Cell Culture 101: Uses, Techniques, & Best Practices

Cell Culture Uses & Applications & Environments

Researchers can use cell cultures for a wide variety of purposes. Cell cultures may include plant, animal, or even human-tissue cells. In the past, cell cultures have been used to carry out experiments in various fields like viral diagnostics, genetic engineering, vaccine production, and cancer research.

Cell cultures consist of cells that have been harvested from living tissue. In addition to the cells themselves, cell culture environments may also contain essential elements to the cells, such as amino acids, vitamins, and carbohydrates. From there, scientists may monitor the behavior and growth of the harvested cells (called “primary cells1”) in certain conditions (with or without added elements like drugs).Primary cells can also be further manipulated to become immortalized cell lines. 

Cell cultures can be subdivided into different groups based on how they are cultivated. First, 3D cell  cultures allow cells to grow in a three dimensional volume that can more closely mimics an in-vivo environment.  On the other hand, 2D cell cultures are attached to a flat surface are, therefore, limited in their ability to grow in contact with other cells in 3D dimensions resembling the in vivo environment., 2D cell cultures are still widely used.

In addition, in adherent cell cultures, cell growth is limited to the surface of an area. Suspension cell cultures, meanwhile, allow the cells to float freely in an environment. What’s more, some cell culture devices are purposefully designed to prevent any cell adhesion occuring along surfaces at all.

Cell Culture Best Practices

One of the biggest problems associated with the process of cell culturing is the danger of contamination. In many instances, labs that suffer cell culture contamination may lose some or even all of their progress made on a given experiment or line of study. So it’s important for professionals and equipment manufacturers to ensure that contaminations occur as seldom as possible. Unfortunately, some contaminants are extremely small and difficult to detect –– like mycoplasma species2.

CELLSTAR® Cell Culture Vessels with Cell-Repellent Surface

For most applications in cell culture, the properties of the vessels to be used are optimized to enhance conditions for cell attachment. With platforms made of polystyrene, this is achieved by using a physical or chemical methods to treat the surface of the vessels. This treatment leads to the incorporation of polar carboxyl and hydroxyl groups to the hydrophobic polystyrene surface resulting in improved and very regular cell attachment. For fastidious cell lines or applications that will stress the cells, pprotein-coatedsurfaces or advanced non-biological surfaces are applied.

Nevertheless, for some applications, a surface that prevents cell attachment is required. These applications include:

• Spheroid culture
• Aggregation of stem cells
• Suspension culture of semi-adherent and adherent cell lines

The CELLSTAR® cell-repellent surface from Greiner Bio-One fulfills these demands. These surfaces are also commonly known as ultra low attachment surfaces (ULA). Achieved through an innovative chemical surface modification, Greiner BioOne’s cell-repellent surface is an ideal substrate for cell culture as it does not degrade or ‘leach out’ under standard cell culture conditions.

All cell culture vessels with a cell-repellent surface are sterilised by irradiation (SAL of 10-3). They are quality controlled for the absence of detectable endotoxins, DNase/RNase and human DNA and show no cytotoxic effects. Evaluation of cytotoxicity is done in accordance with EN ISO 10993-5 with mammalian cell lines. More detailed information concerning general quality aspects can be found on the Greiner Bio-One website and in our product catalog.

To control the performance of the surface of cell-repellent cell culture vessels, attachment of cells is compared to the standard tissue culture CELLSTAR® surface. CaCo-2 cells are seeded at a density of 18,000 cells/cm2 and incubated for 24 hours at 37 °C in an incubator with a 5 % CO2 environment.

After 24 hours of incubation, the medium is removed, the vessels are washed with PBS and EtOH is added to fix adherent cells, if present. The sample vessels are then examined and compared under a microscope with 10-fold magnification (Fig. 1).

Inhibition of cell attachment of semi-adherent and adherent cell lines in vessels with cell-repellent surface

For the culture of suspension cells, surfaces of a strong hydrophobic nature are generally used. With semi-adherent cell lines like macrophages or even adherent cell lines like Vero or CaCo-2 cells, this kind of surface does not reliably prevent cell attachment.

However, if cultivated in vessels with a cell-repellent surface, these cell lines exhibit an almost total inhibition of attachment (Fig. 2). Tested cell lines are listed in Table 1.
 

Culture of spheroids and stem cell aggregates

In pharmaceutical and basic research, two-dimensional (2D) cultures are still predominant. Nevertheless, 2D cultures can only mimic to a limited extent the conditions in physiological tissue where cells are able to interact in a three-dimensional network. Therefore, results generated from 2D cultures have often limited relevance for clinical effectiveness3. The employment of spheroid cultures plays an important role as an alternative approach to better mimic physiological conditions, especially in cancer research. Spheroids are self-assembled spherical cell clusters with different zones (Fig. 3). These zones are characterized by metabolic and proliferative gradients resembling the physical conditions in avascular tumours or micrometastases4,5. As a consequence, experimental data obtained with spheroids are often more significant than data from 2D cell culture experiments. Pluripotent stem cells with their ability to differentiate into cell types of all germ layers (endoderm, mesoderm and ectoderm)6 hold great promise for drug development, therapeutic applications, and basic research. A critical step for the cultivation of stem cells is the formation of non-adherent cell aggregates, called embryoid bodies7. Pluripotent cell types which are able to form embryoid bodies comprise both embryonic stem cells and induced pluripotent stem cells.

For the formation of spheroids and embryoid bodies cell interaction has to be dominant over the interaction of the cells with the surface of the culture vessel used. Standard cultivation approaches comprise the hanging drop method, round bottom, spinner flask, or rotary cell culture for spheroid cultivation3 and static suspension culture for embryoid bodies. However, these approaches have only limited compatibility with automation and high throughput screening. On the other hand, ‘classic’ cell culture vessels like microplates fulfil all the necessary demands for automated handling and imaging. If equipped with a surface effectively preventing cell-surface interactions like the Greiner Bio-One cell-repellent surface, these vessels represent a perfect platform for cultivating spheroids (Fig. 4) and stem cell aggregates (Fig. 5, 6). Tested cell lines for spheroid and aggregate formation are listed in Table 1.

In Conclusion

At Greiner Bio-One we have developed a line of cell culture products designed to reduce the possibility of contamination and protect the viability of cell culture samples. For instance, our 96 well cell culture microplates come in a chimney-like arrangement, which allows each well to stand on its own and thus lower the risk of contamination.

Across the board, Greiner Bio-One offers scientists high-quality cell culture equipment like plates, flasks, and tubes. Our products are sterile, designed for convenience and ease of use, and comply with American National Standards Institute (ANSI) standards for automation. What’s more, we also offer cell culture equipment suitable for both adhesion and suspension cultures –– as well as equipment with cell-repellent surfaces. And our cell culture plates boast lids specifically designed for optimal gas exchange.