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The quality control of cell lines and the prevention,
detection, and cure of contamination
Obtaining the basic materials
Importance of cell culture collections:
1. well characterized, microbe free seed stock
2. prevention of cross-contamination
Resource Center
1.ATCC( American Type Culture Collection
Over 3200 cell lines and hybridoma from 75
species ATCC.com
2 .National Institute of general Medical Science
( NIGM)
5270 cell cultures and 275 DNA samples mainly
derived from patients with genetic and
chromosomal abnormalities
3.European collection of animal cell culture
( ECACC)
1200 cell lines and hybridoma
7000 human genetic and chromosome
abnormality cell lines
4.Riken gene Bank( Japan)
300 cell lines and hybridoma
5.Department of Human and Animal Cell
Culture ( Germany)
100 cell lines and hybridoma
Quarantine and initial handling of cell lines
cultures should be handled in class II
safety cabinet
cultures should be handled in a
Quarantine laboratory separate from main
tissue culture area
a token freeze should be made as soon a
possible
initial characterization and microbial control
should be made
NEW CELL LINES
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TOKEN FREEZE
quality control
QUARANTINE
LABORATORY
---------------------------------------------------------------------MAIN CULTURE SUIT
expand culture
MASTER BANK
quality control
expand one
ampoule
WORKING BANK( 40-100 AMPOULE)
expand one ampoule
TERMINATION OF CULTURES
quality control
Source of contamination
 Operators technique
inadequately trained personal
 Environment
poor laboratory condition
 Use and maintenance of laminar flow
 Humid incubator
 Cold stores
 Sterile materials
 Imported materials and biopsies
 Quarantine non-quality controlled cell lines
Monitoring contamination
 Check by eyes and microscope
 Clean hood and every thing if contamination is
suspected
 Record nature of contamination
 Check stock solution , sterilization procedure if similar
contamination occur
 Do not try to decontaminate unless the contamination is
irreversible
Microbial Contamination
 Sudden change of pH
decrease in bact contamination, increase in fungal
contamination
 Cloudiness in the medium
 Granular appearance between cells( ~x100)
bacterial contamination
Types of Microbial Contamination
Bacteria , Fungi Mold, mycoplasma, prptozoa
bacteria
yeast
mycoplasma
mold
Mycoplasma on
culture cell
2.testing for bacteria, yeast, and other fungi
may be detected by
 turbidity of the medium
 pH change
methods:
Thioglycollate medium
enrichment medium used in qualitative procedures for
the sterility test and for the isolation and cultivation of
aerobes, anaerobes and microaerophiles
e.g. sodium thioglycolate:
comsume O2 and allow growth of anaerobes
microbes
Strict
aerobe
strict
anaerobe
aerotolerant
faculative microaerophile anaerobe
http://www.youtube.com/watch?feature=player_embedded&v=x9
bMS1G1myw
Common sources for microbial contamination
Bacteria
Sources
clothing,skin,hair,aerosol( sneezing pipetting),insecure
caps on media and culture flask
 air current
 humidified incubator
 purified water
 insects
 contaminated cell line
 plants
Fungi( excluding yeast)
 fruit
 damp wood or other cellulose products
 humidified incubator
Yeast
 bread
 humidified incubator
 operators
Mycoplasma
 contaminated cell lines
 serum
 medium
 operator
Detection of Bacteria and Fungi in Cell Culture
http://www.youtube.com/watch?v=_fAVPMbkm78
Mycoplasma
affect the rate of cell growth
 induce morphological change
 cause chromosome aberration
 influence amino acid and nucleic
metabolism
 induce cell transformation
Fluorescence stainning of mycoplasma
 Hoechst 33258 DNA staining method
 PCR detection of mycoplasma
Two step PCR
Universal primer amplify region between
major region gene
( 16S and 23S rRNA)
amplify spacer region of
contaminated mycoplasma
Water control
Positive control + internal control
Positive control
Negative sample+ internal control
Negative sample
Positive sample + internal control
Positive sample
100 bp ladder
Other methods of detecting mycoplasma
1. Mycrobial culture
2. Molecular hybridization
3. 3H thymidine incorporation
3H labeled s.s DNA probe( homologous with mycoplasma)
4. mycoplasma detection kit ( enzyme immuno assay )
5. Myco-tech kit:
cells coculture with 6-ethylpurine deoxyribose

toxic compound forms

cell dies
Ciproflaxacin treatment
Virus contamination
a) virus sources:
 tissue derived viral contamination depend
upon:
 species of origin
 the tissue taken
 the clinical history of the animal and patient
e.g.HBV( 0.2—0.5% of blood donor were
infected)
some virus which can occur in humans
exp. . Herpes simplex virus-1
Herpes simplex virus-2
human cytomegalovirus
Epstein-barr virus
hepatitis B, Hepatitis C
Human herpes virus-6
HIV-1,HIV-2
Human T-cell lymphotropic virus-1
Human T-cell lymphotropic virus-2……………………….
b)Serum derived viral contamination
e.g. Bovine Viral Diarrhoed Virus
 infectious bovine rhinotracheitis
virus
 Parainfluenza
 may be detected by PCR or
fluorescence methods
c) methods of detection
 cocultivation
cell extract of the tested cell lines

cell extract was added into sensitive
cell line( BHK21, WI 38, HeLa, Vero, MDCK,
JM, H9, fresh T cell)
 electron microscope
 In vivo methods
 cell culture assay for murine retrovirus
example:
Murine leukemia Virus( MuLV)
Characters:
i) ectropic( infect only rodent cells)
ii)xenotropic( infect only cells other than
rodent)
iii)amphotropic
 reverse transcriptase assay for retrovirus
detection
precipitate virus particles with PEG

R.T activity assay
 PCR method for detection of BVDV and
other virus
5.Bovine Spongiform encephalopathy
1986, found in contaminated feed stuffs of
cattles
6.Elimination of contamination
a. discard contaminated culture
b.find the source of contamination
c.eliminate source of contamination
 fumigate the whole lab
 swab cell surfaces and equipment
 use of antibiotics
using 10-14 days for at least 3 passages
Antibiotics commonly used in elimination of microbial
contamination
Antibiotics
working concentration
active against
AmphotrricinB
2.5mg/L
yeast and other fungi
Ampicillin
2.5mg/L
bacteria(Gp,Gn)
Cephalothin
100mg/L
bacteria(Gp,Gn)
Ciprofloxacin
10-40mg/L
mycoplasma
Gentamycin
50mg/L
Kanamycin
100mg/L
bacteria(Gp,Gn), mycoplasma
bacteria(Gp,Gn), yeast
mycoplasma removal agent 0.5mg/L
mycoplasma
Neomycin
50mg/L
bacteria(Gp,Gn)
Nystatin
50mg/L
yeast and other fungi
Penicillin-G
100000U/L
bacteria(Gp)
PolymyxinB
50mg/L
bacteria(Gn)
Streptomycin sulphate
100mg/L
bacteria(Gp,Gn)
10mg/L
bacteria(Gp,Gn)
Tetracycline
Authentication
To confirm the identity and origin species of
the cell stocks
1. Isozyme analysis
polymorphic enzyme variants
 catalyze the same reaction but have
different electrophoretic mobility
HeLa cell ???
G6PD:Glucose - 6 – Phosphate Dehydrogenase
LDH:Lactate Dehydrogenase
NP:Nucleoside Phosphorylase
AST:Aspartate Aminotransferase
2.Cytogenic analysis
To establish the common chromosome
complement or karyotype of a species or
cell lines
Giemsa banding( G banding, pH 6.8
giemsa), G11 banding( pH 11 giemsa
stain) , Quinacrine ( Q) banding
3.DNA fingerprinting
Southern blotting hybridization
Southern blotting hybridization
DNA extraction form sample
DNA cut into fragments by
restriction enzyme, separation
by electrophoresis transfer
DNA transfer to nylon membrane
Application of with radiolabelled probe
Develpoment of x-ray film
http://local.testing4dna.com/Local-Cell-Line-Authentication.html
Comparison of the uses of the different cell authentification methods
available
Application
DNA finger printing
cytogenic
analysis
analysis

species determination
individual identification

detection of cell line variation

regulatory authority recognition
Isozyme


Information required for regulatory approval
1. History and genealogy of the cell line
2. Records and storage information on master and
working cell banks
3.Culture requirements
4. Growth characteristics
5. Production and testing facilities
6. Quality control test:
karyology,isozyme analysis,DNA fingerprinting,virus
testing, retrovirus status, test for contaminated DNA,
purification procedure/validation date
characterization of products