Visikol offers a wide variety of cell-based assays, ranging from 2D to 3D and many of these assays draw on Visikol’s expertise in imaging and image analysis. Most live cell imaging assays are performed in 2D such as wound healing and calcium flux assays, however live cell imaging may be appropriate for some 3D assays as well. In particular, 3D invasion assays (angiogenesis assays) and immune cell infiltration assays can be suitable candidates for live cell imaging, but something like a cell viability assay, while able to be evaluated using live cell imaging may ultimately be more suited for a different approach. So, when is an appropriate time to use live cell imaging verses a more traditional fixed-cell approach?

Living Cell Imaging vs. Fixing Cells

The choice of live cell imaging verses fixing cells at set time points is entirely dependent on the type of study being done and the questions being asked. In general, live cell imaging is advantageous when the experiment requires multiple time points, and the desired outputs can’t be adequately determined with one final endpoint or other non-destructive endpoints. For instance, in a wound healing assay (scratch wound assay) to determine how the cells are moving, images need to be taken of the same scratch at the beginning and the end of the study at a minimum, so that differences in how “healed” the wound is can be determined. However, doing the assay this way only gives a snapshot into how the wound healing took place. To get a better idea of what is happening to the cells, and to be able to better quantify things like cellular velocity and cell proliferation, it can become necessary to increase the number of timepoints, where instead of imaging only at 0 hours and 24 hours, a researcher may instead opt to image at 0, 6, 12, 18 and 24 hours. While it is possible to have someone take a sample in and out of an imager for each time point, it becomes burdensome when time points occur outside of standard work hours or when the time points are close together, this is where a live-cell imaging system can come in handy. Live cell imaging systems incorporate incubation, which allows for temperature, CO₂, and humidity levels to be maintained and thus cell cultures can be imaged for days or even weeks.

Live Cell Imaging Considerations

While live cell imaging is useful in some instances, it can become cumbersome, as live cell imaging can take up a lot more microscope time and become cost prohibitive, both in terms of imager time and in the costs associated with the generation of large amounts of data (i.e. storage and analysis). Thus, for longer term experiments it can become necessary to weigh the pros and cons of live cell imaging. For instance, a four-week long study that wants to look at viability over time could be done with a viability dye and live cell imaging, but it would likely be more cost effective to collect supernatant samples over the four weeks and evaluate them for the release of cytotoxicity markers.

Another thing to consider is the target of interest. While there are many live cell dyes on the market that can allow for the visualization of the nucleus, organelles, cytoskeleton etc. without causing harm to the cells themselves this list is not all inclusive and some of the dyes do not hold-up well for long term imaging. An alternative way to visualize targets of interest in cells is through transfection of the cells so that the cells produce a fluorescent version of the target of interest. This can be a useful method, but is often time consuming to achieve stable cell lines for use in a study. Before moving forward with these methods, it should be considered whether fixing cells and using immunofluorescent labeling could accomplish the same outcomes.

Ultimately it is useful to take a step back and think about whether a study truly requires live cell imaging because while it can be key to understanding certain processes it can add significant costs to a study when relevant answers could be obtained with more traditional and less expensive fixed end points. If you would like to learn more about whether live cell imaging is right for you or if you are interested in any of the assays Visikol offers, please reach out.

Figure 1: Differential proliferative response in 2D vs. 3D cell culture

The drug discovery and development process is a long journey that can be quite costly with high rates of attrition. Many drug candidates will fail during clinical trials, leading to the loss of millions of dollars and multiple years of work. Visikol, with its expertise in cell culture models, plays an important role for companies in the early stages of drug discovery and even into the preclinical stage.

In vitro cell culture models and assays offer a wide range of advantages that are beneficial to drug discovery. These assays cost less than running animal studies and can also save time and money by requiring fewer animal studies before going into human clinical trials. There are two categories for these cell culture assays, 2D vs 3D models. Each model has its own advantages and disadvantages depending on what type of evaluation or analysis needs to be performed. One example would be hit screening and lead selection from thousands of compounds. A cell-based assay (2D or 3D) would be beneficial at this stage because it is critical to keep the cost as low as possible for these screenings. Comparing between the 2D and 3D models, it would be more economical to run a 2D cell culture study than a 3D because less cells and time are required for these assays. It is also important to monitor and control reproducibility and quality control within the screening and a simpler 2D model would allow for this while keeping cost lower.

When there are fewer compounds to evaluate, however, and more in-depth characterization is necessary, a 3D model would be the best choice. 3D cell culture models more closely mimic behavior that is seen in in vivo and better represents drug metabolism. Another advantage is protein and gene expression levels in culture resemble the levels that would be found in vivo. An excellent example of these benefits can be seen in the data generated from Visikol’s Cell Proliferation Assay. ER+ breast tumor derived cells (Wood cell line) were plated in both 2D and 3D. Each model was dosed with the same anti-proliferative therapeutics. Cisplatin and Paclitaxel are used as positive controls in this assay and show similar patterns both 2D and 3D models. One therapeutic tested only showed anti-proliferative effects in the 3D model and not 2D. If a choice had been made based solely on the 2D results, this compound would have been dismissed as ineffective.

Whether a simpler model is needed for high throughput drug screening or a more complex model that better mimics the in vivo structure, Visikol has these models and assays to address the Clients’ needs. Contact us to work with our team to identify the best assay and model for your specific research needs.