Since the dawn of stem cell research, beginning with the isolation of stem cells from mouse embryos in 1981 and further gaining momentum with the isolation of human embryonic stem cells (hESC) in 1998, the field has presented both huge potential and extraordinary challenges. Stem cells can be utilized in many ways in both basic and clinical research and may eventually be used therapeutically to treat a variety of diseases including Parkinson’s disease, cancer, diabetes, and spinal cord injuries. There are, however, many technical hurdles that must be overcome before the promise of stem cell research can be realized.
For example, scientists must exhibit exquisite control over stem cell culture in order to ensure that the cells proliferate extensively and differentiate reliably into the desired cell types. The optimization of the cell culture system, including such steps as standardizing the physiochemical environment and removing variability, will be critical in advancing stem cell technology as such factors can impact cell viability dramatically. Furthermore, the ability to offer security for the cultured cells and complete documentation of the experimental process is essential for therapeutic implementation.
While optimization of cell culture protocols is especially important for stem cells, particularly hESCs, the same can be said for any cell type that is rare, expensive, or sensitive, including embryos.
Perfecting cell culture conditions, however, is far from a trivial task. Often a large number of samples are required, making the task difficult or impossible without automation. Additionally, it is important to keep the environmental conditions as constant as possible, necessitating that human handling be kept to a minimum.
New technology, realized in Nikon Instruments’ (www.nikoninstruments.com) BioStation CT, has become available that allows cell cultures to be observed over time while maintaining them in their optimal environment. Additionally, the integrated robotics system manipulates the cells completely within this environment, while software and a terabyte server keep both the data and the cell cultures secure. This technology was used to monitor hES cells and mouse embryos as well as to measure growth rates in cultured cell lines.
The BioStation CT (Figure 1) combines an inverted microscope and a quantitative digital camera system with a complete tissue culture incubator. The instrument contains a culture vessel rack with 30 spaces in order to accommodate a full range of vessels in a variety of formats including flasks, well plates, and culture dishes.
Using integrated robotics, vessels are moved from the culture vessel rack onto the stage of the inverted microscope for both macroscopic and microscopic observation. A color camera is used to take a 1X image of the entire culture vessel, allowing the researcher to observe the color of the media in order to determine pH and providing a way to verify the sample by reading any handwritten notations on the culture vessel.
Microscopic observation ranges in magnification from 2X to 40X and uses Nikon’s ADL phase optics. Additionally, a fluorescence unit with violet, blue, and green LEDs allows three-color fluorescence imaging using a variety of fluorescent dyes and proteins. Images are captured via a quantitative, scientific-grade, monochrome, cooled digital camera and stored on the onboard terabyte server.
Using automated scheduling features incorporated into the software, images of many different samples can be scheduled to be taken over the course of several days without any additional user intervention. The software also contains many tracking and security features, allowing researchers to access only samples they have clearance to access and recording all interactions with the samples in a searchable culture history. Furthermore, data-analysis software with teachable analysis recipes allows experimental image sequence data to be analyzed in a variety of ways.
Figure 2 provides two examples of experimental image data obtained with BioStation CT. In Figure 2A, phase-contrast images of the division of a mouse embryo are shown. The embryo was imaged every 60 minutes at 20X magnification for 72 hours. The four panels of the image show the embryo at the 1-, 2-, 4-, and 8-cell stages.
A human embryonic stem cell culture is shown in Figure 2B. The colony is labeled with GFP- histone 2B and is growing on a mouse fibroblast feeder cell layer. The colony was imaged every 10 minutes in phase contrast and fluorescence at 10X magnification for 48 hours; a single image from near the end of the image sequence is shown. Taken together, this data demonstrates the feasibility of BioStation CT for reliable long-term imaging of valuable, sensitive cell culture types.
While the ability to take images of cells within their ideal environment for several days at a time is critically important for a variety of experiments, data analysis is also essential, especially in determining ideal cell culture conditions. Using data-analysis software, a variety of parameters related to cell growth and viability can be calculated.
HeLa cells imaged for 48 hours at 4X magnification using phase contrast are shown in Figure 3A. The top three panels show the cells initially, then 24 and 48 hours later; the bottom three panels show the same images with the area of the images where cells are growing masked in blue.
Using the software, this masked area is ratioed against the total area to measure confluency and then plotted versus time to yield the growth curve shown in Figure 3B. Growth curves can be plotted for many samples simultaneously observed in the BioStation CT and cultured while varying different parameters in order to determine the best cell culture protocols.
By combining the microscope and imaging system with a tissue culture incubator, it is possible to observe and monitor cells for a long duration with low variability. Furthermore, minimizing human interaction with the cell culture protects the cells from contamination as well as from stress caused by changes in their environment. Not only does this technology increase cell viability, but eliminating variability in the cell culture environment is advantageous to the optimization of cell culture protocols.
Also critical in optimizing cell culture protocols is automation and software that can apply a variety of analysis recipes including those that count cells, measure confluency, and determine growth rates. By integrating all of this functionality into BioStation CT, Nikon Instruments aims to provide tools for improving stem cell culture technology and ultimately realize the promise of stem cell research.