The quality control of cell lines and the prevention , detection, and cure of contamination

1. The quality control of cell lines and the prevention, detection, and cure of contamination

2. Obtaining the basic materials Importance of cell culture collections: 1. well characterized, microbe free seed stock 2. prevention of cross-contamination

3. Resource Center 1.ATCC( American Type Culture Collection Over 3200 cell lines and hybridoma from 75 species ATCC.com 2 .National Institute of general Medical Science ( NIGM) 5270 cell cultures and 275 DNA samples mainly derived from patients with genetic and chromosomal abnormalities 3.European collection of animal cell culture ( ECACC) 1200 cell lines and hybridoma 7000 human genetic and chromosome abnormality cell lines

4. 4.Riken gene Bank( Japan) 300 cell lines and hybridoma 5.Department of Human and Animal Cell Culture ( Germany) 100 cell lines and hybridoma

5. Quarantine and initial handling of cell lines • cultures should be handled in class II safety cabinet • cultures should be handled in a Quarantine laboratory separate from main tissue culture area • a token freeze should be made as soon a possible • initial characterization and microbial control should be made

6. NEW CELL LINES expand TOKEN FREEZEquality control QUARANTINE LABORATORY ---------------------------------------------------------------------- MAIN CULTURE SUIT expand culture MASTER BANK quality control expand one ampoule WORKING BANK( 40-100 AMPOULE) quality control expand one ampoule TERMINATION OF CULTURES

7. Source of contamination • Operators technique inadequately trained personal  Environment poor laboratory condition • Use and maintenance of laminar flow • Humid incubator • Cold stores • Sterile materials • Imported materials and biopsies • Quarantine non-quality controlled cell lines

8. Monitoring contamination  Check by eyes and microscope • Clean hood and every thing if contamination is suspected • Record nature of contamination • Check stock solution , sterilization procedure if similar contamination occur • Do not try to decontaminate unless the contamination is irreversible

9. Microbial Contamination • Sudden change of pH decrease in bact contamination, increase in fungal contamination • Cloudiness in the medium • Granular appearance between cells( ~x100) bacterial contamination

10. Types of Microbial Contamination Bacteria , Fungi Mold, mycoplasma, prptozoa bacteria yeast

11. mycoplasma mold Mycoplasma on culture cell

12. 2.testing for bacteria, yeast, and other fungi may be detected by  turbidity of the medium  pH change methods: Thioglycollate medium enrichment medium used in qualitative procedures for the sterility test and for the isolation and cultivation of aerobes, anaerobes and microaerophiles e.g. sodium thioglycolate: comsume O2 and allow growth of anaerobes microbes

13. Strict aer­obe aero­tol­er­ant anaer­obe strict anaer­obe faculative microaerophile http://www.youtube.com/watch?feature=player_embedded&v=x9bMS1G1myw

14. Common sources for microbial contamination Bacteria Sources clothing,skin,hair,aerosol( sneezing pipetting),insecure caps on media and culture flask  air current  humidified incubator  purified water  insects  contaminated cell line  plants

15. Fungi( excluding yeast)  fruit  damp wood or other cellulose products  humidified incubator Yeast  bread  humidified incubator  operators Mycoplasma  contaminated cell lines  serum  medium  operator

16. Detection of Bacteria and Fungi in Cell Culture http://www.youtube.com/watch?v=_fAVPMbkm78

17. Mycoplasma • affect the rate of cell growth induce morphological change cause chromosome aberration influence amino acid and nucleic metabolism induce cell transformation

18. Fluorescence stainning of mycoplasma  Hoechst 33258 DNA staining method PCR detection of mycoplasma Two step PCR Universal primer amplify region between major region gene ( 16S and 23S rRNA) amplify spacer region of contaminated mycoplasma

19. 100 bp ladder Water control Positive sample Negative sample Positive control Positive control + internal control Negative sample+ internal control Positive sample + internal control

20. Other methods of detecting mycoplasma • Mycrobial culture 2. Molecular hybridization 3. 3H thymidine incorporation 3H labeled s.s DNA probe( homologous with mycoplasma) 4. mycoplasma detection kit ( enzyme immuno assay ) 5. Myco-tech kit: cells coculture with 6-ethylpurine deoxyribose  toxic compound forms  cell dies

23. Ciproflaxacin treatment

24. Virus contamination a) virus sources:  tissue derived viral contamination depend upon:  species of origin  the tissue taken  the clinical history of the animal and patient e.g.HBV( 0.2—0.5% of blood donor were infected) some virus which can occur in humans exp. . Herpes simplex virus-1 Herpes simplex virus-2 human cytomegalovirus Epstein-barr virus hepatitis B, Hepatitis C Human herpes virus-6 HIV-1,HIV-2 Human T-cell lymphotropic virus-1 Human T-cell lymphotropic virus-2……………………….

25. b)Serum derived viral contamination e.g. Bovine Viral Diarrhoed Virus infectious bovine rhinotracheitis virus  Parainfluenza  may be detected by PCR or fluorescence methods

26. c) methods of detection  cocultivation cell extract of the tested cell lines  cell extract was added into sensitive cell line( BHK21, WI 38, HeLa, Vero, MDCK, JM, H9, fresh T cell)  electron microscope  In vivo methods  cell culture assay for murine retrovirus example: Murine leukemia Virus( MuLV) Characters: i) ectropic( infect only rodent cells) ii)xenotropic( infect only cells other than rodent) iii)amphotropic

27.  reverse transcriptase assay for retrovirus detection precipitate virus particles with PEG  R.T activity assay  PCR method for detection of BVDV and other virus

28. 5.Bovine Spongiform encephalopathy 1986, found in contaminated feed stuffs of cattles 6.Elimination of contamination a. discard contaminated culture b.find the source of contamination c.eliminate source of contamination  fumigate the whole lab  swab cell surfaces and equipment  use of antibiotics using 10-14 days for at least 3 passages

29. Antibiotics commonly used in elimination of microbial contamination Antibiotics working concentration active against AmphotrricinB 2.5mg/L yeast and other fungi Ampicillin 2.5mg/L bacteria(Gp,Gn) Cephalothin 100mg/L bacteria(Gp,Gn) Ciprofloxacin 10-40mg/L mycoplasma Gentamycin 50mg/L bacteria(Gp,Gn), mycoplasma Kanamycin 100mg/L bacteria(Gp,Gn), yeast mycoplasma removal agent 0.5mg/L mycoplasma Neomycin 50mg/L bacteria(Gp,Gn) Nystatin 50mg/L yeast and other fungi Penicillin-G 100000U/L bacteria(Gp) PolymyxinB 50mg/L bacteria(Gn) Streptomycin sulphate 100mg/L bacteria(Gp,Gn) Tetracycline 10mg/L bacteria(Gp,Gn)

30. Authentication To confirm the identity and origin species of the cell stocks 1. Isozyme analysis polymorphic enzyme variants  catalyze the same reaction but have different electrophoretic mobility

31. HeLa cell ???

32. G6PD:Glucose - 6 – Phosphate Dehydrogenase LDH:Lactate Dehydrogenase NP:Nucleoside Phosphorylase AST:Aspartate Aminotransferase

33. 2.Cytogenic analysis To establish the common chromosome complement or karyotype of a species or cell lines Giemsa banding( G banding, pH 6.8 giemsa), G11 banding( pH 11 giemsa stain) , Quinacrine ( Q) banding

34. 3.DNA fingerprinting Southern blotting hybridization

35. Southern blotting hybridization DNA extraction form sample DNA cut into fragments by restriction enzyme, separation by electrophoresis transfer DNA transfer to nylon membrane Application of with radiolabelled probe Develpoment of x-ray film

36. http://local.testing4dna.com/Local-Cell-Line-Authentication.htmlhttp://local.testing4dna.com/Local-Cell-Line-Authentication.html

37. Comparison of the uses of the different cell authentification methods available Application DNA finger printing cytogenic Isozyme analysis analysis species determination  individual identification  detection of cell line variation  regulatory authority recognition  

38. Information required for regulatory approval 1. History and genealogy of the cell line 2. Records and storage information on master and working cell banks 3.Culture requirements 4. Growth characteristics 5. Production and testing facilities 6. Quality control test: karyology,isozyme analysis,DNA fingerprinting,virus testing, retrovirus status, test for contaminated DNA, purification procedure/validation date characterization of products