Hands-off approach

Live cell care takes a giant step forward with the development of management, growth and observation

Over recent years, the importance of growing and maintaining stable cell lines has come to the forefront of biomedical science. Perhaps nowhere is this more evident than in the field of regenerative medicine, where the pluripotency of stem cells has gained international attention for their potential use in a wide variety of therapeutic interventions. Recently, for example, it was reported in Germany that patients with advanced liver cancer have had marrow stem cells taken from their hips and injected into their remaining healthy liver tissue. After two years the six of the eight patients who had undergone the procedure were still alive and had regained full liver function.

Stem cells are, however, not the easiest of cells to grow; when maintained in long-term culture, they will often undergo progressive adaptation. Great care needs to be taken to safeguard their environmental conditions and they need to checked at regular intervals. Research indicates that even subtle variations can induce genetic changes in their karotype, which can lead to ensuing complications during research and therapeutic use.

Of course, in addition to stems cells, there are a plethora of other cell lines that are contributing to the advancement of science and medicine. As a result of this increased focus on working with cells maintained in culture, ‘Live Cell Care’ has become a prime concern for scientists and equipment manufacturers alike. Techniques such as fast sweep confocal microscopy and controlled light exposure microscopy have significantly reduced the amount of bleaching that occurs when cells are observed under fluorescence and during extended observation times. This is enabling researchers to gain a far more accurate picture of what is actually happening to cells in vivo.

Despite such recent advances, one major problem still remains – changes to the environment of cells when they are taken from an incubator and placed on a microscope for observation. No matter how carefully the culture flask or dish is transported, the cells are almost certain to encounter some form of disturbance due to accidental rocking actions, which can lead to a changing of shape and/or a detachment of cells. Change in temperature and CO2 concentration can further disrupt cells in culture, impacting the accuracy and reproducibility of experimental data.

These problems were first effectively addressed at the end of 2006, with  the launch Nikon’s  completely integrated ‘hands-off’ system for managing, observing and recording the growth of cells in culture – the BioStation IM. The system combines the precise environmental control capabilities of a high-performance CO2 incubator with the advanced optics needed for drift-free, live-cell imaging, making it ideal for accurate time-lapse readings taken over the course of several days.

With the BioStation CT, it is possible to log in from anywhere around the world to view the progress of cells

While the BioStation IM is a bench-top system specifically aimed at single-user, single experiment applications, Nikon will soon be launching the BioStation CT - a free-standing unit designed specifically for multi-user, multi-experiment work. Its mode of sample handling will remove entirely the need for transporting culture dishes from one location to the next for observation. In addition to an incubator and a research-grade inverted microscope (Nikon TE2000), the BioStation CT will also feature an integrated digital data acquisition and storage system based on the Nikon Digital Sight  CCD camera and a dedicated server. By maintaining environmental conditions at optimal levels, the consistency of cell growth will be significantly improved, leading to reduced intra- and inter-lab variability when the same experimental protocols are followed. Furthermore, the hands-off approach exemplified by the new BioStation will also reduce the scope for contamination. As a result, the BioStation CT should be of great interest to everyone working in regenerative medicine, toxicology, drug discovery, and biologically-based drug production. Considering the complexities and expense of cell validation and toxicology testing prior to HCS/HTS screening, the improved consistency of cell culture gained by using the BioStation CT should make it a very attractive proposition.

To ensure that environmental conditions are precisely controlled and that the stress placed on the cells is kept to an absolute minimum, the incubator section of the BioStation CT will employ a direct heating approach delivered via six separate heaters. It will also use an active injection system to maintain the humidity levels to within fine tolerances (this again serves to reduce the risk of contamination). Several other integral features have also been incorporated to minimise the risk of contamination, such as the small culture vessel loading gate size and the completely hands-off approach to its set-up and operation. All interaction will be via a simple touch screen or remote PC interface, either of which can be used to instruct the system when to observe individual culture vessels. In this manner, it will be possible to run several tests independently on the same or different cell types to, for instance, monitor drug effects over time, but remove scope for inter-operator variability.

For complete, multi-user versatility, the BioStation CT has been designed to handle flasks, dishes and well plates (virtually any vessel fitting the system’s 120mm x 86mm loading stage can be used). When cells need to be observed, vessels will be automatically retrieved from the storage area and transferred to the integral microscope stage. This transfer will be carried out using robotic stepper motors that are virtually vibration-free, avoiding disturbance to cells in culture. Since the cells never leave the controlled environment within the system, there is no need to let them re-equilibrate prior to observation.
Both systems are capable of micro and macro imaging (from 2x to 40x magnification on the CT, or 10x to 80x on the IM), under phase contrast using Nikon APC lenses that provide the highest possible contrast with a near-zero 'halo' for clear viewing of cellular organelles. Both models can also be used to image cells under epi-fluorescence illumination. The BioStation IM comes in two versions, one with a long working distance lens to work with plastic petri dishes and another with a high numerical aperture lens to get the best results with thin bottom glass petri dishes. All interaction with the microscope, including focusing operations and exposure level settings, is managed by the controlling software, ensuring that the highest quality images are always generated.

On the BioStation CT there will also be a ‘bird’s-eye’ view function. This will allow users to see the entire culture vessel, including their own hand written identification schemes on top of the flask or dish, and to detect pH-dependent colour changes and signs of contamination. Viewing is achieved through an integrated digital camera, with images being made available via an internal server within the CT or an external computer linked to the IM. When using the BioStation CT, it will even be possible for users to log in from anywhere around the world to view the progress of cells and check in on recorded results. All they will need is access to a web browser in order to call up the appropriate web page. If their images have been captured in 3D, users will be able to view their samples at different depths within the cell, just as if they were using the microscope by hand.

When users log in to the BioStation CT, they will be immediately presented with the ‘bird’s eye’ view of their culture. Since the time and duration of recording can be pre-programmed and adjusted remotely, there will be no need for users to revisit the laboratory. In addition to the capture and storage of images, the new system can also display the environmental conditions at the time of reading. This integration of images and data will increase the fidelity and reliability of audit trails for GLP and GMP reporting.

When logging on to the software controlling the system, users will always have to enter their ID and password. This will ensure they are only given access to their own samples, results and protocols. Further security and confidence in the results and images is gained from the fact that it is impossible to modify the data once it has been captured by the server.

Among the first scheduled users of the BioStation CT in the UK is a major research establishment working with stem cells, which intends to the apply the system across a variety of research programmes.

In conclusion, the development of the two BioStation variants represents a great step forward for live cell care and signifies Nikon’s commitment to continuously improving the resources, techniques and equipment used in live cell imaging.

By Sundeep Bhandari. Sundeep graduated from the University of Brighton in 1999 with a BSc. (Hons) in Biomedical Sciences. Following graduation, Sundeep was offered a place at Imperial College, London, where he attained his MSc in Human Molecular Biology. He is now European Sales Manager for the BioStation Product Group