Transcript 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
--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
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.
Contaminants (cont.)
Raw Materials
*Cell culture reagents (animal and nonanimal)
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)
Bacteria
Lawn
o
pick one

pick one

Screen for expression of foreign gene
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, 2-8oC.
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.
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
Schrieber’s Protocol
1) Thaw vial quickly in 370C 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 370C/5% CO2
8) After 24 hours, check viability, remove/add 5-6
ml fresh warm media.
References
1.
2.
3.
4.
5.
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.