Agnieszka Kinsner and Sandra Coecke

1. 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 Agnieszka Kinsner and Sandra Coecke

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. HeLa cell contamination of “new” cell lines - HepG2, INT407, 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 Cell Culture: History of “Issues”

4. 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 Standards in Cell Culture

5. 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 developguidelines defining minimum standards in cell and tissue culture, to be calledGood Cell Culture Practice… shouldfacilitate the inter-laboratory comparison of in vitro results … encourage journals in the life sciences to adopt these guidelines...” Start of GCCP

6. Good Cell Culture Practice OECD Draft Consensus Document OECD Task Force ECVAM ICCVAM GLP ECVAM Workshop Report 2005 1999 .................. 2002 2003 2004 World Conference Workshop on GCCP ECVAMTask Force Report ECVAM GCCP Guidance Document Coecke et al. (2005) ECVAM Good Cell Culture Practice Task Force Report 2, ATLA 33, 261-287

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

8. GCCP Guidance Document • Sets the minimum standards for any work involving cells and tissues of human and animal origin • Discusses issues related to: • Characterisation and maintenance of essential features of the in vitro system • Quality control of the systems • Recording and reporting (in-house and in scientific journals) • Safety • Ethics • Education and training

9. Six principles of GCCP • Establishment and maintenance of a sufficient understanding of the in vitro system and of the relevant factors which can affect it • 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 • 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 • Establishment and maintenance of adequate measures to protect individuals and the environment from any potential hazards • Compliance with relevant laws and regulations and with ethical principles • Provision of relevant and adequate education and training of all personnel, to promote high quality work and safety

10. 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

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 thesystem,e.g. morphological evaluation, provenanceand confirmation of genotypic and/or phenotypic characteristics • Stability and functional integrityof the system in relation to its intended use • Purity, e.g. freedom from biological contamination

12. GCCP –principle 1 Authenticity 24Kb HeLa cell contamination of “other” cell lines (Stacey, Dev. Biols., 2000) 5Kb Southern Blot Multilocus Fingerprints probe 33.15

13. GCCP –principle 1 Microbial contamination Mycoplasma: Vampires of Cell Culture! • Cell transformation • Chromosome damage • Physiological changes • Changes in expression of surface • markers • Etc….

14. GCCP –principle 1 Microbial contamination Endogenous viruses

15. GCCP –principle 1 • 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] Stability Reconstituted skin PC12 cell line Primary neuronal culture

16. 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

17. 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., pH, CO2) • Formal cell culture QA role in lab

18. 0 mg/ml tetracycline 2 mg/ml tetracycline 0 10 50 0 10 50 NGF(ng/ml) 53kDa 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 PC12 engineered with p53 TET on/off

19. Six principles of GCCP Principle 3a Document the information necessary to track the materials and methods used and to permit the replication of the work

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

21. Six principles of GCCP Principle 3b Report the information required to enable the target audience to understand and evaluate the work

22. GCCP –principle 3b • 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. Six principles of GCCP Principle 4 Establish and maintain adequate measures to protect individuals and the environment

24. 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

25. Six principles of GCCP Principle 5 Provide relevant and adequate education and training of all personnel to promote high quality work and safety

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. 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

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. More information can be found on the ECVAM web site: http://ecvam.jrc.it Workshop reports Task force reports Mailing list