An introduction to cell culture techniques and challenges
Written by Marlene Frank14. December 2021
"… and the cells lived happily ever after." Read on to make this fairy tale ending come true in your lab! We’ll provide you with tips on reproducibility, contamination, viability and automation to help you overcome the major cell culture challenges.
Table of contents
What is cell culture?
Cell culture refers to the process of growing cells under controlled conditions outside their natural environment. It is an in vitro tool that aids the understanding of cell biology and the mechanisms of diseases. It also plays a role in drug discovery for applications such as drug toxicity testing and pharmacokinetic/pharmacodynamic studies, as well as in personalized medicine.
The American embryologist Ross Granville Harrison developed the first in vitro cell culture technique at the very beginning of the twentieth century, when he successfully grew tissue fragments from frog embryos outside the body.1 Today, cell culture has already helped countless discoveries, such as the development of vaccines against poliomyelitis, measles, mumps and other infectious diseases.
Cell culture techniques
Adherent vs. suspension cultures
There are two basic systems for growing cells: adherent and suspension cultures. Adherent cultures are grown on an artificial substrate, whereas cells grown in suspension are free-floating in the culture medium. While only a few cell types naturally grow in suspension (e.g. lymphocytes), many adherent cell types can be adapted to suspension cultures.
There are two reasons for culturing naturally adherent cells in suspension. The first advantage of suspension cultures is that it's easier to passage the cells, as you don't need to detach them from a culture vessel by enzymatic or mechanical dissociation. Secondly, suspension cultures are easier to scale up, as the cell growth is only limited by their concentration in the medium, not by the available surface area. The major downside of suspension cultures is that they require daily cell counts and viability determination to follow growth patterns, whereas adherent cultures can easily be inspected under a microscope.2
2D vs. 3D methods
Adherent cultures can be further divided into 2D and 3D cultures. In 2D applications, adherent cells are grown in a monolayer system on a flat surface, e.g. in a T-flask.
Due to their simplicity, 2D techniques can’t mimic the cells’ in vivo environment, where they usually grow in three dimensional structures with complex cell-to-cell interactions. This is why some experiments are conducted using 3D cultures, which can be grown using scaffold-based or scaffold-free techniques.
Scaffold-based 3D methods usually involve growing adherent cells in hydrogel scaffolds. Alternatives such as bioceramic, metallic or polymer scaffolds are also used for some applications.3
Scaffold-free techniques are used to grow spheroids by one of three different methods:
- Forced-floating: a cell suspension is loaded into the wells of a low adhesion polymer-coated microplate. The microplate is then centrifuged to force the cells to form spheroids.
- Hanging drop: a cell suspension is loaded into the wells of a hanging drop plate. The suspension will, as the name suggests, hang from the plate in droplets. The cells will aggregate in the tips of these drops and form spheroids.
- Agitation based: a cell suspension is placed in a rotating bioreactor. The cells can’t adhere to the walls due to the continuous stirring, so spheroids are formed.3
Do you want to learn more about the different 3D methods? Check out our application notes:
- Semi-automated medium exchange in Akura™ 96 and 384 Spheroid Microplates with the VIAFLO 96 and VIAFLO 384
- Cell seeding on BIOMIMESYS® Hepatocyte hydrogels with the VIAFLO 96/384 channel handheld electronic pipette
- High throughput, 3D assay development on spheroids in 1536 well plates
- 3D cell culture processing with the VIAFLO 96 and VIAFLO 384 handheld electronic pipette and the Corning® Elplasia® plate
Cell culture challenges
Despite the different approaches and techniques, all experiments have one thing in common: it’s difficult to grow viable cells in the desired quantity to obtain reproducible results. The following sections are therefore dedicated to the four major challenges – reproducibility, contamination, viability and the transition to automation.
According to a survey in Nature, more than 70 % of scientists reported that they failed to reproduce another scientist's experiment, and over half failed to reproduce their own work.4 In cell culture assays, a large proportion of the reproducibility issues come from biological variation between passages or generations of cells. Another huge problem is the misidentification of cell lines, while inconsistencies in culturing parameters play an important role too.
Every time a cell divides, there’s a risk that factors such as random mutations or transcription errors will affect the reproducibility of the experiment. To avoid this, you should create a cell bank at the very beginning of a new project.
Cell banking refers to the process of storing multiple batches of a specific cell type for later use, to avoid factors such as random mutations or transcription errors that compromise reproducibility. The first step is establishing a Master Cell Bank (MBC) by growing a selection of the cell type of interest in a culture, and cryopreserving it in multiple containers. The MBC batches are thawed and used to prepare Working Cell Banks (WBC) later on.
Misidentification of cell lines
The problem of cell line misidentification has been known since the 1960s, when a scientist described HeLa cell contamination of 19 other human cell lines.5 To ensure that your results are reliable and, even more importantly, that you don't draw the wrong conclusions, you should put all new cell lines that enter your lab into quarantine until their origin is authenticated. On top of this, it’s recommended that cell line authentication is repeated before cryopreservation and distribution to other labs, and after the completion of a project. To authenticate a cell line, you should first check if it’s listed in the register of misidentified cell lines. If it's not registered, you still need to confirm its authenticity. For human cell lines, performing short tandem repeat (STR) profiling (DNA fingerprinting) is recommended. Various test methods are available for non-human cell lines, including karyotyping, isoenzyme analysis and mitochondrial DNA typing (DNA barcoding).6
The third factor influencing reproducibility is the culturing parameters.
Oxygen levels, for example, significantly impact the cultured cells. However, the variables impacting oxygenation, e.g. culture chamber specifications or cell density, aren't always documented, and therefore can't be kept consistent.7
Another important culturing parameter is the culture medium. This provides the necessary nutrients, growth factors and hormones, and regulates the pH and osmotic pressure. It is therefore of utmost importance that its composition is always the same. This is especially demanding for culture media formulations supplemented with fetal bovine serum (FBS), the composition of which depends on factors such as the cow's diet, geographical location and time of year. To minimize the impact of FBS on the reproducibility of your results, you should order different batches of serum when your current stock starts to run low and test them to find the closest match. In order to allow others to reproduce your results, you should report how you screened the serum, and record the lot numbers.8
Handling of cells
Most researchers are aware that culturing parameters have a huge impact on their applications, but variations in handling techniques are often overlooked. Read our detailed article on cell culture handling to learn how to further improve reproducibility.
When cells are isolated from tissues to be cultured in the lab, they are no longer protected by the immune system, and are therefore highly vulnerable to contamination.
The first source of contamination is non-living contaminants, such as impurities in media, sera, supplements or water, as well as endotoxins and leachables. Preventive measures include using laboratory-grade water for cell culture experiments, and media, sera, supplements and consumables from manufacturers that provide endotoxin testing certification.2,9 Moreover, it's important that consumables are made from virgin polystyrene or polypropylene, to ensure that plastic additives don't leach into your cell culture.
The second source of contamination is biological contaminants, such as bacteria (including mycoplasma), fungi and yeasts. Learn how to detect and prevent these by reading our extensive article on cell culture contamination.
Cell viability is defined as the proportion of live cells in a sample. Besides the contaminants we've just looked at, there are various other factors that impact cell viability. The environmental conditions – temperature, pH, osmotic pressure, nutrient supply, and O2 and CO2 concentration – are very important. Most of these variables are controlled by the culture medium, and are cell type specific, which unfortunately means that we can't provide specific guidelines.2
As cells are very sensitive to stress, you not only have to keep an eye on the environmental conditions, but also on your liquid handling techniques. More information on the influence of stress, and how to reduce it, can be found in our viability article.
The last factor influencing cell viability is senescence. Most finite cell lines can survive between 20 and 60 divisions before dying, which means that they can only be used reproducibly between 15 and 45 generations.6 After that, it will be necessary to thaw a cryopreserved sample from the cell bank. Continuous cell lines can proliferate indefinitely but, as they are prone to genetic drift, they should be replaced periodically.
Detection assays using colorimetric, fluorescence and bioluminescence approaches are available to measure cell viability. A commonly used colorimetric method is the MTT assay, based on the reduction of yellow tetrazolium salt to purple formazan crystals by viable cells. Check out our application note on how to automate MTT assays to learn more.
Many of the above-mentioned challenges could be solved by automating cell culture workflows. Cell handling, for example, would always be consistent, having a positive impact on the reproducibility and viability. On top of that, automation reduces the risk of contamination from the user.
Despite these advantages, it can be challenging to automate the entire workflow, for budget reasons, because there's no space for robots, or because there aren't enough resources to automate and validate each step at once. But this can be solved! Read our article on how the transition to automation can be eased to learn more.
We hope that we have been able to answer all your questions about cell cultures, and that you now feel more confident in performing reproducible experiments with happy cells. If you have some more time, you should also check out the article How to use a biosafety cabinet. It provides useful tips on aseptic techniques, helping you to keep your cells free from contamination.