Exploring Cell Mechanics – From Ripe Avocados to Sepsis Diagnosis
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Much can be discovered about a cell’s condition and function by measuring its mechanical properties such as deformability. Technology Networks recently spoke with Daniel Klaue, CEO, ZELLMECHANIK DRESDEN, to learn more about the field of cell mechanics. In this interview, Daniel explains why it is important to measure cell mechanics, discusses a novel method of doing so – real-time deformability cytometry – and highlights how this technology could benefit clinical applications.
Anna MacDonald (AM): What is cell mechanics and why is it so important to measure? What can cell mechanics reveal about a cell?
Daniel Klaue (DK): Imagine ten bananas laying in front of you. They have different degrees of ripeness and you wish to pick a ripe one. Easy: you just look at them and if there are green, yellow and brown fruits you will most likely go with yellow. Now the next level: ten avocados of different ripeness… It is difficult to see a difference. So, most likely you will simply take the fruit and gently press it to feel the resistance of the pulp. With your experience you will intuitively know how much force to apply with your fingers to categorize the avocado unripe (hard), ripe (certain softness), over-ripe (too soft). The mechanical properties of the fruit are determined by complex tissue structures. Sensing these mechanical properties allows us to draw conclusions about the state of the fruit.
Now let’s go to the cellular level and raise the question: can the mechanical properties of a single cell tell me anything about the cells state? Yes! The equivalent of the tissue structure in the fruit is the cytoskeleton of the cell (and to a lesser degree, other structural components like the cytoplasm or membranes). The functional link between this highly dynamic filament structure and mechanical properties of the cell has been known for decades. Cell migration or division for example leads to obvious structural changes in the cytoskeleton. Likewise, malignant modifications of the cell are often accompanied by cytoskeletal remodelling (most cancer cells are softer than healthy ones).
Of course, there are countless established molecular markers to study cells, but you either have to work with labels or you have to interpret the signals the cells are giving. In contrast, the mechanical properties are intrinsic (no label necessary) and you directly assess the cell producing or interpreting signals.
Therefore, cell mechanics constitutes a key scientific target for investigating topics ranging from development to disease.
AM: How can cell mechanics be measured? Can you explain how real-time deformability cytometry works and the advantages this method offers?
DK: There are various methods to measure cell mechanics. It is important to distinguish the approaches, because they address different physical properties. Depending on the duration, scale or location of applied forces one measures more elastic or more viscous components, cytoskeleton or nucleus mechanics, global or local properties. Some methods are measuring the forces created by cells, others analyse the reaction of a cell to applied forces and still others interpret particle motion in the cytoplasm.
Real-time deformability cytometry (RT-DC) measures global, elastic properties of the cytoskeleton. Technically, it is a hybrid of flow cytometry and high-speed video-microscopy. Cells are suspended in a buffer and pumped through a channel with a slightly larger cross section than the cells’ diameter. Like in a river, the fluid moves faster in the centre than at the sides. This difference creates forces acting on the cell and results in deformation. A “stiffer” cell will deform less than a “softer” cell when applying the same kind of force. Deformation is measured by analysing the cell’s contour in an image of it in the channel. When assuming that single cells in suspension without applied forces are spherical, this contour is circular. The deformation is now defined as a deviation from a circular shape of the cell contour.
The “real-time” in RT-DC comes from the instant analysis of the cell’s contour when the image is taken. Immediate calculation of cell area, height, width, aspect-ratio, et cetera allows for on-the-fly observation and gating of the data. The algorithm also calculates parameters such as the brightness and brightness-deviation of the cell, giving insights to morphological properties. The icing on the cake is a saved image of every detected event, making it easy to see if the outlier is debris or the rare cell you were looking for.
The biggest advantage of RT-DC is the 10,000-fold increase of the measurement rate compared to other methods addressing cell mechanics (e.g. micropipette aspiration 100 cells/hour, RT-DC 1,000 cells/second). This facilitates the application of a label-free, non-destructive biomarker as a standard in cell biology and clinical research with statistically meaningful numbers of single cell measurements in a few minutes.
AM: Can you tell us about the origin and development of this method?
DK: The method was invented in the laboratories of at the Technische Universität Dresden. Prof. Guck worked with AFM and optically stretching to reveal correlations between cell mechanics and function (or dysfunction) of cells. One major obstacle of those methods was the low throughput (max. several 100 cells/h). The process of measurement was: find a cell, stop to apply forces, release the cell and analyse the measured parameters after the whole experiment. To overcome the most time-consuming steps they developed a method using a continuous flow with continuous forces and on-the-fly analysis: RT-DC.
Dr. Oliver Otto and Dr. Philipp Rosendahl (both now part of ZELLMECHANIK DRESDEN) implemented the idea around 2013. Since then, numerous high impact scientific papers have been published, our start-up has been founded, technical extensions have been developed and the technology is starting to spread.
It is now possible to simultaneously measure physical properties and fluorescence signals of cells (RT-FDC), thus allowing to compare and correlate physical properties of cells to the gold standard in cell biology (fluorescence flow cytometry). It recently became possible to dynamically track cell deformation along the channel and study the time-dependent dynamics of deformation (dRT-DC). Sorting by mechanical properties is already running in the lab of Prof. Guck and will be commercially available soon. This all shows that the method is well established and we will keep on pushing the technology to further limits and distribute it world-wide.
AM: What applications can this method particularly benefit?
DK: Our mission is to utilize RT-DC in a medical setting. Potential scenarios comprise diagnosis and therapy-monitoring for various disease indications, ranging from inflammatory diseases like pneumonia to leukemic cancers.
ZELLMECHANIK DRESDEN focusses on sepsis. Sepsis is one of the most common causes of death in the industrialized world after heart disease and cancer although the awareness is not as strong yet. And sepsis kills fast: the mortality risk in sepsis increases by 8% per hour. There is a lot of research and development going on to quickly identify the infection (cause, location, bacteria, virus, fungus, …) to swiftly initiate the matching therapy. However, with the existing methods this still takes up to several days which in many cases is fatal. Meanwhile the patient is treated with broad-spectrum antibiotics (that don´t help in the case of a viral infection). There is a great need for rapid diagnosis and permanent treatment monitoring of the disease.
Therefore, it is indispensable to find a biomarker that a) significantly shortens the time for diagnosis of the sepsis-causing agent (diagnostic marker) and b) that shortens the time from diagnosis to effective verification of the applied therapeutic agent (monitoring marker). We believe that biophysical properties of cells of the immune system can constitute this biomarker. RT-DC allows us to characterise thousands of individual cells from just a drop of blood. Since the different types of blood cells show different properties (size, deformation, brightness, …), it is easy to identify the different populations (without labelling). We call the result a mechanical hemogram.
Specific states of the immune system are reflected in specific patterns of the mechanical hemogram. Thus, also changes in the immune system will reflect in changes of the mechanical hemogram (first confirming experimental results are already published). It is known that e.g. lymphocytes react primarily to viral infection, neutrophil granulocytes react to bacterial infection and that the complications of a sepsis are caused by a strong (over)-reaction of neutrophils. We believe that these cellular reactions happen earlier, than those on a macro level, such as body temperature, heart rate, and physical comfort. Using RT-DC we can quickly access this information for live monitoring of therapeutic success. This will also help to reduce the exhaustive use of antibiotics.
If any of our customers finds another medical application of RT-DC (in cancer, quality control of blood transplantation, infectious diseases, …) we are happy to individually adapt our technology. Our precise mission is to help health care providers to improve their diagnostic and prognostic decisions for sustainable therapies.
AM: Your vision is to turn the technology into a medical device present in all hospitals within 10 years. What challenges do you anticipate along the path to achieving this? What impact could the technology make to the future of diagnostics and healthcare?
DK: It will be difficult to develop a medical device in line with all regulations, for different markets, at an economical price, reimbursable by health insurance. But this is solvable, by working together with experienced partners. Really challenging will be to prove the clinical applicability and increased value in sepsis therapy management. We have to perform clinical trials that convince all the key players such as medical professionals, decision makers for purchases in the hospital and health insurances. And then there is the strong competition in the medical device market. Maybe it will be even more challenging to stand against those established players in the field.
Having a test that reveals a compelling statement on the improved or declined immune status under therapy could greatly impact the outcome of sepsis. It can reduce the tremendous costs of intensive care units for treatment and accommodation of sepsis patients. In addition, improved therapy management of infectious diseases also touches on the subject of unnecessary use of, and development of resistance against, antibiotics.
RT-DC in a medical device will most likely not completely replace other diagnostic technologies. It will improve the overall process. As the differential blood count is standard today, we are going to make it standard to perform a mechanical hemogram… about 10 years from now.
Daniel Klaue was speaking to Anna MacDonald, Science Writer for Technology Networks.