1 / 19

Making a Master or Working Cell Bank

Master Cell Bank (MCB) . Established from a single cloneRepresents a cell reserve

Lucy
Download Presentation

Making a Master or Working Cell Bank

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

    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 Bank Transformation or Transfection Introduce Foreign Gene that expresses Protein Product: (bacterial transformation) pick one pick one ?? ? ? Screen for expression of foreign gene

    13. Grow Cells to 90% Confluence

    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 Cells Working 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.

More Related