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Making a Master or Working Cell Bank

Making a Master or Working Cell Bank. Bioman 2009 July 27 to July 30 Rochester, NY Presenter: Dana M. Hopkins Wm. Davies, Jr. Career & Technical HS. Master Cell Bank (MCB). Established from a single clone Represents a cell reserve “Frozen in Time” --Preserves characteristics

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Making a Master or Working Cell Bank

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  1. Making a Master or Working Cell Bank Bioman 2009 July 27 to July 30 Rochester, NY Presenter: Dana M. Hopkins Wm. Davies, Jr. Career & Technical HS

  2. Master Cell Bank (MCB) • Established from a single clone • Represents a cell reserve “Frozen in Time” --Preserves characteristics --Prevents contamination and deterioration • Produced in accordance with regulatory standards (21CFR 610)

  3. Cell line characterization • Testing objectives of cell line --Confirm identity (expression construct) --Confirm purity (contamination) --Confirm genetic stability (coding region) • Quality assurance established from master bank to end-of-production/post production cells (EPC/PPC)

  4. Safety testing of cell banks • Eliminates/minimize adventitious agents to the biopharmaceutical --bacteria --mycoplasma --fungi --viruses

  5. Source of Contaminants • Cell Substrate • Endogenous viruses • Exogenous microbial contaminants • Source material screening -Human (HIV, HBV, HCV, CJD, etc) -Animal (TSE sources, species-specific viruses.

  6. Contaminants (cont.) • Raw Materials *Cell culture reagents (animal and non- animal) • Environment • Water • Air • Human/Technicians

  7. Regulatory Documents • CBER/FDA: Points to consider in cell line characterization • CBER/FDA: Points to consider in manufacturing and testing • European Pharmacopeia (EP) • US Pharmacopeia (USP) • Japanese Pharmacopeia (JP)

  8. Validated In-house Guidelines • Species Identity: Confirmed by iso-enzyme analysis and cross-species contamination • Species Banding Pattern: Confirmed by agarose gel electrophoresis • DNA Fingerprinting: Karyology

  9. Purity Testing • Sterility: --Bulk harvest/cell banks tested for bacterial & fungal contamination. • Mycoplasma: --Two methods recommended by inoculation in broth and agar.

  10. Purity Testing • Adventitious viruses --Invitro assay with indicator cell lines --Invivo assay with embryonic chicken eggs • Retrovirus (rodent cell lines): --XC plaque assay --S+L- focus assay

  11. Purity Testing • Transmission Electron Microscopy (TEM) • Reverse transcriptase assay: --Product enhanced RT (PERT) --Unique enzyme in retroviruses --PCR sensitive to cDNA enzyme

  12. Building the Master Cell BankTransformation or Transfection • Introduce Foreign Gene that expresses Protein Product: (bacterial transformation) pick one pick one    Screen for expression of foreign gene o o oo o o Bacteria Lawn

  13. Grow Cells to 90% Confluence Images Courtesy of Corning Inc

  14. Cell Preparation for Freezing • Check cell line for stability and contaminations. • Refeed cells to ensure log phase of growth. • Label cryotubes with cell line, cells/vial, date, MCB • Count cells with a hemocytometer. Use trypan blue for viability.

  15. Freezing Media • 60 ml DMEM, 40 ml FCS, 1 ml Penicillin-Streptomycin Filter through 0.2u filter Aliquot in 15ml conical tubes for long storage at -80, or refrigerator for shorter periods of time. • For 100% DMSO: 4ml pH adjusted medium (DMEM) 1ml sterile 100% DMSO 0.1ml versene (prevents clumping of cells) Filter through 0.2u filter

  16. Cell Prep. Cont. • Transfer cell suspension to centrifuge tubes. • Centrifuge at 1000rpm fo 5 minutes, 2-8oC. • Siphon off all the medium. • Slowly add chilled freezing medium to yield 1 x 107 cells/ml. • Resuspend cells by gently pipetting. • Place tubes on ice. Dispense cells, 1 ml/vial. • Parafilm cryovials and place in -80 freezer.

  17. Mammalian Cells • Trypsinize adherent cells and pellet • For each 100 mm dish, resuspend pellet in half the volume of freezing medium. (if freezing 10 vials, add 5 mLs media) • 20% DMSO made fresh daily • Add dropwise DMSO to 10% final vol. • Final suspension: DMEM with 20% FCS, 10% DMSO, cells from 100 mm dish. • Chill cells on ice in centrifuge tube before dispensing in pre-chilled, 1 mL vials

  18. Thawing Mammal CellsWorking Cell Bank • Points to Consider: • Cells should be thawed rapidly and then diluted slowly into warm growth medium. • Transfer one 1ml vial to 50ml centrifuge tube. Slowly (dropwise) add 10ml warm medium. • Pelleting DMSO cells may harm fragile cells. • DMSO is toxic to cells. Change media quickly. • DMSO is OSHA sensitive. Replace with glycerol if at all possible

  19. Schrieber’s Protocol • Thaw vial quickly in 370C water. • Transfer cells to sterile, 15ml centrifuge tube • Add FBS in 1 minute increments: 50ul/1min;100ul/1min; 200ul/1min; 400ul/1min; 800ul/1min 4) Centrifuge for 5 minutes at 1000rpm 5) Aspirate supernatant, resuspend in 5-6ml warm media. 7) Transfer to T-25 flask, incubate at 370C/5% CO2 8) After 24 hours, check viability, remove/add 5-6 ml fresh warm media.

  20. References • Shama, B., “Manufacturing of Low Molecular Drugs”, Contocor, Raritan, NJ, 2005. • Blackwell, JV, “Mycoplasma-Recent developments in Detecting and Preventing Bioreactor Contaminants”, ISPE annual meeting, Scottsdale, AZ, Nov. 2005 • Cooper, J., ECACC, “A cell banking process for the provision of cryo-preserved, “assay ready” cells for drug discovery programs. 16 July, 2009. • http: www.newlab: Cell line characterization. • http://cerhb.ufl.edu/pdf/edcenter/cellbanking.

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