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Keeping Cells Happy – Topics in Cell Health Maintenance and Viability S te v e n B u d d , M . S . , M . B . A P r o d u c t L i n e B u s i n e s s S p e c i a l i st , ATC C S e p te m b e r 2 9 , 2 0 1 6 About ATCC Founded in 1925, ATCC is a non-profit organization with headquarters in Manassas, VA World’s premiere biological materials resource and standards development organization Established partner to global researchers and scientists ATCC collaborates with and supports the scientific community with industry-standard biological products and innovative solutions Strong team of 400+ employees; over onethird with advanced degrees 2 Outline Cell health topics Overview Complete growth media Cryopreservation Cell growth and propagation Contamination and aseptic technique Cell authentication 3 Overview of cell health Cell culture practices that involve good cell health techniques cover a wide range of topics, including: Measuring and monitoring growth Good aseptic technique Addressing cryopreservation issues Cell line authentication 4 Complete growth media Classical cell culture media Media ingredients Additives Animal sera 5 Classical cell culture media Eagle’s Minimum Essential Medium (EMEM; ATCC® 30-2003™) Early formula for mouse L cells Amino acid composition to match mammalian cells Dulbecco’s Modified Eagle’s Medium (DMEM; ATCC® 30-2002™) Twice the amino acid concentration as EMEM, four times the vitamin concentration Iscove’s Modified Dulbecco’s Medium (IMDM; ATCC® 30-2005™) Additional amino acids as DMEM, suited for lymphocytes and hybridomas Kaighn’s Modification of Ham’s F-12 (ATCC® 30-2004™) Designed for primary cells with or without serum 6 Classical cell culture media DMEM/ F12 Medium (ATCC® 30-2006™) Mixture of DMEM and Ham’s F-12, used in a wide range of cell types Proliferation assays McCoy’s 5A (ATCC® 30-2007™) Hepatoma cells and primary cells RPMI-1640 (ATCC® 30-2001™) Ideal for Lymphocytes Leibovitz’s L-15 (ATCC® 30-2008™) For use without CO2 7 Media ingredients Sodium bicarbonate H2CO3 H2O + CO2 H2O + CO2 NaHCO3 H+ + HCO3H+ + 2HCO3- HEPES buffer Can buffer without CO2 enrichment Good for working under the hood Phenol Red Monitors pH of media Yellow = acidic Purple = basic May mimic action of steroid hormones. Sodium Pyruvate Helps maintain metabolism 8 Additives Nonessential Amino Acids Can be added to reduce the metabolic burden on cells L-Glutamine (ATCC® 30-2214™) Present in ATCC classical cell culture media Relatively stable in bottles kept at 4°C - 8°C Glutamine degradation increases ammonia toxicity Generally not recommended to “spike” media with L-Glutamine Antibiotics and Antimycotics Penicillin-Streptomycin, Gentamicin Sulfate Amphothericin B Generally not recommended 9 Animal sera Fetal Bovine Serum (ATCC® 30-2020™) Fetal Bovine Serum, Embryonic Stem Cell Qualified (ATCC® SCRR-30-2020™) Very rich in growth factors, most common choice Heat inactivation: Not Advised Calf Bovine Serum (ATCC® 30-2030™) Lower concentrations of growth factors, good for contact inhibition studies Horse Serum (ATCC® 30-2040™) Collected from closed herds, lot-to-lot consistency, no bovine viruses 10 Media usage considerations Maintain cells in the same media Media variability Possible osmotic shock When transferring to new media: Use 1:1 mix (50% old, 50% new media) 1:2 mix 1:3 mix 1:7 mix ATCC® Animal Cell Culture Guide 11 Cryopreservation procedure Overview Cryoprotectants and media preparation Freezing cells in a controlled-rate chamber Long-term storage 12 Cryopreservation principles Ideal Negative effect on viability High Ice formation Solute concentration Low 0.1 1.0 Rate of cooling (°C/min) 10 High levels of ice formation and increased solute concentration have a negative impact on cell viability Optimal cooling rate for cell viability is 1°C/min to 3°C/min 13 Cryoprotectants Cell type Cryoprotectant Temperature Number of cells Animal cells DMSO (5-10%) or Glycerol (5-10%) -140°C 106 to 107/mL Bacteria Glycerol (5-10%) -80°C 107/mL Yeast Glycerol (10%) -140°C 107/mL Protozoa DMSO (5-10%) or Glycerol (10-20%) -140°C 105 to 107/mL Plant cells DMSO (5-10%) and Glycerol (5-10%) -140°C 3% to 20% cell volume Animal viruses (free) None -80°C NA Animal viruses (infected cells) DMSO (7%) -10°C 106/mL 14 Media preparation Classical cell culture media – DMEM, EMEM, RPMI-1640 (for suspension cells) 5-10% DMSO 20% fetal bovine serum (FBS) or bovine serum albumin (BSA) • Additional cryprotectant properties • Necessary for post-thaw cell survival ATCC Serum-free Freezing Media (ATCC® 302600™) All in one media 10% DMSO with proteins and additives for cell survival 15 Freezing cells -70°C Controlled rate freeze chamber -1°C/min cooling rate A few hours to 24 hours -140°C Liquid nitrogen tank 16 Freezing cells CoolCell® LX (ATCC® ACS-6000™) Reliable -1°C/min cooling rate 4 hours in -70°C freezer Comfortable to touch No alcohol use or maintenance Can be used with all cell types Insulated polyethylene material Thermoconductive alloy • Verified use with organoids 17 Low temperature storage For the best security, always store your cells in liquid nitrogen freezers 18 Low temperature storage Mammalian cells Long-term storage should be below -140°C -140°C for an indefinite length of time -80°C for less than 1 year Vials should be stored in a liquid nitrogen unit above the volume of liquid at the bottom of the tank This temperature should be between -140°C and -180°C -150°C -178°C -196°C 19 Cell growth and propagation Population doubling level Measuring cell viability and growth • MTT Assay • XTT Assay 20 Population doubling level 21 Growth and viability Quantitative evaluation of cell proliferation rate and response to external factors that affect cell viability Uses tetrazolium salts in a colorimetric method for evaluating cell populations MTT Cell Proliferation Assay (ATCC® 30-1010K™) • Tetrazolium MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) XTT Cell Proliferation Assay (ATCC® 30- 1011K™) • Tetrazolium XTT (sodium 2,3,-bis(2-methoxy-4-nitro-5-sulfophenyl)-5[(phenylamino)-carbonyl]-2H-tetrazolium) 22 Population doubling level MTT Reaction XTT Reaction MTT salt is reduced within cellular matrix to Formazan, lysed with detergent to solubilize crystals XTT salt is reduced to Formazan at cell membrane with PMS agent Media turns PURPLE Media turns ORANGE Detergent Reagent Formazan MTT NADH NAD+ NADH NAD+ 23 Growth and viability Determining Optimal Cell Counts Plate, in triplicate, a serial dilution of 1 x 106 to 1 x 103 cells per mL (96-well plate) MTT Assay XTT Assay Add MTT Reagent Add XTT Reagent + Activation Agent Incubate 2-4 hours – add Detergent Incubate 2-4 hours Incubate 2-4 hours or overnight Determine optimal number of cells to use Repeat assay with experimental factors – compare absorbance at optimal cell volume Plots absorbance versus cell number 24 Growth and viability 25 Contamination and aseptic technique Sources of contamination Aseptic technique Mycoplasma contamination Cross-contamination 26 Sources of contamination Types Microbial – Bacteria, mycoplasma, fungi, viruses Cellular – Cross-contamination Signs (bacteria and fungi) Turbid media Rapid decline in pH – color change Morphological changes Filamentous structures 27 Sources of contamination Contaminated Resources Cell Lines Media, Sera, Reagents Nonsterile equipment Personnel and equipment Poor culturing practices Dust and aerosol Physical Causes Improperly maintained autoclaves/ovens Faulty laminar flow 28 Aseptic technique Make it difficult for microorganisms to invade culture vessels Sealed cultured vessels Vented cap flasks Disposable aspirators and pipette tips Cell culture hoods with good laminar flow Do not use as a storage area! Spray media bottles / reagents with alcohol 29 Aseptic technique Use small volumes of reagents at a time Use clean lab coats and protective clothing AVOID USING ANTIBIOTICS IN MEDIA! Can contribute to chronic contamination Rarely completely prevents contamination Toxic to cells 30 Mycoplasma contamination Not easily detected, cannot be seen by microscopy Chromosomal aberrations Disruption of nucleic acid synthesis Changes in membrane antigenicity Inhibition of cell proliferation and metabolism Decreased transfection rates Changes in gene expression profiles Cell death 31 Mycoplasma contamination PCR-based kit Detects any of the 60 most common mycoplasmas Kit Components/Procedure Buffer Mix of primers specific to mycoplasma Positive control • DNA from mycoplasma Collect/pellet cells Cell lysis Touchdown PCR Run gel/stain 32 Cross-contamination Leads to the replacement of the original cell line with the contaminant Causes Multiple cell lines under the hood at the same time Failure to change out pipettes Receiving cell lines from other labs 20% of scientific publications use misidentified cultures 50% of preclinical research is not reproducible 33 ATCC Short Tandem Repeat (STR) Profiling Ensures your cells are what you think they are STR profile of your cell line Comparison of your cells against ATCC STR Profile database Electropherograms supporting the allele calls at each locus Comprehensive interpretation of results 34 Summary points Cell culture • Select appropriate media for your cells • Understand issues/considerations for adding additional ingredients • Consistently use same media whenever possible Cryopreservation • Use a reliable rate-controlled cooler • Keep mammalian cells at -140°C for long term • Keep cells in the vapor phase in liquid nitrogen tanks Contamination control • Follow aseptic technique • Don’t rely on antibiotics/antimycotics • Test for mycoplasma • Make sure to authenticate cell lines 35 Disclaimers © 2016 American Type Culture Collection. The ATCC trademark and trade name, and any other trademarks listed in this publication are trademarks owned by the American Type Culture Collection unless indicated otherwise. 36 Thank you for joining today! Register for more ATCC “Excellence in Research” webinars, or watch recorded webinars, at www.atcc.org/webinars. November 3, 2016 12:00 PM ET The Biology of Anaerobic Bacteria and Predominant Propagation Practices Allison Faust, Senior Biologist, ATCC Nancy Krueger, Senior Biologist, ATCC Please email additional questions to: [email protected] 37