Cultivating Consistency: Tips and Tricks for Successful Cell Cultures

Cell cultures are a central part of life science research programs, and culture techniques are continuously evolving. Properly optimizing workflows and utilizing the latest best practices are the keys to obtaining consistent results and advancing research.

Cell Authentication

Estimates suggest that as many as half of all cell lines are misidentified. Proper and regular authentication is the key to avoiding this pitfall.

Planning Future Cell-based Experiments:

• Choose the cell line carefully, ensuring that it accurately reflects the biology of interest, can withstand the planned experimental manipulations, and is able to generate reproducible results.
• Obtain your cells from a trusted source with proper documentation of the cells’ identify and quality controls.
• Don’t share or borrow cell lines informally. Insist on authentication and formal documentation.
  

Developing a New Cell Line:

• Follow the ethical and legal requirements, including patient consent, pertaining to the acquisition of tissue for cell lines.
• Keep detailed records about the line’s origin, including: clinical information about the donor or patient, if applicable; cell type, source, lot numbers of all reagents used; images of the culture; and methods of genetic modification or reprogramming.
• Preserve tissue samples for subsequent confirmation of origin. In the case of cells derived from a disease, also preserve a tissue sample for histopathology as well as a sample of normal tissue for comparison.

Cells Currently in Use:

• Check the International Cell Line Authentication Committee (ICLAC) registry of misidentified cell lines.
• Validate cells using DNA genotyping, not phenotyping, at least once per year, ideally every six months. Comparison of short tandem repeats (STRs) or single nucleotide polymorphisms (SNPs) with available databases are the two most cost effective and acceptable genetic methods for intra-species comparison.

Culture Preparation

A successful culture requires proper sterile technique and the use of other best practices.

Best Cell Culture Practices:

• Proper training is essential. Generate standard operating procedures (SOPs) for all culture techniques and make sure everyone in the lab knows how to properly implement them.
• Purchase high-quality reagents and supplies with documented quality control from reputable sources. Record lot numbers for later troubleshooting.
• Perform regular sterilization, calibration, and servicing of all equipment, including pipettes, culture hoods, incubators, microscopes, and autoclaves.
• Quarantine new cell lines until after authentication to safeguard against contamination.

Sterile Filtration:

• The membrane choice depends on sample characteristics, composition, and volume. Both the solute and the dissolved molecules or particles of the sample being filtered must be chemically compatible with the filter membrane.
• Sample agitation and adjusting the flow rate per unit area of filter surface can improve throughput and help prevent particulates from aggregating on the membrane surface.
• Clarification and prefiltration is best accomplished with a 0.45 μm pore size.
• For fast filtration of most media and buffers use a polyethersulfone (PES) membrane with a 0.45 μm pore size.
• For sterilizing media containing serum and other protein use a PES membrane with a 0.22 μm pore size.
• For removal of mycoplasma use a PES membrane with a 0.10 μm pore size.
• For very low protein binding and sterilizing solutions with high value biomolecules use a polyvinylidene difluoride (PVDF) membrane with a 0.2 μm pore size.

Cell Freezing:

• Label stock vials completely and unambiguously.
  
• Freeze stock samples at the lowest passage number possible.
• Utilize the proper freezing technique for
  each cell line. Most require slow cooling,
  though some (e.g. stem cells) prefer ultrarapid
  freezing.
  
• Liquid vs. vapor phase nitrogen storage — while liquid phase storage allows for lower temperatures and a longer response time to storage issues, it has a higher risk of contamination and cracked/bursting vials. Vapor phase nitrogen is a great alternative to store cells and provide lower risk for contamination.
  
• To safeguard against loss, store important stocks in more than one location and thaw one vial to assess cell viability soon after cryopreservation.

Cell Growth

Keeping cells healthy by mimicking their natural environment is no easy task. Vigilance is key.

Under the Hood:

• Never work with more than one cell line at a time and don’t share bottles of media between cell lines.
• Prepare and filter media on the same day it will be used.
• Pay special attention to serum quality. When possible, buy sera in bulk and test each batch to ensure it supports cell growth.
• To combat genotypic and phenotypic instability, characterize cells regularly and replace from frozen stock frequently.

Detecting Microbial Contamination:

• Screen for infections (especially the most common, mycoplasma, which doesn’t produce visible signs of contamination) at regular intervals.
• Don’t routinely use antibiotics to control contamination, as they can mask signs of infection. In antibiotic-free media, signs of bacterial, yeast, and fungal infection include cloudiness as well as color and pH changes.

Cell Analysis

Monitoring and quantifying cell health and function is imperative to yield biologically meaningful data.

Cell Counting:

• Count your cells often to examine your cell culture.
  
• The Coulter principle is the most precise method for counting cells, where cells flow, one by one, through an aperture within an electrical sensor.
  
• Data generated by Coulter principle-based cell counters not only deliver precise cell counts but also display average cell size and population-distribution information.
  
• Another benefit of using a Coulter principle-based cell counter is that you’ll also obtain a histogram of the size distribution of your cell population, which can tell you how monodisperse your sample is.
  
• Make sure the cells you are counting are well mixed, or you’ll end up with local variations in cell density that could introduce error into your cell counts.
  
• Use a cell counter that doesn’t involve loading a chamber, and you won’t need to worry about air bubbles.
  
• Transferring cell samples can introduce errors. Using a handheld, automated cell counter without leaving the culture hood is the best way to avoid variability and obtain an accurate snapshot of your culture.
  
• Check your instrument specifications when preparing a sample and make sure your cell diameter and starting dilution are compatible with your counting method.

Quality is Paramount When it Comes to Cell Culture Supplies.

We have over 20,000 proven tools to help you cultivate consistency:

• Thousands of authenticated cell lines and primary cells
  
• High-flow rate sterile filtration systems
  
• Application-tested cell freezing solutions
  
• High-quality liquid media, serum, and supplements/growth factors
  
• A diverse line of cultureware and specialty culture inserts and plates

For all your cell culture needs, visit /US/enSigmaAldrich.com/cellculture

Recommended Reading

1. Geraghty RJ et al. Guidelines for the use of cell lines in biomedical research. Br J Cancer. 111:1021-1046 (2014).
2. Yu M et al. A resource for cell line authentication, annotation, and quality control. Nature. 520:307-311 (2015).

SNAP i.d. and Muse are are registered trademarks of Merck KGaA, Darmstadt, Germany.

References

1. Geraghty RJ, Capes-Davis A, Davis JM, Downward J, Freshney RI, Knezevic I, Lovell-Badge R, Masters JRW, Meredith J, Stacey GN, et al. 2014. Guidelines for the use of cell lines in biomedical research. Br J Cancer. 111(6):1021-1046. https://doi.org/10.1038/bjc.2014.166

2. Yu M, Selvaraj SK, Liang-Chu MMY, Aghajani S, Busse M, Yuan J, Lee G, Peale F, Klijn C, Bourgon R, et al. 2015. A resource for cell line authentication, annotation and quality control. Nature. 520(7547):307-311. https://doi.org/10.1038/nature14397