Faster Cell Counting

Cell Counting

by Jeffrey M. Perkel

If you've ever worked with cell cultures, you probably know the agony of manual cell counting. It's a fact of life in the cell culture hood: Before you can run an experiment, you need to know how many cells you have.

The standard procedure is ridiculously simple: Harvest cells, mix an aliquot with trypan blue, load the hemacytometer slide, put it onto the microscope stage, and count. And count. And count.

If you work with many different cell lines, or if you're running many experiments, you could find yourself doing this procedure with depressing regularity. It's not so much that it's difficult – it isn't – but it is time-consuming, tedious, and error-prone.

That's because hemacytometer counting, while technically easy, isn't as simple as it sounds. Cells can clump or distribute unevenly across the slide surface, making the counts imprecise. Also, it is sometimes difficult to distinguish live cells (which exclude trypan blue) from dead cells (which do not), which lends a dose of subjectivity and irreproducibility to the process. Finally, and most importantly, the whole procedure depends on accurately counting enough cells to get an accurate count of the starting sample – a requirement that tends to make the procedure mentally draining, especially when multiple cultures must be processed.

"When we talk to customers, often it's the postdoc or grad students who count cells, and when they have to count many cells, they are almost scared of [those] days," says Veronika Kortisova Descamps, Product Manager at Bio-Rad Laboratories.

John Harper, senior sales consultant at Beckman Coulter, says, "I run across customers that say counting cells is such a pain that people stop counting them altogether."

Fortunately, Bio-Rad, Beckman Coulter, and others have developed automated solutions to overcome these problems. Though invariably more expensive than a hemacytometer, what these devices offer in speed and accuracy, for many researchers, more than makes up for the cost. Speaking of one such device, Harper says, "[It] is very easy to use, and it's a way to do a very tedious task, and when you automate it, you remove all the chances of human error or subjectivity."

Automated cell counters come in three basic flavors: devices based on electrical impedence, devices based on trypan blue exclusion, and flow cytometers.

Electrical impedence is the principle on which the venerable Coulter Counter (first released in 1949) is based. The process (formally called the "Electrical Sensing Zone method") works like this: The instrument probe, which is placed in a vial of cells, contains a small hole or aperture through which the cells can pass. There are electrodes on either side of the aperture, such that a current flows across them. As cells are drawn through the aperture, they momentarily block the current, producing an electrical spike that the instrument detects.

"The amplitude of the pulse is proportional to the three-dimensional volume of the cell," Harper explains. "So what the instrument is doing is counting pulses and assessing the 3D volume of each pulse."

The result is a histogram of particle size versus count, which can be gated so that the final cell tally reflects only particles of a specified range. Typically, the device's operating range covers the whole of eukarya – from 1 to 120 microns, in the case of the Z2 Coulter Counter, and 8 to 25 microns for Millipore's Scepter, a portable instrument akin to a souped-up Pipetman.

With a familiar form factor and just two buttons, the newly released Scepter (which began shipping in March), "is very intuitive," says product manager Grace Johnston. The "tip" consumable, she explains, contains the probe, so no instrument clean-up between counts is necessary.

"We wanted to bring the handheld portable concept of cell counting to the hood," explains Johnston. "Instead of having to go to a large instrument [outside the hood], you could bring your instrument to your culture hood."

Also based on this principle is Roche's CASY line of benchtop instruments. According to Steven Hurwitz, Marketing Manager in the Applied Science Division of Roche Diagnostics, Casy cell counters supplement the Coulter principle with "Electrical Current Exclusion," which enables the device to distinguish live from dead cells based on the differential electrical membrane properties of live and dead cells.

Able to distinguish live cells from dead cells and cell debris, the Casy "is like a Coulter Counter with viability analysis," Hurwitz says.

The Casy notwithstanding, counters based on the Coulter counting principle cannot for the most part distinguish live from dead cells. For that, researchers need image analyzers based on trypan blue exclusion.

Most such systems, including Life Technologies™' Countess®, Roche's Cedex XS, and Bio-Rad's TC10 (which will be released this month) use disposable slides, which the system images with a CCD camera to produce a count – a direct digital analog of the hemacytometry workflow. Others, including Beckman Coulter's Vi-CELL instrument and Roche's Cedex Standard and HiRes models, are flow based, drawing sample into a capillary and counting the cells as they pass a detector.

"When you are analyzing something on a flow basis, it enables you to count a lot more of the sample than if you were to count something that's static, like a slide," says Harper. Slide-based systems, like hemacytometers, typically count perhaps a few hundred cells, he explains. "But Vi-CELL, being a flow based method, we'll generally count between 1,000 and 4,000 cells."

Whether they use slides or are flow-based, the advantage of all trypan blue systems, says Bio-Rad's Kortisova, is that they can distinguish live from dead cells. Bio-Rad's TC10 even images at multiple focal planes, just to ensure that seemingly dead cells really are dead. In addition, imaging-based systems generally enable users to actually visualize the counted cells, instead of relying on a histogram, and even to store the raw data.

"If someone comes back six months later and asks you to verify your data, with the Vi-CELL you can go back very easily and say, here it is," says Harper. "You can't do that with manual counting."

But there also is a downside to these methods, says Mike Olszowy, flow cytometry group leader at Life Technologies. "There are some schools of thought that trypan blue may not be the best [counting method], because some membrane-damaged cells may take up less dye that others and may not be counted as dead; and because, trypan blue may ultimately cause dead cells to swell and explode. So dead cell discrimination is more effectively done using fluorescence."

Fluorescent cell counting is the modus operandi of the final class of automated counters, flow cytometers.

But not just any flow cytometer can multitask as a cell counter, says Olszowy. Most flow cytometers aren't designed to draw a precise volume of fluid and count only that volume; instead, they count a specific number of events, regardless of the volume required to reach that threshold. So, they can provide relative counts (that is, what proportion of cells are, say, helper-T cells), but not absolute counts.

"A [conventional cytometer] can be programmed to stop once it has analyzed a defined number of cells, but you don't know the volume, and if you don't know the volume you cannot know the concentration," Olszowy explains. Life Technologies' Attune™, however, uses a syringe pump system that can be precisely controlled, starting and stopping at a defined volume. As a result, if the system counts 10,000 cells, it can report that number and the volume of cells in which it counted them, and thus, the absolute concentration of cells in the sample.

Millipore's Guava personal cytometer can also count cells directly.

Of course, unlike the other instrument classes described above, flow cytometers are not dedicated cell counters. Instead, they are multipurpose instruments used to characterize cell populations. But any flow cytometer can provide absolute counts if the user also employs specially designed counting "beads," Olszowy notes. These beads act effectively as "spike-in" controls, from which the user can calculate the absolute cell numbers in the starting sample.

When choosing an automated cell counter, variables to consider include cost (prices for the counters described here run the gamut from $2995 for the single-function Scepter to $99,000 or so for the multipurpose Attune) and sample volume requirements, as well as the need to assess cell viability. The good news is, with so many options available, users really cannot make a bad decision. After all, any automated solution is going to be better than old-school hemacytometry.

Says Wenlan Hu, senior marketing development manager for Countess, "Any automated cell counting process is trying to address the painful points in the process, because it is very tedious to count cells by hand."