Mycoplasma contamination in cell culture

Together with cross contamination, mycoplasma contamination is the worst problem we have in cell culture! Besides that, mycoplasmas are the most malicious microbiological contaminations we know. Why ? Because we can't see them in the light microscope, they don't float or really overgrow the cultures and this is why they don't kill our cultures quickly. That might sound rather nice but actually the consequence is that we don't easily realize that they are present. Many cultures are mycoplasma contaminated already for decades without us realizing it. And to even top that, they come with us to the lab each and every day as we carry them on our skin and mucosa.

The worst thing about them, however, is that - for some strange reason - they seem to be able to convince us that only other people have mycoplasma contamination and that tests are superfluous as we would of course realize if we ever had a problem. You don't believe that ? Well, at present only around 30% of cell culture labs test for mycoplasmas!

That this is not a good idea, what effects mycoplasma contamination has on your culture and how to test is what we explain on these pages.

Zellkultur mit Mycoplasmen-Kontamination

The following topics are covered on this page:

Mycoplasma classification

Effects on cell cultures

Incidence of mycoplasma contamination

Sources of contamination

Mycoplasma test methods

Mycoplasma detection and test kits

EuPharm listed species

Species found in cell cultures

Mycoplasma classification

Mycoplasma are bacteria and belong to the class Mollicutes (soft skin) as - naturally they do not possess a cell wall. Originally, they were thought to be viruses and later were named PPLO (pleuropneumonia-like organisms). Today this term is only rarely used.

As Mycoplasma species have no cell wall they are constitutively resistant to all cell culture antibiotics that inhibit cell wall synthesis, like penicillin, streptomycin and gentamycin.

In addition, the lack of a cell wall also allows mycoplasma species to pass through 0,2µm membrane filters. The exerted pressure squeezes the mycoplasma cells through the pores even though they are usually larger than the 0,2µm.In the image you see a mycoplasma cell on the left and a normal bacterial cell with a red cell wall on the right.

Effects on cell culture

All mycoplasma species are parasites! This means they have to live on or in other cells and they steal nutrients and other metabolites from cells and cell culture medium. Additionally, they secrete toxins that harm or poison the cells. Below you find a list of effects described in literature:

Inhibition of cellular proliferation due to nutrient consumption
Reduction of medium pH due to accelerated glucose degradation
Arginine depletion and inhibition of cellular protein synthesis
Inhibition of differentiation
Activation of immune cells (lymphocytes)
Changes in membrane integrity
Changes in protein expression patterns
One species is thought to penetrate into the cytoplasm
Chromosomal aberrations
Reduction of virus production in production cell lines

Incidences of mycoplasma contamination in cell cultures

International cell banks, like DSMZ, ECACC or ATCC usually test their cultures on a regular basis. Some of them in addition to some companies publish the results of ring tests. These usually are a good indictaor for the general incidence of mycoplasma contaminations.

ost tests by cell banks currently state 15-35% of samples as being contaminated
Some studies (regional) show rates up to 80%
Usually, industrial production lines show much less contamination compared to university research cultures
There is a correlation between incidence and testing. Labs that test usually have no or little contamination.
At present only 30-50% of university labs test their cultures
Less than 20% test regularly

Sources of mycoplasma contamination in cell culture

Mycoplasma contamination can be categorized into two main categories: a) direct and b) indirect contamination. A direct contamination means that accidentally contaminated reagents are added to cells because they have not been tested (FCS, growth factors, coating proteins). Indirect contamination is mostly due to mistakes in aseptic technique. Thereby, skin germs or contamination from other cultures are introduced into a cell culture.

Lack of hygiene allows human skin and mucosa mycoplasmas to get into contact with gloves or pipettes and from there they get into our cultures by another mistake in aseptic technique
If we handle two cultures in parallel, we run the risk to transfer mycoplasmas from one to the other culture
At RT, mycoplasma species survive in medium droplets for about 6 weeks
Mycoplasmas can transfer from cryo tube to another one in liquid nitrogen

Mycoplasma detection methods

Mycoplasma detection can be achieved by several different test methods:

Microbiological culture method (only industry or GMP due to complexity)
Indicator cell method using DAPI (research and GMP)
PCR detection (research and GMP)
PCR hybridization (only GMP due to price)
ELISA (low sensitivity only research)
Luminescence (low sensitivity only research)

Among these methods are two that are most recommendable due to price, ease, specificity and sensitivity and these are the DAPI stain and PCR. A DAPI or Hoechst stain is easy and fast even though it is harder to read-out. The PCR test is the state-of-the-art technique as it combines - in the best case - ease of performance, speed, price, sensitivity and specificity. Below we have crudely outlined how - in a research lab - you can perform mycoplasma tests using one of these two methods.

PCR detection

In the PCR example, we show how to test an FCS batch that was newly purchased. First you prepare a known clean cell culture. To this, you add medium containing the new FCS batch. Now you grow and passage the cells (best without antibiotics) for at least a week to give the potential mycoplasma contamination time to grow. Longer culture periods would be safer. Then, you take a media sample from this culture. After boiling for approximately, 5min the sample is ready for testing using a PCR kit like the ones listed below. After 3-4 hours you get the result as bands on a gel.

Zellkultur Mycoplasmen-Nachweis PCR

When performing a PCR detection of mycoplasmas in a GMP production surrounding, this very crude and basic protocol has to be changed in several aspects and controls have to be included. This is why research level kits have a true sensitivity of roughly 1.000 CFU/mL while GMP final product testing uses tests validated to have a sensitivity of 10 CFU/mL for all relevant species.

Detection by DAPI stain

The second example outlines the DAPI stain. You start off as described above with adding the test sample to a clean culture and culturing for at least 7 days. Then. you discard the medium, you wash and fix the cells with formaldehyde and stain them with DAPI or Hoechst solution. Now you can read out the result using a fluorescence microscope under the UV setting. As this method is much less sensitive it is better to prolong the culture period to 14-21 days in order to be really sure.

Zellkultur Mycoplasmen-Nachweis DAPI

To use this test in a GMP production surrounding not much more than the cultivation period has to be changed. Of course controls have to be included.

Mycoplasma detection and test kits

Supplier	Method/DL/GMP	Species / Test result type	Price	Remarks: Research kits
Example video	PCR	28 (yes/no answer) = M. orale, M. arginini, M. hyorhinis, A. laidlawii, M. fermentans, M. pneumoniae, M. synoviae, (EuPharm 7 of 7-9) M. hominis, M. penetrans, M. pirum, U. urealyticum, M. pulmonis, M. falconis, M. arthritidis, M. spermatophilum, M. opalescens, M. primatum, M. maculosum, M. bovis, M. cloacale, M. hyosynoviae, M. salivarium, M. faucium, M. genitalium, M. bovigenitalium, M. sp. ovine/caprine, M. agalactica, M. timone,	<6,63	Positive control: yes Internal control: yes This is a strongly simplified and therefore, very fast and safe PCR kit. The tubes contain most reagents in lyophilized form. Only the master mix (which already includes the loading buffer), polymerase and sample have to be pipetted. This drastically reduces the number of pipetting steps. In addition, the positive control is already added to the tubes in red which helps to avoid false positive results and cross contamination. 3 pipetting steps per sample. The test kit is not available any more, but still shwos the priciple. In our MOOC, we have used this kit to demonstrate the method of PCR in a Video .
Minerva	PCR-Gel <1000-2500 CFU GMP: -	28 (yes/no answer) = M. orale, M. arginini, M. hyorhinis, A. laidlawii, M. fermentans, M. pneumoniae, M. synoviae, (EuPharm 7 of 7-9) M. hominis, M. penetrans, M. pirum, U. urealyticum, M. pulmonis, M. falconis, M. arthritidis, M. spermatophilum, M. opalescens, M. primatum, M. maculosum, M. bovis, M. cloacale, M. hyosynoviae, M. salivarium, M. faucium, M. genitalium, M. bovigenitalium, M. sp. ovine/caprine, M. agalactica, M. timone,	<10,34	Positive control: yes Internal control: yes Simplified kit as described above with lyophilized reagents in tubes. VenorGeM Advance
Sigma	PCR-Gel <1000-2500 CFU GMP: -	19 (yes/no answer) = M. arginini, A. laidlawii, M. orale, M. fermentans, M. hyorhinis, (EuPharm 5 of 7-9) M. salivarium, M. hominis, M. agalactiae, M. arthritidis, M. bovis, M. cloacale, M. falconis, M. faucium, M. hyosynoviae, M. opalescens, M. primatum, M. pulmonis, M. spermatophilum, M. timone	<9,96	Positive control: yes Internal control: yes Simplified kit as described above with lyophilized reagents in tubes. LookOut^®
Supplier	Method/DL/GMP	Species / Test result type	Price	Remarks: No internal control
R&D Systems	Hybridization- ELISA ~15 - 2500 CFU GMP: -	8 (yes/no answer) = A. laidlawii, M. orale, M. arginini,* M. hyorhinis, M. fermentans (EuPharm 5 of 7-9)* M. hominis M. pirum M. salivarium	~4	Positive control: yes Internal control: no With this kit you detect mycoplasma rRNA in a colorimetric ELISA. The assay cross reacts with Lactobacillus and therefore, is not mycoplasma-specific. In addition the sensitivity is lower. MycoProbe^®
Agilent	PCR-Gel n.a. GMP: -	Description states "most" (yes/no answer, 5 identified)	~9,8	Positive control: yes Internal control: no This kit only contains the primers. No polymerase or master mix are includ. After a PCR, 5 species can be identified in a restriction digest run on a gel. The product information does not state what is meant with "most" species. Inquire and compare to the lists below! The internal control provided is a mycoplasma DNA and therefore only serves as a PCR control. Mycoplasma Plus Primer set
Supplier	Method/DL/GMP	Species / Test result type	Price	Remarks: Research kits
Greiner BioOne	PCR- Mikroarray <10 CFU GMP: +	all known species (yes/no answer), (EuPharm 9 of 7-9) 40 (identifiziert)	>560	Positive control: yes Internal control: yes Pharma-Kit developed and used as a final product release test. It is fully validated by the supplier and contains all necessary and several additional controls. Due to the double method principle, all known mycoplasma species can be detected with a yes/no answer and additionally 40 species are identified on the species level. In addition to the PCR equipment, other machinery has to be purchased to run the test. Widely used in pharma industry. Cytoinspect^TM
Roche	PCR-Gel <10 CFU GMP: +	all known species (yes/no answer), (EuPharm ? of 7-9)	>500	Positive control: yes Internal control: yes This regular PCR Pharma-Kit uses universal PCR primers that should react with all known mycoplasma species. It gives out only yes/no answers but on the other hand only PCR equipment is used. This kit was developed by Genentech for testing Roche products and has then been launched as a test kit. The validation of this kit has some flaws but it was approved for Roche production by the EMA. Revalidation is strongly recommended. MycoTool

* Price is price per sample for a kit size ~ 25 samples.

Mycoplasma species listed in the EuPharm.:

The European Pharmacopoiea precisely lists how to test for mycoplasma contamination in sterile products and which species to use for validation and verification. Depending on what you test, the detection limit has to be 10-100 CFU per mL. Depending on the product, one of these control species is run as a positive control in all batch tests. Purified mycoplasma DNA should never be used as a GMP positive control as it does not realistically reflect the results obtained with intact mycoplasma as a positive control.

A. laidlawii
M. fermentans
M. hyorhinis (where cell-culture enrichment is used, a fastidious strain such as ATCC 29052 is included)
M. orale
M. pneumoniae or M. gallisepticum
M. synoviae (where there is use of or exposure to avian material during production)
Mycoplasma arginini
Spiroplasma citri (where there is use of or exposure to insect or plant material during production).

The most important and relevant species for culture

There are several test systems and test kits on the market. Several of these detect only a limited number of species. Make sure that the species below are in the detection range (specificity and sensitivity) of the kit you purchase. There are several kits that are cheap and have a wide range of detection.

M. orale
M. hyorhinis
M. arginini
M. fermentans
M. salivarium
M. hominis