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

expand TOKEN FREEZE quality control

QUARANTINE

LABORATORY

----------------------------------------------------------------------MAIN CULTURE SUIT

expand culture

MASTER BANK quality control

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ampoule

WORKING BANK( 40-100 AMPOULE) quality control

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TERMINATION OF CULTURES

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

mold

mycoplasma

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

http://www.youtube.com/watch?feature=player_embedded&v=x9bMS1G1myw

Strict aer obe

strict anaer obe faculative microaerophile

aero tol er ant anaer obe

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

http://www.youtube.com/watch?v=_fAVPMbkm78

Detection of Bacteria and Fungi in Cell Culture

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

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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 contaminationAntibiotics 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 bacteria(Gp,Gn), mycoplasma

Kanamycin 100mg/L 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)

Tetracycline 10mg/L bacteria(Gp,Gn)

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 ???

NP:Nucleoside Phosphorylase

G6PD:Glucose - 6 – Phosphate Dehydrogenase

AST:Aspartate Aminotransferase

LDH:Lactate Dehydrogenase

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

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

Southern blotting hybridization

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 Isozyme

analysis analysis

species determination individual identification

detection of cell line variation

regulatory authority recognition

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