How it Works: Miniaturizing Automated Cell Counting

Problem: Cell counting is necessary when seeding and passaging cells or when preparing experiments for cell-based assays. Cell counts can be used to monitor the health of cultures and rates of cell proliferation, as well as assess immortalization for transformation. Therefore, it is critical that cell counts be accurate, consistent and fast—particularly when monitoring quantitative cellular responses. Despite the need for speed and accuracy, the vast majority of cell counting is accomplished by use of a hemocytometer—a technique first introduced in the late 1800s. While hemocytometry is inexpensive, the many steps required to complete a cell count are tedious and can result in inaccurate counts. Sources of error include uneven cell distribution in the sample; too many or too few cells in the sample; subjective judgments as to whether a given cell falls within the defined counting area; contamination of the hemocytometer; variation in how a sample is loaded; and differences in user technique. Researchers can also count cells using automated instruments such as vision- based counters, flow cytometers, or systems that incorporate Coulter technology, though the cost of these instruments can be prohibitive.

Solution: The latest innovation in cell counting combines the ease of automated instrumentation and the accuracy of Coulter technology in an affordable, hand-held format. The Scepter™ cell counter from Millipore incorporates Coulter impedance-based particle detection in a miniaturized format. The instrument, which is the size of an automated pipette (Figure 1), incorporates analog and digital hardware for sensing, signal processing, data storage, and graphical display. The disposable sensor is engineered with a microfabricated, cell-sensing zone that enables discrimination by cell size and cell volume at sub-micron and sub-picoliter resolution. Cell population statistics are displayed as a histogram directly on the Scepter cell counter’s screen. Cell counts are conducted by attaching a sensor and inserting it into the cell sample.

Depressing the plunger of the Scepter cell counter activates a pump that draws fifty microliters into the sensor’s microchannel. The sample flows through a nylon mesh to break up any clumps and then passes through an orifice outfitted with an electrical field. As cells pass through the orifice, changes in resistance are recorded and translated into a cell count. At the same time, the amount of flow impeded as cells pass through the orifice is translated into cell volume. Cell diameter is then extrapolated from the volume recorded.

The Scepter cell counter detects each cell passing through the sensor’s orifice and calculates cell concentration and displays a histogram of cell volume or cell diameter. The time required to perform cell counts using Scepter is generally less than twenty seconds.

In order to compare the linearity and precision of the Scepter cell counter with alternative techniques, nineteen cell types were counted using Scepter; an automated vision-based system; a full-size Coulter-based system; and a hemocytometer. Results showed that cell concentrations measured by the Scepter cell counter closely matched theoretical cell concentrations with high linearity. Counts completed with the Scepter cell counter were shown to be more precise than vision-based counting and hemocytometry, displaying smaller standard deviations and smaller average coefficients of variation.

In addition to counting cells, the Scepter instrument also provides insights into the health of cell populations. For example, a researcher can compare histograms of the diameters of well-maintained cultures of to a new culture. The histograms of well-maintained cultures will show a relatively high quantity of cells with the expected diameter, whereas the histogram of a neglected culture will be dramatically shifted. Changes in the histogram can also reveal the mix of live and dead cells within a culture, as well as the diameter (and relative health) of cells in an early passage compared to a later passage.

For more information, visit www.millipore.com/scepter.