Push Past Your Limits: Extended Live Cell Imaging

A broad range of biomedical research and pharmacological applications rely on the identification, study, and behavior of cellular conditions and processes. Observation and experimentation on living cells is vital to understanding, diagnosing, and successfully treating human diseases. Life science research, drug discovery, and biopharmaceutical production are all demanding new solutions to meet their pressing requirement to work with and study live cells for periods of time extending from days to weeks. Effective in vitro studies are playing an increasingly important role in many areas of disease research, drug discovery, and therapeutic applications.

Although the requirement for improved in vitro technology has existed for many years, biomedical researchers and pharmaceutical companies have tended to take a conservative role in the adoption of these new technologies. The complexity of today’s diseases and the skyrocketing cost of drug discovery is driving change and leading many research scientists and pharmaceutical company executives to embrace a new technology paradigm.

Cell Viability

Cell viability assays have long been a workhorse tool for cell biologists. The proliferative state of a cell population has also become an important parameter in drug discovery research, particularly in evaluating cancer therapeutics and in assessing the health of a cell population as part of absorption, distribution, metabolism, and excretion studies. In drug discovery, cell viability often is one component of a group of related assays that assess not only proliferative activity but also cell viability, metabolic activity, cell cycle phase, cell toxicity, and apoptosis. Together, the information derived from these assays can indicate whether a cell population that has been exposed to an experimental stimulus is healthy or dying, actively dividing or in stasis, or has committed to an apoptotic pathway.

In vitro cell culture model systems and screening assays for cell death have become important tools for determining the chronology of events resulting in apoptosis. In vitro model systems have also been proven useful in screening for inhibitory or stimulatory compounds that affect specific enzyme systems or the general process of cell death and apoptosis.

Understanding of the basic mechanisms that underlie apoptosis will point to potentially new targets of therapeutic treatment of diseases that show an imbalance between cell proliferation and cell loss. In order to conduct such research, techniques and tools to reliably identify and enumerate death by apoptosis are essential.

Market-Driven Demands

What are the factors driving new technologies and more rapid adoption of these solutions at leading government research centers, biotechnology firms, and pharmaceutical companies?

Miniaturization.

Pharmaceutical and biotechnology companies are seeking ways to obtain more accurate results while reducing the amount of expensive reagents consumed per experiment. To meet this need, a significant number of vendors have developed low volume well plates, slide-based containers, and lab-on-a-chip (LOC) products. Low volume well plates are able to reduce reagent usage but have minimal impact on the accuracy or quality of the data collected during an experiment. LOCs are micro-fabricated devices designed for various types of chemical and cellular analyses and can generally be classified either as microfluidic (based on microchannel networks) or microarray (based on well plate-type formats or microchips). In key areas of drug discovery, such as chemical syntheses, screening of compounds and preclinical testing of drugs in living cells, microfluidic tools can make a useful contribution, and indeed represent an improvement on existing technologies. Novel reaction, manipulation and analytical steps can be performed with microfluidic systems that are not accessible to other approaches providing new information about biological systems.

Accuracy.

Scientists are looking for easier to use and more reliable instrumentation to improve the accuracy of the data being captured. Until now, there has been no effective and easy-to-use technology that allowed laboratory researchers to observe live cells and to acquire longitudinal data from those same living cells over an extended period of time. A large number of pharmaceutical and biotechnology companies are beginning to use more cell-based assays to obtain more accurate data. Information closer to the real life model can be obtained using these assays when compared to information obtained from other conventional assay types. Watching cells and biological molecules such as DNA change, move, develop, and interact with other cells and biological molecules may clarify many of the unanswered questions about how living organisms work. Most of the instrumentation available today limits the observation time of live cells and hinders the ability to solve some of today’s complex research questions.

Efficiency.

Scientists are also looking for technologies that will not only improve the throughput of screening, but also reduce the time and labor involved in these tests. Secondary screen technologies oftentimes handicap progress because the researcher must spend large amounts of time trying to reproduce environments outside of the body. This approach fosters tremendous levels of inefficiency and in most cases the observation time of live cells in these simulated environments is still quite limited.

Flexibility.

Because of ever-increasing costs, laboratory managers and scientists are seeking equipment that can perform a variety of functions with a smaller footprint than room sized equipment. However, as part of that miniaturization, they still want the ability to utilize their existing investment in capital equipment whenever possible.

Performance.

Scientists are looking for technologies that are fully integrated, which involves putting microseparations and other control and communications components together in a package that is biocompatible, compact, and reliable. No longer is it sufficient to shop for best-in-breed components and perform the complex systems integration in the scientist’s own laboratory. And, once the system is installed, it is critical that multiple experiments can be run simultaneously with the fewest number of environmental variables.

Re-usability.

Laboratory managers and pharmaceutical executives alike are seeking ways to lower their costs. Many of today’s laboratory supplies have been designed to be disposable adding significantly to the bio-hazard waste and requiring large amounts of storage space. Some vendors are providing supplies made out of glass that can be autoclaved and reused several times, saving on overall cost of materials, which leads to a lower total cost of ownership of the new instrumentation.

New capabilities.

Scientists are finding that many of their experiments require longer cell life in order to achieve the results they are looking for. Cells have a limited life span in a well plate and many live cell assays require a life span of days to weeks. Along with the requirement for longer cell life is the need for time-lapse imaging capabilities where scientists can automate image acquisition throughout the multi-day experiment for post processing and data analysis. Stem cells may hold the potential to repair or replace tissue cells damaged in many devastating diseases. They also have the capacity to self-renew by cell division and differentiate into mature, specialized cells. Differentiation takes place over an extended period of time, demanding technology solutions that utilize limited amounts of valuable material and can keep the stem cells alive for multi-day experimentation.

The role of environmental controls, microfluidics and imaging

Scientists seek to understand how treatment with potential therapeutic compounds will affect specific cellular processes; such as, protein and gene expression, cell division and multiplication, and cellular self-destruct programs. Live cell analysis is extremely important in bio-pharmaceutical production, where judging the viability and growth rate of cells is essential for optimizing cell culture media and production parameters. Keeping cells alive for extended periods of time while capturing images requires an incubator-type environment for the microscope platform. The environmental control system should control temperature, CO 2 and humidity, and the microfluidics system should provide a controlled flow of nutrients to keep the cells alive for the duration of the experiment.

One path to extended live cell imaging

Nanopoint’s cellTRAY ® Imaging System was designed to take up far less room than conventional instruments and enable microscope-sized analysis. The system creates new standards of precision and levels of efficiency for the study of individual and small groups of live cells, enables new approaches for multiple cell analysis and simultaneous processing, and supports multi-day time lapse imaging of live cells. The cellTRAY Imaging System can be used for a variety of applications, including RNAi knockdowns, “split” GFP reporter assays, cell morphology, clone expression studies, apoptosis studies, karyotyping, oncology studies, stem cells, and time release drug studies.

Nanopoint’s cellTRAY is based on lab-on-a-chip technology using fabrication methods similar to those found in the semiconductor industry. The base substrate is glass which increases the number and types of applications a cellTRAY can be used for. Glass offers low thermal conductivity and optical clarity, which is ideal for imaging applications in the biological sciences. The cellTRAY also utilizes the standard slide holder on the microscope stage, allowing it to be used with any existing laboratory equipment that supports slides.

The microfluidics cellTRAY is a micro-scale live cell containment device with parallel isolated regions enabling 10 distinct experiments while minimizing the volume of fluid to 3-4µL per region. The cellTRAY allows biologists to hold multiple cells in an ordered array enabling automated processing and simultaneous monitoring of a large matrix of cells. Cells can be observed before and after replication allowing the researcher to monitor the response of cells to other stimuli. The re-usable cellTRAY has the dimensions of a standard microscope slide (1” x 3”) and contains an array of 8 x 300µm open wells per region which provide discrete, uniform sample populations along with replicate wells for statistical analysis. Cells attach themselves to the cellTRAY surface slightly below the flow entrance to minimize shear stress. Each cellTRAY is packaged in a cellTRAY Dish that enables easy loading, incubation, and cell culturing.

The manifold, mounted on the microscope stage, contains integrated temperature feedback and an airport enabling regulated CO2 flow. The hinged glass cover on the manifold is also used to minimize contamination while still allowing easy access to the cellTRAY. The software regulates the flow of fluids by syringe pumps that lead to and from a cell media reservoir and waste bottle. The infusion ports, contained in the system manifold, enable parallel isolated experiments utilizing different fluids in each of the 10 regions of the cellTRAY. Since each of the regions on the cellTRAY can be individually addressed, different reagents can be easily administered to each one independently. The syringe pumps are capable of delivering a variety of flow rates, programmable by the user, with a flow resolution of 100nL/sec. The volume of fluid in a single syringe will cover a four hour experiment; and the volume is automatically replaced from the reservoir, extending the experimental time to multiple days. 

The future is now

The cost of drug discovery and development continues to skyrocket with no end in sight. The population of baby boomers with health related issues is growing. New diseases are challenging and scientists must unravel the underlying causes of these complex diseases in order to develop appropriate therapeutic treatments.

One factor has remained unchanged since the beginning of time, however – cells are the basic units of life. Biological processes inside cells determine how we function and how diseases operate. Learning about these processes through observation and analysis is vital to understanding, diagnosing, and successfully treating human diseases. Capturing images of the real-time behavior of live cells over extended periods of time may unlock some of the mysteries of life. Now is the time for scientists and pharmaceutical executives to search out and adopt new technologies and solutions to meet their challenges.

Reference:

Jerry M. Adams, “Ways of Dying: Multiple Pathways to Apoptosis”, Genes and Development, 2003, 17, 2481-2495.