Presentation on theme: "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."— Presentation transcript:
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 --Prevents contamination and deterioration Produced in accordance with regulatory standards (21CFR 610)
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)
Safety testing of cell banks Eliminates/minimize adventitious agents to the biopharmaceutical --bacteria --mycoplasma --fungi --viruses
Contaminants (cont.) Raw Materials *Cell culture reagents (animal and non- animal) Environment Water Air Human/Technicians
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)
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
Purity Testing Sterility: --Bulk harvest/cell banks tested for bacterial & fungal contamination. Mycoplasma: --Two methods recommended by inoculation in broth and agar.
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
Purity Testing Transmission Electron Microscopy (TEM) Reverse transcriptase assay: --Product enhanced RT (PERT) --Unique enzyme in retroviruses --PCR sensitive to cDNA enzyme
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 o oo o Bacteria Lawno
Grow Cells to 90% Confluence Images Courtesy of Corning Inc
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.
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
Cell Prep. Cont. Transfer cell suspension to centrifuge tubes. Centrifuge at 1000rpm fo 5 minutes, 2-8 o C. Siphon off all the medium. Slowly add chilled freezing medium to yield 1 x 10 7 cells/ml. Resuspend cells by gently pipetting. Place tubes on ice. Dispense cells, 1 ml/vial. Parafilm cryovials and place in -80 freezer.
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
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
Schriebers Protocol 1)Thaw vial quickly in 37 0 C water. 2)Transfer cells to sterile, 15ml centrifuge tube 3)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 37 0 C/5% CO 2 8) After 24 hours, check viability, remove/add 5-6 ml fresh warm media.
References 1.Shama, B., Manufacturing of Low Molecular Drugs, Contocor, Raritan, NJ, Blackwell, JV, Mycoplasma-Recent developments in Detecting and Preventing Bioreactor Contaminants, ISPE annual meeting, Scottsdale, AZ, Nov Cooper, J., ECACC, A cell banking process for the provision of cryo-preserved, assay ready cells for drug discovery programs. 16 July, http: Cell line characterization.www.newlab 5.http://cerhb.ufl.edu/pdf/edcenter/cellbanking.