Laura Lane has worked as a health and science journalist since 1997. She received her master's degree in biology from Stanford University. Since then, she has written for the Dallas Morning News, the Contra Costa Times, Shape magazine, WebMD, Yoga Journal, Diagnostic Imaging, the International Medical News Group, The Scientist, Bio IT World and Biocompare.
Barry Stripp probably wouldn’t get very far without today’s advanced cell-isolation techniques. Stripp’s research hinges on isolating cells and studying them for their ability to repair and renew lung tissue. Using monoclonal antibodies to mark out different cell types, Stripp, who is director of the new Lung Stem Cell Program at Cedars-Sinai Medical Center in Beverly Hills, Calif., relies on “technologies that allow you to interrogate a large battery of markers,” he says.
From there, he looks at which cells stimulate the clonal expansion required for regenerating lung tissue. It can be quite tedious, experimenting with an essentially endless number of marker combinations, different levels of enrichment and various fractionation techniques by hand.
The laborious nature of such work hasn’t gone unnoticed, as evidenced by a marketplace full of products that enable users to separate their cell samples faster and more efficiently. And that, as Stripp proposed, “would allow you to more precisely define the phenotype; it would move you forward more rapidly.”
Fast and (hands-) free
Nothing speeds up a task quite like automation. With that in mind, companies have designed instruments that do the work while their users attend to other important matters.
“We have to provide quick solutions,” says Adrian Arechiga, global product manager for cell-separation technology at Miltenyi Biotec. “By expediting [cell separation,] researchers can spend the bulk of their time on downstream analysis.”
Arechiga’s company specializes in magnetic bead-based cell separation. Antibodies specific for the cells to be isolated are conjugated to the beads. Those beads are then incubated with the cell sample, capturing the cells of interest. Exposure to a magnetic field separates those target cells from the rest of the material.
Normally, this process is performed manually. But with the company’s autoMACS® Pro Separator, users can simply load their samples and walk away. The instrument takes it from there, performing all the necessary pipetting and separation steps. The MultiMACS™ Cell24 Separator doesn’t come with automated pipetting, but it does separate 24 samples in parallel. Integrated with Tecan’s Freedom EVO®, the MultiMACS Cell24 can automate all the liquid handling for multiple sets of 24 samples, or between one and nine samples with the new Single Column Adaptor, which is available for both manual and automated platforms.
For researchers who aren’t ready to shell out hundreds of thousands of dollars for big liquid-handling platforms, Arechiga recommends starting at lower price points. The autoMACS Pro, which comes with automated liquid handling, starts at $46,500. If the features of the MultiMACS seem to fit your needs better, he recommends starting with that unit and then considering automation later on.
STEMCELL Technologies’ offering in the arena of automated cell separation is the RoboSep™. The $50,000 unit operates with magnetic beads called RapidSpheres™, which are engineered to separate mouse cells in 15 minutes. The company recently released a new selection of isolation kits with beads conjugated to mouse antibodies, says Cyrus Edjulee, product manager of cell separation.
“It works in as little as 15 minutes, from the time of single-cell suspension to isolated cells,” he says.
Fast and fantastic
Magnetic based separations offer speed, simplicity and the ability to separate bulk quantities of cells, says Marty Bigos, director of the Flow Cytometry Shared Resource at Stanford University Medical Center. Its capabilities are even beginning to be harnessed in the clinic, where physicians use separated cells to treat patients.
The downside, however, is that you can only separate the cells based on one parameter, Bigos says. “The technology is pretty broad.”
He and companies like Miltenyi Biotec and STEMCELL suggest that magnetic technology could be used pre-enrichment, followed by additional separation, if necessary, with flow cytometry.
Although the general technology for flow cytometers has remained largely the same for decades, incremental advances have enabled researchers to go further in their discoveries. One of these advancements is the increasing number of markers that the instruments can simultaneously recognize. Beckman Coulter’s MoFlo® Astrios™ EQ, for example, available this fall, can differentiate 32 different markers in one sort.
Many of the latest flow cytometers can also be integrated with liquid-handling platforms for sample preparation and downstream analysis. In addition, flow sorters can simultaneously process more samples. For example, the Astrios EQ offers not only six-way sorting but also combines this with multiple sort capability to provide maximal flexibility in sample recovery.
The Astrios EQ also reflects the trend towards maximizing sample utility. More and more flow cytometers are designed to separate cells without losing sample, which sometimes can be irreplaceable. With these instruments, you can collect both sorted cells as well as any aborted cells that may not have passed the sort logic.
“This ensures that you get the most out of the available sample, particularly when purity and/or recovery of your desired sort populations is concerned,” says Robert Sleiman, Global Product Manager for flow cytometry sorters at Beckman Coulter Life Sciences.
Fast and (usually) flawless
For both flow cytometers and magnetic-based instruments, increased efficiency also comes in the form of convenient kits and preprogrammed protocols.
“Automated systems nowadays are very straightforward,” Arechiga says. “You just program what you want to do and walk away. We’ve taken a lot of the guesswork out of the game and made it really simple.”
That is, if you’ve followed the directions, Edjulee adds. He explains that some researchers accustomed to traditional methods combine those methods with the kit’s protocol and then complain about reduced cell viability after separation. But when Edjulee investigates, he discovers that these customers also added ammonium chloride to the samples, a traditional technique to lyse red blood cells that can stress the cells.
“Don’t do it,” he says. “Just follow the directions.”
Furthermore, Edjulee says, resist the urge to save money by diluting reagents. “The amount we give you is to be sure that you appropriately label all cells.”
Overall, you’ll get better results with better starting material. Prepare your cells correctly and carefully. Extract extraneous debris, such as tissue fibers. And ensure that your cells are healthy from the start, if you want healthy cells at the end.
“For any experiment, garbage in means you get garbage out,” Edjulee says.
After mindful preparation of cell samples, you can be confident that the cell-separation instruments of today can take on the most demanding projects.
“What we have today probably exceeds most researchers’ needs,” Beckman Coulter’s Sleiman says. “Is it perfect? It’s close. Does it need to be perfect? Probably not ... but why stop, there?”
Image: Miltenyi Biotec autoMACS Pro Separator.