Culture Collections

 Frequently Asked Questions: Cell Lines

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Why is vapour phase LN2 recommended rather than liquid phase LN2?

If vials are immersed in liquid phase LN2 there is a risk of LN2 seeping into the vial; this could result in cross-contamination and an increased risk of the vial exploding when thawed.

 

What Quality Control (QC) tests do ECACC cell lines undergo?

The following tests are carried out:

  • cell count, viability and plating efficiency
  • sterility testing for the detection of bacteria and fungi
  • mycoplasma testing (using a minimum of two out the following three different methods: PCR, Indirect Hoechst DNA stain, culture isolation)
  • cell line authentication
    • human cell lines: cells will undergo STR profiling.
    • animal cell lines: cells were previously authenticated using isoenzyme analysis (for speciation) and DNA fingerprinting, but due to technological advances they now undergo DNA barcoding.
    • hybridoma cell lines: cells will undergo antibody isotyping.

A Certificate of Analysis, showing the results of these tests, is available for each lot number of cells.

 

Can I use cell lines obtained from colleagues?

Although it can be tempting to source cell lines from colleagues they may inadvertently supply you with contaminated or misidentified cell lines. If they can provide you with confirmation that the cells are mycoplasma free and the DNA profile has been confirmed to be as expected you can have more confidence.  If not, is it worth taking the risk? 

 

How can I make sure that I keep my cell line safe in a shared laboratory?

Effective segregation of cell lines is essential to safeguard the credibility of your work especially in shared laboratories. In order to do this we would recommend the following steps:

  • make sure the microbiological safety cabinet is clutter free and cleaned before you begin working on your cells. This can be done with 70% IPA on a daily basis.
  • only work with one cell line at a time in the cabinet. Once you have finished working on the cell line spray down the cabinet with 70% IPA, allow it to settle and then set up the cabinet for the next cell line. This reduces the chances of cross contamination.
  • keep separate and clearly labelled bottles of media and all other reagents for each cell line
  • aliquot media and reagents from stock bottles for use in the cabinet where possible. Discard any unused media and reagents; never return this to the stock bottles
  • practice good aseptic techniques
  • if possible, keep flasks segregated in the incubators
  • keep accurate and detailed records

If you suspect that your cells may have become cross contaminated, contact us to arrange for authentication testing of your cells.

 

What does seeding density mean?

Seeding density refers to the number of cells per cm2 (for adherent cell lines) or per ml (for suspension cell lines) seeded into a flask when setting up a culture.  Check the subculture routine information available on the product detail page on our website and the data sheet included with your order.

 

I do not have a CO2 fed incubator – can I grow the cells without it?

If the subculture routine specifies CO2 is required then it is necessary to provide a CO2 supply.  If you do not have a CO2 fed incubator you can manually gas the flasks using gas cylinders with the appropriate mix of air and CO2. The requirement for CO2 is linked closely to the type of culture medium used.  Some culture media, such as L15, do not require a supply of CO2 

 

The subculture routine states that CO2 is not required.  However, I only have access to a shared incubator that has a CO2 supply – can I use this anyway?

You can use the incubator to incubate at the correct temperature but you must use sealed flasks (rather than flasks with vented caps) or put the flask in a sealed plastic box and then into the incubator to exclude the CO2.

 

Why are my cells not adhering to the culture flask?

There may be several reasons why. Firstly, check the expected growth mode of the cell line i.e. is it an adherent cell line or a suspension cell line? This information is available on the product detail pages on our website and the data sheet included with your order. 

If the cell line is an adherent line there may be a problem with the culture.  The majority of adherent cell lines will adhere to the substrate within a few hours after seeding.  If the cells have not adhered 24 hours after resuscitation this would indicate that the culture may be non-viable.  However, a few adherent cell lines can take some time to adhere, particularly immediately post resuscitation e.g. the HEK 293 cell line can take up to seven days to adhere post resuscitation. 

Secondly, consider whether you are using the correct type of cell culture flask; there are several types of cell culture plastic available. Cell culture treated plastic for use with adherent lines and ultra-low attachment flasks, which enables cells to be cultured in spheroid culture.  Non-treated flasks are ideal for cell lines that grow in suspension. It is not always obvious which type of flask is which; make sure that you are using the one best suited to the cells you are culturing.

To determine if the cells are dead – do not rely solely on the visual appearance of the cells observed using an inverted microscope.  In addition, perform a viable cell count e.g. using trypan blue stain, a haemocytometer and an inverted microscope or an equivalent viable cell counting method.

 

Can I use antibiotics in the cell culture medium?

We do not generally recommend the use of antibiotics in cell culture work as they can mask a low level contamination and poor aseptic technique. The ampoule of cells which you received either directly from us or via one of our distributors will not have been cultured using antibiotics. However, we understand that many laboratories do use antibiotics although this is not considered to be best practice. Below is a table of antibiotics most frequently used during cell cultures.

Antibiotic

Working Concentration

Activity Against

Amphotericin B

2.5 ug/ml

Fungi, yeasts

Ampicillin

2.5 ug/ml

Bacteria, gram negative and gram positive

Ciprofloxacin

100 ug/ml

Mycoplasma

Neomycin

50 ug/ml

Bacteria, gram negative and gram positive

Penicillin

100 U/ml

Bacteria, gram positive

Streptomycin

100 ug/ml

Bacteria, gram negative and gram positive

Tetracycline

10 ug/ml

Bacteria, gram negative and gram positive

Table 1. Common antibiotics used in cell culture. Information from ‘Culture of Animal Cells: A manual of basic technique’. Fifth Edition by R. Ian Freshney.

A combination of Penicillin (100U/ml), Steptomycin (100ug/ml), and Amphotericin (2.5ug/ml) is effective against the most common forms of cell culture contamination; bacteria (gram positive and gram negative), yeast, and fungi; when used at the concentrations stated.

Please note: Antibiotics should not be added to freeze media as during the freezing of the cells the antibiotics will become concentrated and result in a toxic effect on the cells

 

How do I culture serum free cell lines?

Cell lines adapted to serum free medium can be more difficult to resuscitate and establish in culture in comparison to cell lines cultured in the presence of serum. For cell lines which have been adapted to serum free or animal component free media, cell viability may be poor on resuscitation and may initially decrease further. It is important to start serum free cultures at a relatively high cell density. Full recovery and establishment of a proliferating cell line may take up to two weeks.

When ordering this type of cell line from us we recommend that you read the subculture routine given in the product detail pages on our website where we recommend a specific protocol for the resuscitation and culture of these cells; this is the protocol which is routinely and successfully used here at ECACC.

 

How will my DNA order be supplied and how do I handle it upon receipt?

DNA products are supplied at either +4°C or frozen; consult the data sheet in your delivery. Frozen DNA products should be stored at -20°C upon receipt if not to be used immediately.

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What’s the best way to keep my laboratory clean?

In order to maintain a clean and safe working environment tidiness and cleanliness are key. All spills should be dealt with immediately and you should routinely clean all work surfaces both inside and outside of the microbiological safety cabinet, the floors, and all other pieces of equipment i.e. centrifuges. 

Humidified incubators are a particular area for concern due to the potential for fungal and bacterial growth in the water trays. This will create a contamination risk that can only be avoided by regular cleaning. All major pieces of equipment should be regularly maintained and serviced by qualified engineers, for example: 

  • Microbiological safety cabinets should be checked every six months to ensure that they are safe to use in terms of product and user protection. These tests confirm that the airflow is correct and that the HEPA filters are functioning properly  
  • Incubator temperature should be regularly checked with a NAMAS (National Accreditation of Measurement and Sampling, UK), or equivalent, calibrated thermometer and adjusted as necessary  
  • Incubator CO2 and O2 levels should also be regularly checked to ensure the levels are being maintained correctly.

 

What biosafety level are ECACC cell lines? Why?

ECACC cell lines are routinely handled at Biosafety Level 2 (unless a higher containment level is specified). This is in accordance with the guidelines provided by the UK Advisory Committee on Dangerous Pathogens; any cell line has the ability to harbour an as yet unidentified adventitious agent.

We provide a generic Material Safety Data Sheet (MSDS) which covers the cell lines from our catalogue. The information contained in this document will help you to carry out your own Biological Risk Assessment covering your intended use for your cells in your own laboratory. Once you have assessed the potential risks involved, then, after consultation with your own safety advisors you can decide whether you are happy to reduce the level of containment at which you handle the cells.

 

More information can be found on our technical support web page. We also and run several cell culture training courses throughout the year.

As well as the information available online customers can request a free copy of the Fundamental Techniques in Cell Culture Laboratory Handbook. This compact laboratory handbook, produced in partnership with one of our distributors Sigma Aldrich (now a part of of Merck KGaA, Darmstadt, Germany), provides a wealth of information from the sourcing of cell lines, safety and laboratory design to aspects of cryopreservation and quality control.

 

Still have a question? Contact our technical team:

Email: culturecollections.technical@phe.gov.uk

Tel: +44 (0)1980 612684

 

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