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AUTHENTICATION and CHARACTERIZATION of ANIMAL CELL LINES: TOWARDS BEST PRACTICES in CELL CULTURE Yvonne A

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AUTHENTICATION and CHARACTERIZATION of ANIMAL CELL LINES: TOWARDS BEST PRACTICES in CELL CULTURE Yvonne A AUTHENTICATION AND CHARACTERIZATION OF ANIMAL CELL LINES: TOWARDS BEST PRACTICES IN CELL CULTURE Yvonne A. Reid, Ph.D. Manager/Scientist, Cell Biology Program, ATCC February 27th 2014 ATCC • Founded in 1925, ATCC is a non-profit organization with headquarters in Manassas, VA • ATCC serves and supports the scientific community with industry-standard products and innovative solutions • World’s leading biological resource center and provider of biological standards • Broad range of biological materials . Microorganisms . Cell lines . Derivatives . Bioproducts 2 OutlineMinimum Requirements for Reproducible Results Monitoring cell morphology Optimizing cell growth Applying the ‘Seed Stock’ concept Using cells at low passage or PDL Checking for microbial contamination Confirming species (misidentification or cross-contamination) Confirming characteristics/functionality History of misidentified cell lines – impact on research Reducing cellular and microbial contamination Receiving a cell line into the laboratory 3 Monitoring cell morphology Fibroblast-like Lymphoblastoid-like Epithelial-like Senescing cells Adherent Suspension Adherent β- galactosidase • Monitor morphology: shape, membrane structure, optical properties – at various stages of cell growth . Detecting senescing cells: larger, vacuolated, more heterogeneous . Detecting overt microbial contamination 4 Optimizing cell growth Growth profile 1.00E+05 PDT:33 hr • Recommended inoculum 2 PDT:21 hr • Population Doubling Time (PDT) 1.00E+04 • Optimum concentration range for Cells/cm PDT:20 hr maintenance and subculture • Observe for changes in growth rate (crisis) 1.00E+03 0 1 2 3 4 5 6 7 Days in Culture 5 Applying the ‘Seed Stock’ concept to manage cell banks Donor vial • Microbial contamination check (bacteria, fungi, Mycoplasma, viruses) • Species verification (interspecies, intraspecies) • Post-freeze recovery • Post-freeze viability • Functional/characterization test Pre-Master Cell Bank • Growth Curve Master Cell Bank • Microbial contamination check (MCB) • Species verification • Post-freeze recovery • Post-freeze viability • Doubling time Working Cell Bank • Functional test (WCB) Customer 6 Managing your cells – apply the ‘Seed Stock’ concept Seed stock (MCB) Lung cells Working Cell Bank (WCB) Lymphocyte cell Experiment (assay) Pancreatic cells 7 Advantages of the ‘Seed Stock’ concept • Ensures safe stocks of early material (Master Cell Bank) • Reduces passage number (prevents genotypic and phenotypic drift) • Prevents lot-to-lot variability • Reduces the chance of cellular and microbial contamination • Ensures reproducibility of experimental data HEK-293 cells stained with beta tubulin 8 Use cell lines at low passage number • Mimics tissue of origin • Reduce phenotypic drift • Reduce genotypic drift • Reproducible results Lymphocyte 9 Effects of high passage number Several characteristics were tested to determine the effect of various passage numbers on Syrian hamster embryo (SHE) cells Characteristic Low passage no. Intermediate passage no. High passage no. (3) (43, 48) (64) Plating efficiency Doubling time Cell growth Chang-Liu, CM et al. Cancer Letters. 26;113:77-86. (1997) 10 Test for microbial contamination Bacteria and Fungi • Microbiological culture (Aerobic, Anaerobic) • PCR HeLa contaminated with E. coli Mycoplasma • Direct agar culture • Indirect Hoechst stain • PCR M. hyorhinis infection Viruses • Cytopathic effect (CPE) • Indirect immunofluorescent antibody (IFA) • Enzyme immunoassay (EIA) • PCR CPE of human herpes virus 11 Tests for bacterial/fungal contamination Medium Temperature Atmosphere Incubation Detection time Brain Heart Infusion 26°C/37°C Aerobic 14 days Wide variety of fastidious bacterial (BHI) Thioglycollate broth 26°C/37°C Aerobic 14 days Obligate aerobes (O2); Obligate anaerobes (no O2 to grow) Facultative anaerobes (growth with or without O2 ) Trypticase Soy broth 26°C/37°C Aerobic 14 days Wide variety of aerobic and facultative (TSB) anaerobic microorganisms (S. aureaus, B subtilis, P. aeruginosa, C. albicans, A. niger) Blood agar 37°C Aerobic 14 days Wide variety of fastidious microorganism Anaerobic (bacteria) Sabouroud broth 26°C/37°C Aerobic 14 days Molds, yeasts, fungi (Aspergillus, Saccharomyces, Candida) HTYE broth 26°C/37°C Aerobic 14 days Water-borne organisms 12 Consequences of bacterial and fungal infection • Interferes with the growth rate of cells (by depleting medium of nutrients) • Cell-cell interaction (interaction of NK cells and dendritic cells • Interferes with invasion assay • Interferes with protein synthesis, RNA synthesis and DNA synthesis Cells infected with Mycoplasma 13 Tests for Mycoplasma contamination Mycoplasma • Small bacteria without cell wall • Growth to a high concentration without visible morphological changes Direct • Alter cell growth characteristics Agar Culture • Easily detected . Indirect Hoechst stain method . Direct agar method . PCR 14 Tests for Mycoplasma contamination Direct agar (positive) Direct agar (negative) Indirect Hoechst stain* (positive) Indirect Hoechst stain (negative) * FDA Points to Consider (POC) 15 Tests for Mycoplasma contamination PCR 200 bp 1. 100 bp DNA ladder 6. M. arginini 2. A. laidlawii 7. M. hominis 3. M. pirum 8. M. orale 4. M. hyorhinis 9. M. fermentans 5. M. salivarium 10. 100 bp DNA ladder Universal primers designed specific for the 16S rRNA coding region in the Mycoplasma genome will detect a wide variety of Mycoplasma species 16 Consequences of Mycoplasma infection • Interferes with the growth rate of cells • Interferes with lymphocyte transformation • Induction of morphological alteration (CPE) • Alters DNA, RNA, and protein synthesis • Interferes with the selection of mutant cells • Induces chromosomal aberrations (e.g., M. orale, and M. arginini) • Depletes essential amino acids (e.g., arginine from culture medium) • Interferes (inhibit or enhance) with viral replication Cells infected with Mycoplasma • Alters host plasma membrane (interferes with receptor studies) 17 Test for viral contamination Detection of human viruses by qPCR DNA virus RNA virus Human Papilloma Virus (HPV) Human Immunodeficiency Virus (HIV) Epstein-Barr Virus (EBV) Hepatitis C Virus (HepC) Cytomegalovirus (CMV) Hepatitis B virus (HBV)) 10610 510 410 310 210 HPV EBV Sensitivity test: amplicons separated on agarose gel; qPCR 18 Consequences of viral infection • Induces morphological changes (cytopathic effect – CPE) • Changes metabolic activity of cells • Interferes with receptor studies • Health hazard Note: Special safety precautions should be employed when working with cells from humans to avoid possible transmission of viral infection from the cultures to the technician. Contact your safety office for additional information on appropriate practices for your institution. 19 Confirm species and identity of cell line • Cytochrome C Oxidase subunit 1 Barcode of Life (COI) for interspecies identification Barcoding for species Identification • STR analysis (DNA profiling) for intraspecies identification and authentication of human cell lines 20 Confirm interspecies identity of cell line Cytochrome Oxidase I (COI) Assay Amplification of ~650 bp (base pairs) target DNA sequence in the mitochondrial gene for cytochrome c oxidase subunit 1 (CO1) 21 Short Tandem Repeat (STR) analysis for intraspecies identification of human cell line DNA location Degree of repetition Number of loci Repeat unit length Satellite DNA (centromere) 103 to 107 1 to 2 2 to several thousand bp Minisatellite DNA 2 to several hundred Many thousands 9 to 100 bp (telomere) Microsatellite DNA (STRs); 5 to about a hundred 104 to 105 1 to 6 bp randomly scattered STR profiling a method for cell line authentication! 22 STR analysis for human cell line identity • Target sequence consists of microsatellite DNA • Typically use 1-2 ng DNA • 1 to 2 fragments; discrete alleles allow digital record of data • Markers distributed throughout the genome • Highly variable within populations; highly informative Probability of two cell lines having identical STR profile at 18 markers is 1 x 10 -15 23 Outline of STR profiling procedure Resolve PCR fragments (Capillary electrophoresis) Extract DNA Size PCR Fragments (GeneScan software) PCR amplified sample Convert PCR fragment sizes to PowerPlex 18D System) alleles (Genotyper software) Spot onto FTA® paper Create reference database • Curate • Global comparisons 24 STR polymorphism Homozygous Heterozygous TATC TATC 8 11,12 Cell line A Cell line B 25 Human cell line identification: STR analysis 2 unrelated cell lines (separate individuals, female in origin) K562 WS1 D5S818 D13S317 D7S820 D16S539 vWA THO1 Amel. TPOX CSF1PO K562 11, 12 8 9, 11 11, 12 16 9.3 X 8, 9 9, 10 WS1 13 12 9, 10 10, 11 17, 18 8, 10 X 8, 9 10, 13 26 Human cell line identification: STR analysis 2 related cell lines (same individual; male in origin) HAAE-2 aortic artery HFAE-2 femoral artery D5S818 D13S317 D7S820 D16S539 vWA THO1 Amel. TPOX CSF1PO HAAE-2 12,13 11,12 8,10 12,13 14,18 7,9 X,Y 10,11 10,11 HFAE-2 12,13 11,12 8,10 12,13 14,18 7,9 X,Y 10,11 10,11 27 Gender is important for identification (amelogenin gene) male AMELX AMELY female AMELX gene contains a 6 bp deletion in the intron 1 28 STR analysis to monitor genomic stability Donor Token (Pre-MCB) Seed (MCB) Distribution (WCB) 29 Various cell lines function confirmed ATCC® CRL-1712.1™ ATCC® CL-173™ ATCC® CRL-1712.1™ (PC-12 Adh) rat ATCC® CL-173™, 3T3 L1, mouse pheochromocytoma cells form beta-tubulin preadipocyte, induced with in the presence of NGF dexamethasone and
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