in vitro culture

1. Alternatives 2007 –Workshop, Ankara, 12-13 November 2007 1
Agnieszka Kinsner and Sandra Coecke
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…..
• 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

4. 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 5
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, 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 .................. 2002 2003
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 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. 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
• 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
• Stability and functional integrity of the system in relation to its
intended use
• Purity, e.g. freedom from biological contamination
GCCP – principle 1

12. Alternatives 2007 –Workshop, Ankara, 12-13 November 2007 12
KB
KBcl2
WISH
(D)
WISH
(M)
L132
(M)
L132
(D)
CHANG
LIVER
INT407
(D)
INT407
(M)
15-C4
HeLa
(CCL2)
HeLa
229
HeLa
Ohio
Hep2
Hep2
clB
Hep2C
HeLaB
HeLa
S3
23Kb
9.5Kb
6.6Kb
2.3Kb
1
3
2
4
5
HeLa cell
contamination
of “other” cell
lines
(Stacey, Dev.
Biols., 2000)
Southern Blot
Multilocus
Fingerprints
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
• Chromosome damage
• Physiological changes
• Changes in expression of surface
markers
• 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
• 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]
GCCP – principle 1
Stability
Primary neuronal
PC12 cell 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, calibration and monitoring of critical equipment
(influence on cell growth/product – e.g. temp., pH, CO2)
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 10 50
0 mg/ml
tetracycline
2 mg/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
• Species/strain of donor animal
• Tissue origin
• Sex of donor animal
• Age of foetuses or animals
and numbers used
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:
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
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