What is cell culture?
cell culture
Cell culture refers to the process of growing cells under controlled conditions outside of their natural environment. It is an in vitro tool that facilitates understanding of cell biology and disease mechanisms. It also plays a role in drug discovery, such as drug toxicity testing and pharmacokinetic/pharmacodynamic studies, as well as personalized medicine.
American embryologist Ross Granville Harrison developed the first in vitro cell culture techniques in the early 20th century, when he successfully grew tissue fragments from frog embryos in vitro. Today, cell culture has helped countless discoveries, such as the development of vaccines against polio, measles, mumps, and other infectious diseases.
Adhesion and Suspension Culture
There are two basic systems for cell growth: adherent culture and suspension culture. Adherent cultures are grown on artificial substrates, while cells in suspension cultures float freely in the medium. While only a few cell types grow naturally in suspension (e.g. lymphocytes), many adherent cell types can be adapted to suspension culture.
There are two reasons for culturing naturally adherent cells in suspension. The first advantage of suspension culture is that cells are easier to passage because you do not need to enzymatically or mechanically dissociate them from the culture vessel. Second, suspension cultures are easier to scale up because cell growth is limited only by their concentration in the medium, not by the available surface area. The main disadvantage of suspension cultures is that they require daily cell counts and viability assays to follow growth patterns, whereas adherent cultures can be easily examined under a microscope.
2D and 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, such as in a cell culture flask.
cell culture
Due to its simplicity, 2D technology cannot mimic the in vivo environment of cells, which typically grow in three-dimensional structures with complex cell-to-cell interactions. This is why some experiments are performed using 3D cultures that can be grown using scaffold-based or scaffold-free technologies.
Scaffold-based 3D methods typically involve growing adherent cells in hydrogel scaffolds. Alternatives such as bioceramic, metal or polymer scaffolds are also used for some applications.
Scaffold-free techniques are used to grow spheroids by one of three different methods:
Forced Float: Load the cell suspension into the wells of a low-adherence polymer-coated microplate. The microplate is then centrifuged to force the cells to form spheroids.
Hanging drop: The cell suspension is loaded into the wells of a hanging drop plate. As the name suggests, the suspension will hang on the plate as droplets. Cells will aggregate at the tips of these droplets and form spheres.
Agitation-based: The cell suspension is placed in a rotating bioreactor. Due to the constant agitation, the cells were unable to adhere to the walls, thus forming spheroids.
Cell Culture Challenge
Despite the differences in methods and techniques, all experiments have one thing in common: it is difficult to grow live cells in the required numbers to obtain reproducible results. Therefore, the following sections will be devoted to four challenges - repeatability, contamination, feasibility and the transition to automation.
reproducibility
According to a survey by Yongyue Medical, more than 70 percent of scientists reported that they failed to reproduce another scientist's experiments, and more than half failed to reproduce their own work. In cell culture assays, most reproducibility problems arise from biological differences between cell passages or generations. Another big problem is the misidentification of cell lines, and inconsistencies in culture parameters also play an important role.
biological variation
Factors such as random mutations or transcription errors have the potential to affect the reproducibility of experiments each time a cell divides. To avoid this, you should create a cell bank at the beginning of a new project.
cell bank
Cell banking refers to the process of storing batches of specific cell types for later use to avoid factors such as random mutations or transcription errors that affect reproducibility. The first step is to establish a master cell bank (MBC) by growing selected cell types of interest in culture and cryopreserving them in multiple containers. MBC batches are thawed and used later to prepare working cell banks (WBCs).
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. To ensure that your results are reliable and, more importantly, that you do not draw false conclusions, you should quarantine all new cell lines entering the laboratory until their origin has been verified. Most importantly, it is recommended to repeat cell line qualification prior to cryopreservation and distribution to other laboratories and after the project is completed. To verify a cell line, you should first check if it is listed in the misidentified cell line registry. If it is not registered, you still need to confirm its authenticity. For human cell lines, short tandem repeat (STR) analysis (DNA fingerprinting) is recommended. A variety of testing methods are available for non-human cell lines, including karyotyping, isoenzyme analysis, and mitochondrial DNA typing (DNA barcoding).
Culture parameters
A third factor affecting reproducibility is culture parameters.
For example, oxygen levels can significantly affect cultured cells. However, variables that affect oxygenation, such as chamber size or cell density, are not always documented and therefore cannot be kept consistent.
Another important culture parameter is the medium. This provides essential nutrients, growth factors and hormones, and regulates pH and osmotic pressure. Therefore, the most important thing is that its composition is always the same. This is especially critical for medium formulations supplemented with fetal bovine serum (FBS), whose composition depends on factors such as the cow's diet, geographic location, and time of year. To minimize the effect of FBS on the reproducibility of results, you should order different batches of serum when current stocks start to run low and test them to find the closest match. In order for others to reproduce your results, you should report how you screened the serum and record the lot number.
cell culture
Cell processing
Most researchers know that culture parameters have a huge impact on their applications, but changes in processing techniques are often overlooked.
Pollution
When cells are isolated from tissue and grown in the lab, they are no longer protected by the immune system and are therefore extremely vulnerable to contamination.
The first source of contamination is abiotic contaminants such as impurities in culture medium, serum, supplements or water, as well as endotoxins and leachables. Precautions include the use of laboratory-grade water for cell culture experiments, and media, serum, supplements, and consumables from manufacturers that offer endotoxin testing certification. Additionally, consumables must be made of virgin polystyrene or polypropylene to ensure plastic additives do not seep into your cell culture.
The second source of contamination is biological contaminants such as bacteria (including mycoplasma), fungi and yeast.
feasibility
Cell viability is defined as the proportion of viable cells in a sample. In addition to the contaminants we've just seen, there are various other factors that affect cell viability. Environmental conditions—temperature, pH, osmotic pressure, nutrient availability, and O2 and CO2 concentrations—are very important. Most of these variables are controlled by the medium and are cell type specific, which unfortunately means that we cannot provide specific guidelines.
Since cells are very sensitive to pressure, you should pay attention not only to environmental conditions, but also to your liquid handling techniques.
A final factor affecting cell viability is senescence. Most finite cell lines survive 20 to 60 divisions before dying, which means they can only be reused between 15 and 45 generations. Afterwards, the cryopreserved sample needs to be thawed from the cell bank. Continuous cell lines can proliferate indefinitely, but because they are prone to genetic drift, they should be replaced regularly.
Detection assays using colorimetric, fluorometric, and bioluminescent methods can be used to measure cell viability. A commonly used colorimetric method is the MTT method, which is based on the reduction of yellow tetrazolium salts to purple formazan crystals by living cells.
96-well plates
automation
Many of the challenges described above can be addressed by automating cell culture workflows. For example, cell handling will always be consistent, positively impacting reproducibility and viability. Most importantly, automation reduces the risk of user contamination.
Despite these advantages, automating entire workflows can be challenging due to budget reasons, lack of space for robots, or because there are not enough resources to automate and validate each step at once. But this can be solved!
