I began the interview by asking Paul about how the use of real-time live-cell analysis is permitting cell analysis in ways that weren’t previously possible and what he sees as the biggest benefits of real-time live-cell analysis. Paul explained that live cell experimentation is typically a dynamic process with cell changes occurring over the entire course of the experiment. Traditional analysis methods only collect data at the end of an experiment. Real-time live-cell analysis using a dedicated device adds a kinetic dimension to the information collected. Furthermore, development of non-perturbing labeling reagents and label-free imaging techniques now permit researchers to acquire information over extended time periods. The ability to collect this kinetic data allows for a complete understanding of the experimental system.
He went on to say that real-time live-cell analysis provides a temporal context on its own, but can also be combined with end point analysis and contemporaneous in-situ measurements to provide a greater understanding about the experimental system. Paul described an example of an immune cell killing workflow protocol developed by Sartorius scientists that utilized IncuCyte for the imaging coupled with temporal cytokine measurements while the imaging was occurring. Then end point confirmation of T cell activation using the IQ flow cytometer was conducted. By using this combination of techniques, morphological and cytotoxic changes in target cells were imaged while measuring temporal cytokine production followed by specific identification of subsets of activated T cells by flow cytometry.
Next we discussed keeping up with the continually evolving uses of real-time live-cell analysis and the recent publication of Sartorius’ 3rd edition of the Live Cell Analysis Handbook. Paul shared some of the highlights of the new edition, including optimization and characterization of neuroimmune cell models especially human iPSC derived models and how this continues to be a focus for cellular neuroscientists. He shared that in the chapter on kinetic assays there is a suite of assays that enable the study of structure and function of neurons and neuroimmune cells including new data. The IncuCyte can measure as neurons become active and form connected networks via repeated in-situ measurement of calcium flux. Using a genetically encoded calcium indicator and purpose built image analysis tools, scientists can measure culture in 96 well plates for days, weeks, even months, while cells stay stationary in the incubator. This will greatly improve the productivity of scientists who are developing differentiation protocols for iPSC derived neuronal models.
I enjoyed the section on analysis approach and the steps involved in image processing to ultimately lead us to analysis and actionable data, so I asked Paul to provide an overview. He said that the image processing workflow has the necessary steps for processing and analyzing images. When collecting so many images it is not uncommon to have thousands of images per experiment. Image processing must be systematic and repeatable. Paul explained how digital snapshots are collected, and then image processing is used to clean up the data. Last, image analysis is used to extract usable information for analysis. Large numbers of images requires operations and automation be applied to many images in a single experiment. Sartorius has developed specific assays and automation for IncuCyte’s image processing workflow while still maintaining microplate throughput.
Another section that I enjoyed was the new section on neuronal cell models. I asked Paul to provide some information about this section as well. He said that this section of the handbook covers fully characterizing differences between patient specific diseased cells and normal cells. For this application, scientists need to continuously analyze the same sample population of cells to detect both significant and subtle cell changes. In this application, real-time live-cell analysis provides a powerful tool for discovery of novel neurotherapeutics.
Next, I asked Paul to describe the traditional tools being used to measure areas such as neuronal cell health, morphology and function and the limitations with these methods. He said that many of the traditional technologies can’t measure long term changes in biological behavior. End point analysis provides no insight into dynamic changes and interactions of a population of cells in the nervous system. In addition, the sample prep for this analysis can damage sensitive neuronal cells and compromise the study.
I asked how live-cell imaging addresses these limitations and Paul said that real-time live-cell analysis was designed for live cells. Instead of fixing and killing cells for end point measurement, real-time live-cell analysis uses non-invasive technology and reagents to monitor cells over time. This permits the evaluation of the long-term changes of a cell population over time and provides the considerable advantage of characterizing the function of the nervous system, something end point analysis technologies can’t do.
We then discussed more specifically how the IncuCyte live-cell analysis methodology enables the study of neuronal cell dynamics. Paul said that live-cell analysis has evolved to detect fast kinetic activity such as neuronal activity by capturing calcium oscillation using unique move mode acquisition. Thus enabling a look at how cell activity changes over time. Scientists need to capture this information longitudinally without perturbing the biology. It can take months for these cells to become mature and activity. Complex neuronal activity measurements over time inform researchers on when to assess treatment paradigms in these cell cultures.
I followed up by asking what this means for current research opportunities. He said that at Sartorius they will continue to focus on development of stem cell research and use of patient specific cells. The ability to monitor these cells and how they change and interact allows researchers to breakdown the complex nature of the human brain. Sartorius’ goal is to continue to develop new tools and applications to enable new discoveries.
I closed by asking if there was anything he would like to add for listeners. Paul said that there are now over 2,000 peer-reviewed publications using the IncuCyte. Sartorius looks forward to expanding the real-time live-cell analysis portfolio with new techniques, like the ones featured in this 3rd edition of the handbook.
To learn more, please see Live-Cell Analysis Handbook
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