<b>Enhanced Environmental Control Of Live Cell Microscopy Advances Breast Cancer Recurrence Research</b><br/><br/>

Enhanced Environmental Control Of Live Cell Microscopy Advances Breast Cancer Recurrence Research

Evironmental control is vital to the use of live cell video microscopy as a prognostic tool for early-stage breast cancer recurrence.

by Ed Sullivan

Figure 1. When there is a need for complete isolation of the specimen or the ability to perform more advanced modes of microscopy during long-term time-lapse studies, a closed- system chamber is required.

As the average stage of breast cancer at diagnosis decreases due to improved screening techniques, the study of early stage breast cancer (stages I and II) via microscopy becomes all the more significant. Environmental controls that are often deficient hinder the quality of results available to live cell researchers.

"I believe there are not enough groups looking at live cells because the process is difficult," says Jean Latimer, Ph. D., a leading researcher in breast cancer at the University of Pittsburgh's Hillman Cancer Research Center. "However, there are now more advanced ways of culturing cells, that when combined with an improved micro-environmental control system, has allowed my group to produce much longer time-lapse movies using this technique plus an associated scoring system we developed. We're getting very legitimate trends from the output. And it is certainly possible that other researchers could benefit from the more advanced culturing techniques, cell behavior scoring protocols and micro-environmental control technology."

Breast cancer affects more than 180,000 Americans each year, and kills approximately 46,000 according to the American Cancer Society. More than 40,000 surviving women annually find that their cancer has come back.

While the 10-year relative survival rate has improved in the last decade, a minimum of 7.5 to 35% of patients with stage I breast cancer will recur within that time, respectively. Current clinical practice often fails to identify the cohort that is susceptible to recurrence. One of the aims of Dr. Latimer's studies is to utilize live cell video microscopy to study breast tumor primary cultures in order to subdivide tumors of the same stage, and improve the ability to predict the potential for recurrence in individual tumor samples. In this effort Dr. Latimer's laboratory has developed a novel way of culturing mammary epithelial cells based on her experience in growing mouse embryo explants. This method involves a paradigm shift from traditional cell culture to concepts more closely related to tissue engineering.

Figure 2. FCS2 design features allow the specimen, adherent cells on a coverslip, to be maintained safely in a temperature-controlled, user-definable-flow, optical environment.

An important component of that paradigm shift is the ability to maintain the viability of live cells and accuracy of viewing them. For that purpose Dr. Latimer's laboratory uses the FCS2 live-cell micro-observation system (Bioptechs Inc., Butler, PA). The FCS2 is one of two basic systems that Bioptechs offers for live cell microscopy. Those systems address the two basic forms of specimen containment and micro-environmental control, the open culture dish and the closed-system chamber.

"The open dish or Petri dish, the most common method of micro observation, was originally designed for the growth and gross observation of bacteria. This type of dish was then available and used in early live cell experiments, but it was not optimal for today's imaging requirements," explains Dan Focht, Bioptech's president. "Because the dish's surface was not optically compatible with high resolution imaging, it produced degraded or distorted images. The use of an external peripheral heater transferred heat to cultures inefficiently, which was unhealthy for live cells and caused the focus of the microscope to drift. The internal volume of the dish was large, thereby increasing surface evaporation, not to mention a dilution of the cells factors. These environmental conditions compromised live cells on the microscope and induced shortened life spans."

For those reasons Bioptechs developed the micro-environmental system (Delta T), an open dish system that is designed for live cell, time-lapse imaging on any microscope. Using a No. 1.5 cover glass as its bottom, and with an underside coated with transparent, electrically conductive material, the Delta-T dish offers high-resolution viewing with optimum thermal control via an electronic feedback control system. The system also prevents unwanted ambient light from entering the specimen, and provides fluid perfusion and/or CO₂ control to stabilize osmolarity. The system is also available with numerous accessories to accommodate a variety of specimen types. This technology is compatible with all modes of microscopy on both upright and inverted microscopes.

Figure 3. This is a thermographic image of the FCS2 showing thermal uniformity across the entire field.

When there is a need for complete isolation of the specimen or the ability to perform more advanced modes of microscopy during long-term time-lapse studies, a closed-system chamber is required. Closed systems, or parallel plate chambers, are often constructed by placing a gasket between two glass surfaces, creating a fluid optical cavity. Historically this cavity would be perfused with the addition of a set of ports. This typical configuration results in a turbulent jetstream that tends to dislodge specimen cells. This problem became the impetus for development of the FCS2 closed system, the model used by Dr. Latimer's team. The perfusion jetstream quandary is solved by a patented "microaqueduct" perfusion technique, which incorporates perfusion grooves into one of the optical surfaces of the optical cavity. "The grooves produce a smooth, controlled laminar flow that will not disrupt the cells" explains Focht. "To fully optimize the system there is an electrically-conductive thermal control coating on the outer surface of the FCS2 microaqueduct slide, thus adding thermal uniformity even during experiments with periods of no perfusion flow." With the addition of the Bioptechs Objective heater system for high numeric aperture applications, the FCS2 has become the prime micro-environmental system for long-term time-lapse studies, such as those Dr. Latimer's lab performs.

These FCS2 design features allow the specimen, adherent cells on a coverslip, to be maintained safely in a temperature-controlled optical environment for inverted and upright microscopes that is compatible with all modes of microscopy, including: low- and high-N.A., transmitted, brightfield, phase, DIC, and reflected modes of fluorescence as well as with confocal microscopy.

Figure 4. Underside of the FCS2 in visible light.

Some live cell researchers have chosen to "box" the whole microscope by encasing it with Plexiglas in order to warm the stage and specimen. In the process they have to maintain 5% CO₂ and humidify the scope and enclosure to reduce evaporation. "It's not that this approach does not work," Focht says. "But in addition to the fact that it is awkward to work in a box it is a very expensive proposition. Not only is the box expensive, but the humidified CO₂, costly in itself given the volumes involved, promotes corrosion of the microscopes components, which is a very pricey consequence."

About the author

Ed Sullivan, a Hermosa Beach, CA-based writer, has covered healthcare and high technology topics for over 25 years.

More information about micro-environmental control is available from Dan Focht at info@bioptechs.com.

Bioptechs, Inc.
877-548-3235
www.bioptechs.com