The Live Cell Array

Researchers at Tufts New England Medical Center are conducting research on beta cells and how those cells can be affected by environmental stimuli. The cells are cultured and placed into a newly developed slide known as the LiveCell array. The LiveCell Array is a small slide that has tiny wells designed to hold the cells. The wells, which come in two sizes, 10 micrometers and 100 micrometers, hold the cells in position so that single cells can be studied over time. Figure three shows a close up of the tiny micrometer size wells. The perfusion system will be designed to utilize the new LiveCell Array technology.

Figure 3 The live cell array and the small micrometer size baskets are shown close up.

The advantage of using the LiveCell array is that it gives scientists the ability to look at a large amount of individual cells and see how different solutions affect their performance. Images, like that shown in Figure Four, help scientists to study the effect of solutions on living cells.

Figure 4 Cells residing in the microscopic baskets

On one side of the array, liquid can be added. That liquid is then pulled across the cell baskets by capillary action. The cells get nutrients and other agents from the liquid through diffusion. The LiveCell Array is small and portable, allowing for easy imaging using a microscope and CCD camera. The images are then analyzed using the MetaMorph image processing software. One of the main uses of the program is to create a region of interest around individual cells being analyzed and obtain the average intensity of each region. As seen in both Figure 1 and Figure 3, the cells have been manipulated to fluoresce when their mitochondria are working correctly. Among other functions, the image analysis determines which cells are functioning properly and which are not.

Currently, the state of the art involving maintaining and visualizing cells in the laboratory does not involve cycling of glucose. Researchers can keep beta cells alive and operational for about an hour by subjecting them to a concentration of glucose in an incubator. The beta cells can be sustained for the short term using this method, but they are unable to operate at optimal levels. This technique does not mimic the body’s sinusoidal glucose conditions in vivo as closely as they should and therefore limit how long the research can go. There have been some perfusion systems developed to cycle the glucose concentration, but they are large and bulky and are not feasible with the small scale required when working inside an incubator or with the CCD camera and microscope. Cytometry is also used in labs to visualize cells and its most common forms are flow, bulk and static. [3] In order to visualize the cells, a laser beam is passed through a cell and the scatter is detected.[4] The current drawbacks to the cytometry is that cells can only be visualized/ studied once and then discarded because they must be killed in order to be viewed. This does not allow for longevity studies of the same cells and does not allow scientists to tests cells as they progress through their lifecycle in response to what the researcher is testing.

Goals

Overall goals

Our goal was to devise an automated perfusion system that has the ability to regulate the glucose levels to which the beta cells are exposed to aiding in the research being conducted at the Tufts Medical Center. We hope that the perfusion system will show superiority over the conventional manual method. The system would be able to keep cells alive for days at a time by cycling the glucose being perfused onto the cells improving the in vivo conditions that researchers are mimicking in the lab. The system would also provide cells with other nutrients they need in order to be cultured. We hope to design the system to utilize the two Cell Array configurations. The first is the typical slide array on a rectangular slide. The second is a modified slide array that sits on the bottom of a confocal petri dish.

We set out to develop this automated perfusion system so that it fits within the confines of an incubator to keep the cells at their optimal temperature. The user would be able to control the flow rate between 5-14 and 50-700 µL/min. The user would also be able to manage the times at which the cells will be exposed to certain solutions all the while keeping the cells at 98.6 °F. This accomplishment alone would greatly aid beta cell researchers and increase the longevity of their work, but subsequent projects will be developed following the initial prototype. Later work will include integration with a current CCD camera data acquisition system as well as temperature and CO 2 controls to more fully mimic the in vivo environment.

Design Specific Goals

• Create a wireless automated perfusion system for both the cell array and confocal cell array dish
• Perfuse cells with multiple sources of media for several days
• Operate in cell culture incubator and on the microscope slide during imaging
• Cyclic variation of media concentrations (e.g. glucose concentrations)