation of cells and tissues and retrieval of vials from frozen storage Biological: Proper use of laminar-flow cabinets GCCP – principle 4

25. Alternatives 2007 –Workshop, Ankara, 12-13 November 2007 25 Provide relevant and adequate education and training of all personnel to promote high quality work and safety Six principles of GCCP Principle 5

26. Alternatives 2007 –Workshop, Ankara, 12-13 November 2007 26 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 GCCP – principle 5

27. Alternatives 2007 –Workshop, Ankara, 12-13 November 2007 27 Ensure that all work is performed in an ethical, responsible and accountable manner, and in compliance with relevant laws and regulations Six principles of GCCP Principle 6

28. 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 involved:  Basic research  Testing procedures in diagnostics, pharmacology, regulatory toxicology  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 29 More information can be found on the ECVAM web site: http://ecvam.jrc.it Workshop reports Task force reports Mailing list