CCBPE Lec 5: Contamination

List some common sources of cellular contamination

(list at least 5)

• Continuous culturing of working cell banks

• Use of feeder cells

• Mislabeling of culture flasj

• Working with multiple cell lines at once

• Using one reservoir of growth medium for multiple cell lines

• Unintentional inoculation of original cell line with an invasive cell line (by transfer/drip)

• Creation of aerosol in BSC

Sources of Contamination in Media

• Lot‐to‐lot variations
• Human error and mistakes
• Expired/deteriorated reagents + failure to check
• Poor quality reagents

Fluroescent light in contamination

• Breaks down HEPES (buffer), Riboflavin, & Tryptophan

  → H2O2 and free radicals produced (toxic to cells)

Endotoxins source

Lipopolysaccharide of Gram Negative Bacteria

Contamination issues surroudning endotoxins

• toxic if found in injectable products

• negatively affect cell performance

What causes chemical contamination in incubators?

• Contaminated gases due to poor choice of cylinders, unlcean tubing, valves, and regulators

• Residual agents in cleaning and disinfection

Why is bacteria commonly encountered as a contaminant?

• Ubiquity

• Size

• Fast growth rates

Why is mycoplasma so difficult to detect?

• extremely small size

• low visibility until high density is reached

Effect of mycoplasma contamination

• Affect transfection success

• Reduced cell growth (due to increased arginine demands)

• Affect cytokine production

• Afect chromosomal aberrations

• Affet cell membrane processes

How to detect mycoplasma?

• Culture method (w/ special nutrient media)

• Electron microscopy

• PCR

• DNA Fluorescent staining

• Enzyme immunedetection

Advantages of PCR test in mycoplasma detection

• Using conserved 16s rRNA sequences

• Rapid

• Minimal sample handling → reduced risk of contamination

• HIghly sensitive (100-1000 CFU/ml)

* Detects band at 500bp (characteristic of mycoplasma)

DNA Fluorescent staining process in detecting mycoplasma DNA

• Culture cells in absence of antibiotics

• Incubate 3-5 days and stain with Hoescht 33258 or DAPI

• Mycoplasma detected by fluorescent blue granules in the cytoplasm

Describe enzyme immunedetection in mycoplasma detection

• Polyclonal antibodies against mycoplasma antigens

• Sensitivity: 10⁴ - 10⁶ PFU/ml

Detection of viruses

1) Electron microscopy

2) Immunostaining with panel of antibodies

3) ELISA
4) PCR w/ approrpiate viral primers

How to conduct decontamination to eradicate Bacterial/Yeast/Mycoplasma infection

• Wash cells w/ high conc. antibiotics and antifungal agents

• Subculture at lowest cell density possible

What is the disadvantage of using morphology of cells for characterisation?

• Cell morphology changes from 20% confluence to 100% confluence

• Plasticity of cellular morphology in response to culture conditions

Describe karyotyping in cell characterisation

• Detection of gross abnormalities in chormosomes

• Identify species, sex

Steps:

1) Metaphase arrest by colcemid/vinblastine

2) Swelled (w/ hypotonic soln)

3) Fixed (w/ methanol-acetic stain)

4) Spread

5) Stained (giesma)

6) Observe chromosomes from single cell (count at least 50 metaphase spreads in order to rule out lower rates of mosaicism)

Describe STR Profiling in Characterisation of cells

• STR is a microsatellite region of the DNA

• consists of a unit of 2-13 nucleotides repeated several times in a row on a DNA strand

• STR analysis measures the exact no. of repeating units

Describe Immunofluorescence in Characterisation of cells

Direct method:

• (Primary) Antibodies are linked to a fluorescent probe

• Used directly to localise antigen in sample of interest

Indirect method:

• Cell is probed to primary antibody

• Primary antibody is detected by Secondary antibody conjugated by fluorescent probe

Describe Cytoskeleton markers in Characterisation of cells

• Use of antibodies specific to cytokertins/ other cytoskeleton types

Describe Isoenzyme analysis in Characterisation of cells

• Looks at a range of enzymes present in almost all cell lines, but demonstrate heterogeneity between species (seen by electrophoresis)

• Separation by electrophoresis gives different distribution patterns

• Isoenzymes used:

  1) LDH

  2) G6PD

  3) NP

  4) MDH

(Isoenzyme Analysis) Mouse/Chinese Hamster

Peptidase B (PepB)

(Isoenzyme Analysis) Human Vero

Maltate Dehydrogenase (MDH)

(Isoenzyme Analysis) Chinese Hamster/Syrian Hamster

Maltate Dehydrogenase (MDH)

(Isoenzyme Analysis) Human/Chinese Hamster

Lactate Dehydrogenase (LDH)

(Isoenzyme Analysis) Human/Mouse

Lactate Dehydrogenase (LDH)