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Mycoplasma are too small to be seen in a light microscope. Unlike other bacteria, they don´t visibly change the culture medium. Therefore it is critical to routinely test cultures. When choosing a test method the following aspects should be considered:  

  • Throughput (How many samples do you need to test at once?).
  • Desired turnaround time (How long can you wait for the results? A longer time increases the risk of the contamination spreading).
  • Access to the required lab equipment.

Get an overview of the advantages and disadvantages of mycoplasma detection methods with this PDF file.

You can find more information on cell culture contamination at www.eppendorf.com/cellexperts.

Mycoplasma DNA stain - Cell Handling Eppendorf

Cell culture work often implies the cultivation of various cell lines for different ongoing experiments. The simultaneous nourishment of different cell lines in a biosafety cabinet might save some time during a busy day in the laboratory but dramatically increases the risk of cross-contamination and can thus seriously impair the entire experiment. Therefore, some aspects should be considered when working with different cell lines:

  • Work with only one cell line at a time in the biosafety cabinet.
  • Use labeled aliquots of media and reagents for each individual cell line.
  • Disinfect the interior of the biosafety cabinet after a work session with one cell line.
  • Run the laminar flow for a few minutes between work sessions with different cell lines.

Check www.eppendorf.com/cellexperts to get some more tips for how to handle cells during daily cell culture practice.

Process cross contamination - Cell Handling Eppendorf

It is common practice to share cell lines with external laboratories and collaborators. But these cell lines might come with an incalculable contamination or misidentification risk. Therefore, it is highly recommended to verify quality and origin to obtain further reliable data with these cell lines. Consider the following procedure when adding a foreign cell line to your cell culture:

  • Quarantine the new cell line (separate incubators, biosafety cabinets or even cell culture laboratories depending on the individual laboratory equipment).
  • Quality tests for any microbial contaminants like bacteria, fungi, yeast, and mycoplasma.
  • Compare with the list of known misidentified cell lines using the ICLAC database.
  • Identity tests for cross-contamination or misidentification to verify the authenticity of the cell line.

Using your own tests to verify cell lines can save you valuable time, efforts and costs.

More information about how to check for quality and identity of cell lines can be found at www.eppendorf.com/cellexperts.

Man in front of incbuator quarantine - Cell Handling Eppendorf

Have you ever wondered how mycoplasma get into your cell culture? A frequent source of this invisible plague are previously contaminated cultures that are used in the same laboratory. When a new cell line from an outside source arrives, it should be quarantined until the absence of mycoplasma and any other type of contaminant is proven.
Mycoplasma can be transferred from infected reagents via aerosols which are created when pipetting warm liquids. Using filter tips and working with one cell line at a time in the biosafety cabinet reduces the risk of spreading the contamination throughout the cell culture laboratory.

A major source of mycoplasma are the people working with the cells. One of the most predominant mycoplasma species isolated from cell cultures is Mycoplasma orale, which is commonly found colonizing the human oral cavity and oropharynx. By sneezing, coughing and talking, mycoplasma can find their way into our cell cultures. Therefore, avoid speaking when sitting at the cleanbench or standing in front of the open incubator and strictly follow recomendations for aseptic techniques.

As mycoplasma are very small and flexible due to their lack of a cell wall, they are able to penetrate membrane filters used for sterilization of cell culture media, sera, and other reagents, especially  when filtering under pressure conditions. Furthermore, mycoplasma are able to survive in a dried state for several days and can easily be transferred in the shape of aerosols and particles. In case of an acute mycoplasma contamination in the laboratory, you should consider all surfaces, personal protective equipment and lab equipment as potentially contaminated and take strict disinfection measures.

More information about cell culture contamination can be found at www.eppendorf.com/cellexperts.

Mycoplasma - Cell Handling Eppendorf
David M Phillips/ Science Source/ Getty Images

Cell lines purchased from reputable commercial or non-profit suppliers such as cell banks are carefully tested for their identity and absence of contaminants. This guarantees a comfortable safety when starting the cultivation of these cells. In contrast, incoming cell lines obtained from external laboratories and collaborators have to be analyzed independently with regards to their quality and identity before accepting them for general use.

But regardless of the source, consider the following aspects when cultivating a new cell line:

  • Establishment of a two-tiered cryopreservation system (Master Cell Bank and Working Cell Bank including cryopreservation at the lowest passage).
  • Detailed documentation (unique cell line name, origin, characteristics, quality and identity control testing, list of cryopreserved stocks).
  • Quality and identity tests at regular intervals (microbial contaminations, cross-contamination or misidentification).

Visit www.eppendorf.com/cellexperts to get more detailed information on how to proceed with new cell lines in cell culture.

When working at the laminar flow hood there are a few different ways of handling caps and lids of sterile vessels. When holding caps in hand while pipetting make sure that you don´t touch the inside of the cap. Especially for bigger cell culture flasks this requires practice and routine. When putting the cap aside place it on the rim if possible. This ensures that nothing falls into the cap due to careless movements and prevents the risk of contamination from particles on the basement plate. Place plate and dish lids either inside-down or inside-up at a position with minimal movement to minimize the risk of contamination.

Putting a lid at a higher position such as on a pipette tips box, however, provides no additional protection against contamination. Only open sterile vessels immediatly prior to use and reclose them immediatly after use. There is a certain air flow within the hood that will keep the work area as germ-free as possible.

Do not fill the interior with too many items to ensure uniform air flow. Furthermore, avoid unnecessarily quick movements and air turbulence, e.g. caused by open flames (Bunsen burner). Disturbances to the air bring particles and germs into unnecessary movements.

Learn how to fight cell culture contamination on www.eppendorf.com/cellexperts.

When working with small volumes evaporation of culture medium during incubation and disturbance of the atmosphere in the incubator can become a critical factor. Especially in 96-well plates with a working volume of 100-200 µL evaporation can lead to accumulation of media components, thereby affecting cell metabolism.
Filling these wells with medium without cells creates a more stable micro-environment in the plate and therefore minimizes the edge effect. In addition, opening the incubator while incubating the plates should be reduced as much as possible to not disturb the atmosphere inside.

Find out more tips how to avoid the edge effect in our Application Note and visit www.eppendorf.com/cellexperts to learn more about increasing the reproducibility of your cell-based experiments.

DMSO (Dimethyl Sulfoxide) is a polar, aprotic organic solvent that is commonly used as a cryoprotectant because of its membrane penetrating and water displacement properties. It is added to cell culture media to reduce ice formation and thereby prevent cell death during the freezing process.

Empiric studies report that during an initial freezing phase a temperature reduction of about 1°C/min is optimal. In vitro, DMSO at concentrations higher than 10% (v/v) is reported to induce membrane pore formation and apoptosis through caspase-9 and -3 activation. Toxic effects have also been reported at 1% (v/v) or higher. The response will depend on the type of cell or cell line.

Time is thus a critical factor when you freeze and thaw cells, because you want to minimize their exposure to DMSO. A trypan blue test can help to detect the cells' viability after thawing.

Hematopoietic cells, embryonic stem cells and many hybridoma cell lines seem to be particularly vulnerable to DMSO, as it is also a differentiation inductor.
Learn more about increasing the quality of your cell culture www.eppendorf.com/cellexperts.

Serum is a common supplement in media used for cultivating animal cells. Fetal bovine serum (FBS), also known as fetal calf serum (FCS), is the most widely used. It contains vital nutrients, hormones and growth factors which stimulate cell growth. Other serum components serve as binding proteins which promote cell adhesion in vitro.

Although the use of serum is well established in cell culture, it remains the most undefined component of culture media. Serum batches usually show qualitative variations, and the lack of uniformity in composition introduces high lot-to-lot variability. In order to avoid this variability affecting cell-based experiments, it is recommended to test different serum batches for their ability to support growth of a certain cell type and to stock up on a suitable batch.

It is also possible to request that serum suppliers reserve appropriate volumes of a suitable batch. When the batch is used up, testing should be repeated to identify the next suitable batch.
Since serum is an animal-derived reagent, the risk of microbial contamination arises. This risk can be minimized by obtaining serum from suppliers that follow strict quality testing protocols. 

A variety of chemically defined, serum-free media and synthetic serum substitutes is available from different suppliers. These represent an alternative to the use of serum and can enhance the long term reproducibility of experiments.

In a cell culture lab, the biological safety cabinet is meant to be a sterile (contamination-free) working area where cell culture vessels and tubes with sterile reagents can be opened without the risk of being contaminated.

The reality in many labs looks different: the hood is cluttered with pipettes, tip boxes, tube racks, waste containers and all the stuff people think they might need at some point during the workflow.

Having every item needed for the experiment at hand, but not overcrowding your work space, is the key. Every piece of equipment and every movement causes turbulences within the laminar air flow that is supposed to keep contaminants outside the hood.

So don’t overcrowd your work space and avoid hectic movements with your arms. Never block the air vents and ensure that constant air flow is maintained. And keep in mind that using a Bunsen burner in the hood does not give any extra protection. On the contrary, the open flame disrupts the laminar airflow and therefore eliminates the protection and safety of the working space.

Daily monitoring of cell cultures using phase contrast microscopy enables you to recognize any unusual changes at an early stage. Each day that you examine your cells for confluency, take a closer look at your cells according to the following checklist:

  1. What does the medium look like?
    Increased turbidity, as well as a rapid color change of medium containing phenol red as a pH indicator, strongly suggests a contamination. Watch out for bacteria, fungal colonies floating on the medium surface as well as ovoid bright particles between the cells, indicating a yeast contamination.

  2. Do cells proliferate?
    Cells undergoing mitosis assume a round shape and partly detach from the surface. The occurrence of these cells in your cell culture is a sign of a healthy culture.

  3. Do you see cells swimming in the medium?
    An increased number of cells swimming in the medium can be a sign of a cell culture that has become overly confluent and which actually needs to be passaged. Cells start to die off and detach from the surface. Depending on the extent of die-off, a healthy culture may not be recoverable.

  4. Vacuoles or granules?
    Cytoplasmic vacuolation, as well as granules around the nucleus, indicate an unhealthy culture due to cellular damage, oxidative stress etc. Change of medium might be sufficient to remedy the problem, whereas in other cases cultures will have to be discarded due to serious causes (e.g. microbial contamination, senescence of the cell line, or inadequate medium or serum).

Check your cell culture by microscope on a daily basis! It just takes a few minutes and contributes to the maintenance of consistent and optimized culture conditions, resulting in high reproducibility of your experimental data.

In biological systems, high reproducibility of results is much harder to achieve than in physical or chemical experiments. Several factors contribute to the variation between individual results:

  • The quality and age of cells
  • The sterility of the cultures
  • The consistency and quality of equipment and media used
  • The individual person performing the experiment
  • The duration of the experiment

In addition to these factors, the incubator plays a crucial role as it provides the ideally uniform culture conditions needed for optimal and standardized cell growth. The homeostasis inside the incubator provides and influences three factors of the cell environment:

  • A precise temperature control
  • A CO2 atmosphere that regulates the pH in bicarbonate-buffered culture media
  • A humid atmosphere to limit evaporation from the culture vessels 

This homeostasis, however, is disturbed every time the inner incubator door is opened as the air inside the incubator flows out. Afterwards, all three factors need to recover to the set specifications which might take several minutes. In order to counteract the unavoidable effects of opening the doors, you should consider the following:

  • Avoid opening any door for longer than 30s
  • Shorten the number of times the door is opened as much as possible
  • Limit the angle the door is opened
  • Use an incubator with divided doors which greatly reduces the disturbance and saves gas consumption
  • Consider a second incubator when the number of times the door is opens exceeds the acceptable amount
  • Consider a small additional incubator for sensitive long-term experiments

Sterility of cultures is a continuous challenge in cell culture. Germs may be transferred involuntarily by many different means:

  • Contaminated cell stocks
  • Cross contamination of cell stocks in liquid nitrogen
  • Errors in aseptic technique
  • Lack of strict general hygiene
  • Malfunction of equipment
  • Non-sterile plastic ware, media and supplements

In addition to these factors, the incubator plays an important role as it may increase or decrease the risk of culture contamination. Even though incubators do not provide a “sterile” environment, one can reduce the risk of contamination when following the below rules:

  • Remove contaminated cultures from the incubator immediately 
  • Keep door opening times as short as possible 
  • Use incubator with split inner doors
  • Immediately clean any spills inside the incubator
  • Exchange the water frequently with autoclaved, distilled water 
  • Clean the incubator thoroughly once a month using an appropriate disinfectant 
  • Unscrew ventilators and other parts (if your incubator is equipped with) that often hide fungi for cleaning
  • When fungal contamination occurs check the incubator thoroughly 
  • If possible, regularly decontaminate/sterilize using the respective function after cleaning
  • Use HEPA filters in the incubator gas inflow if necessary
Answer by Dr. Nadine Mellies, Application Specialist Cell Handling at Eppendorf:

Pluripotent stem cells (PSCs) are per se prone to differentiate spontaneously in culture. Controlling the balance between their expansion and differentiation can be challenging, but reliable maintenance in the undifferentiated state is critical for stem cell propagation. When maintained properly, spontaneous differentiation should be <5%. PSCs can be distinguished easily from differentiated cells, which show less-defined edges, loose morphology, dark areas, and/or exhibit fibroblast- or endothelial-like morphology. Those differentiated areas must be removed from the culture. Using fully chemically defined media with appropriate hormones and growth factors can reduce the risk of serum-induced spontaneous differentiation of PSCs. Spontaneous differentiation can be further minimized with appropriate split ratios to balance the culture confluency, while cell overgrowth will trigger spontaneous differentiation, and result in loss of pluripotency and differentiation potential. Regular monitoring of PSC morphology and careful handling are therefore mandatory to maintain high-quality PSC cultures.
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