Presentation on theme: "Making a Master or Working Cell Bank"— Presentation transcript:
1Making a Master or Working Cell Bank Bioman 2009July 27 to July 30Rochester, NYPresenter: Dana M. HopkinsWm. Davies, Jr. Career & Technical HS
2Master Cell Bank (MCB) Established from a single clone Represents a cell reserve “Frozen in Time”--Preserves characteristics--Prevents contamination and deteriorationProduced in accordance with regulatory standards (21CFR 610)
3Cell 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)
4Safety testing of cell banks Eliminates/minimize adventitious agents to the biopharmaceutical--bacteria--mycoplasma--fungi--viruses
5Source of Contaminants Cell SubstrateEndogenous virusesExogenous microbial contaminantsSource material screening-Human (HIV, HBV, HCV, CJD, etc)-Animal (TSE sources, species-specific viruses.
6Contaminants (cont.) Raw Materials *Cell culture reagents (animal and non- animal)EnvironmentWaterAirHuman/Technicians
7Regulatory DocumentsCBER/FDA: Points to consider in cell line characterizationCBER/FDA: Points to consider in manufacturing and testingEuropean Pharmacopeia (EP)US Pharmacopeia (USP)Japanese Pharmacopeia (JP)
8Validated In-house Guidelines Species Identity: Confirmed by iso-enzyme analysis and cross-species contaminationSpecies Banding Pattern: Confirmed by agarose gel electrophoresisDNA Fingerprinting: Karyology
9Purity Testing Sterility: --Bulk harvest/cell banks tested for bacterial & fungal contamination.Mycoplasma:--Two methods recommended by inoculation in broth and agar.
11Purity Testing Transmission Electron Microscopy (TEM) Reverse transcriptase assay:--Product enhanced RT (PERT)--Unique enzyme in retroviruses--PCR sensitive to cDNA enzyme
12Building 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 geneo ooo ooBacteriaLawn
13Grow Cells to 90% Confluence Images Courtesy of Corning Inc
14Cell 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, MCBCount cells with a hemocytometer. Use trypan blue for viability.
15Freezing Media 60 ml DMEM, 40 ml FCS, 1 ml Penicillin-Streptomycin Filter through 0.2u filterAliquot 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% DMSO0.1ml versene (prevents clumping of cells)
16Cell 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.
17Mammalian 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 dailyAdd 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
18Thawing 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
19Schrieber’s Protocol Thaw vial quickly in 370C water. Transfer cells to sterile, 15ml centrifuge tubeAdd FBS in 1 minute increments:50ul/1min;100ul/1min; 200ul/1min; 400ul/1min; 800ul/1min4) Centrifuge for 5 minutes at 1000rpm5) Aspirate supernatant, resuspend in 5-6ml warm media.7) Transfer to T-25 flask, incubate at 370C/5% CO28) After 24 hours, check viability, remove/add 5-6 ml fresh warm media.
20ReferencesShama, 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. 2005Cooper, J., ECACC, “A cell banking process for the provision of cryo-preserved, “assay ready” cells for drug discovery programs. 16 July, 2009.http: Cell line characterization.