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Cell Culture Contamination Control

Cell Culture Contamination Control: What Are Mycoplasma?

The Stealthy Invader





What Are Mycoplasma and Why Are They so Feared?


Mar 26, 2024 | Informative Articles


 

 Featured in this article:

• What is mycoplasma contamination?
• What happens when a cell culture is contaminated by mycoplasma?


In the world of microbiology, there exist tiny organisms causing big trouble – mycoplasma.
Mycoplasma are the smallest prokaryotic organisms and the most common contaminants in cell cultures. They can easily pass through sterile filtering systems and are resistant to commonly used antibiotics. Mycoplasma contamination is a serious problem for academic and biopharmaceutical laboratories due to its impact on cell properties and safety of biological products. A contamination often remains undetected and pose significant challenges to cell culture experiments, potentially comprising data integrity and research outcomes. It is estimated that mycoplasma are present in almost every lab and that about 5 to 30 % of the world’s cell lines are contaminated with mycoplasma, causing enormous costs and effort [1].


In this article we give a brief overview of mycoplasma contamination. Learn about the biology of mycoplasma and the consequences of a mycoplasma contamination to understand how cells are contaminated.

What is Mycoplasma Contamination?  The Biology of Mycoplasma

 
Mycoplasma Structure

Mycoplasma are the smallest self-replicating bacteria (about 0.15 - 0.3 µm) and belong to the class of mollicute [1]. In fact, mycoplasma and mollicutes are commonly used as synonyms. Unlike other bacteria they lack a cell wall, makes them capable of assuming various shapes and resistant to commonly used antibiotics. Due to their flexible membrane and small size mycoplasma are able to pass through filtering systems.

They cannot be detected visually - neither by the naked eye nor by microscope. Even highly contaminated cell cultures do not show any turbidity or other obvious symptoms.

What makes mycoplasma so special?

• Absence of cell wall
• Flexible membrane
• Invisible
• Pass through filters
• Resistant to common antibiotics

 

 

Mycoplasma lack a cell wall, making them resistant to commonly used antibiotics. The flexible membrane allowing them to shrink to diameters < 0.45 µm and pass through filtering systems.

 

What happens when a cell culture is contaminated by mycoplasma?  Consequences of a mycoplasma contamination

Mycoplasma species are known to infect a variety of eukaryotic cells, including human, animal and plant cells.

When a cell culture becomes infected with mycoplasma, it can lead to various detrimental effects on the cells and their growth. Mycoplasma, being simple parasitic organisms, rely on the host cells for their metabolic needs. Initially, they attach to the host cell and then can merge with its membrane. Following this, mycoplasma replicate rapidly, overgrowing significantly the host cells.

Unlike other bacterial contaminants, mycoplasma contamination doesn't typically cause visible changes in the growth medium. However, its impact on cell cultures can be profound and can lead to:

• Altered cell metabolism
• Inhibition of cell growth and proliferation
• Reduced transfection efficiency
• Changes in gene and protein expression
• Chromosomal abnormalities
• Impair nucleic acid synthesis
• Apoptosis

 

These effects can vary depending on the cell line and the species of mycoplasma involved. Nevertheless, mycoplasma contamination poses a serious challenge to cell culture research, as it can compromise the reliability, reproducibility and validity of experimental results.

The presence of mycoplasma in cell culture has different side effects, including:

• Loss of time
• Loss of money
• Loss of valuable cells
• Potential dissemination of inaccurate findings through publications
• Misleading publications
• Biosafety concerns

If a contamination remains undetected, contaminated cultures can endanger scientific data, delay research projects and jeopardise the safety of biopharmaceutical products. Therefore, it's crucial for researchers to be vigilant in detecting and preventing mycoplasma contamination in cell cultures.

The impact of mycoplasma contamination in cell culture can be profound and represent a serious problem for academic and biopharmaceutical laboratories due to its impact on cell properties and safety of biological products.

 

References

[1] Nikfarjam L, Farzaneh P. Prevention and detection of Mycoplasma contamination in cell culture. Cell J. 2012 Winter;13(4):203-12. Epub 2011 Dec 22. PMID: 23508237; PMCID: PMC3584481.

Venor® Mycoplasma qPCR

Venor® Mycoplasma qPCR offers a validated, real-time PCR-based solution for the rapid and reliable detection of mycoplasma in cell cultures, biopharmaceutical products and ATMPs.

Minerva Biolabs

Catalogue No.DescriptionPack SizePriceQty
  • Change to:
  • £
111-9025Venor® Mycoplasma qPCR25 rxns €834.90 Quantity Add to Order
111-9100Venor® Mycoplasma qPCR100 rxns POA Quantity Add to Order

Description

Mycoplasmas are among the most significant contamination risks in cell culture and biopharmaceutical manufacturing (e.g., ATMPs). EP 2.6.7 requires a validated testing method for the release of such products.

The Venor® Mycoplasma qPCR Kit is specifically designed for use in QC and GMP laboratories. Compared to classical culture methods, it reduces detection time from several weeks to ≤ 3 hours and enables reliable release testing of biopharmaceuticals and ATMPs. The PCR-based method offers the advantage of detecting both culturable and non-culturable mycoplasmas, making it superior to traditional culture tests.

Detection is based on amplification of a highly conserved 16S rRNA coding region in the mycoplasma genome, enabling reliable identification of all mycoplasma, acholeplasma, and spiroplasma species listed in EP 2.6.7, as well as over 130 additional mollicutes. By combining reverse transcriptase and DNA polymerase, both RNA (as cDNA) and DNA are amplified, significantly increasing sensitivity. The assay has been validated according to current regulatory requirements EP 2.6.7 (Edition 12.2) and in compliance with ICH Q2(R2), meeting criteria for specificity, robustness, and sensitivity with detection limits of ≤ 10 CFU/ml and < 100 GC/ml. Positive control as well as internal and process controls further ensure the reliability of results and provide maximum security in routine testing.

The external positive control with defined concentration, as required by the new EP 2.6.7, can be obtained directly from us. Our 100GC® Mycoplasma Standards serve both as reference material for validation and as an external positive control in routine testing.

Specifications

Application Mycoplasma detection
Application areas
  • QC release of biopharmaceuticals and ATMPs
  • QC of cell cultures
Contents
  • Mycoplasma Mix (lyophilized)
  • Positive Control (lyophilized)
  • Internal Control (lyophilized)
  • PCR grade Water
Compliance EP 2.6.7
Validation report available
PCR type RT-qPCR
Compatible devices qPCR cyclers
Detection limit (LOD) ≤ 10 CFU/ml
< 100 GC/ml
Detection spectrum ≥ 130 mollicutes
Control system
  • Positive control
  • Internal control
Number of reactions
  • 111-9025: 25 reactions
  • 111-9100: 100 reactions
Price per reaction
  • 111-9025: €31.96
  • 111-9100: €28.90
Required equipment
and consumables
  • qPCR cycler (FAM™ and HEX™ alternatively VIC™ or JOE™)
  • PCR tubes
  • centrifuge for 1.5 ml PCR tubes
  • pipettes
Storage 2-8 °C; ≤ –18 °C after opening or rehydration
Shipping Ambient temperature
Shelf life (max.) 18 months

Regulatory Disclaimer

For use in quality control/manufacturing process only.

If you cannot find the answer to your problem then please contact us or telephone +44 (0)1954 210 200

Applications & Benefits

  • EP 2.6.7 (12.2) compliant
  • Fast results in ≤ 3 h with minimal hands-on time ≤ 30 min
  • Easy handling and clear result interpretation
  • Highest sensitivity through detection of both RNA and DNA

If you cannot find the answer to your problem then please contact us or telephone +44 (0)1954 210 200