| scale cell-disruption technologies. |
BioSpec Products Inc.
Cole-Parmer Instrument Co.
Glas-Col Apparatus Co.
Omni International Inc.
Parr Instrument Co.
Sonics and Materials Inc.
"The method selected will depend on its capability to process samples of a certain size or to be able to process multiple samples in a reasonable period of time," says Timothy R. Hopkins, a biochemist and president of BioSpec Products Inc. in Bartlesville, Okla. "Other considerations are the availability, cost, and general utility of the disruption equipment.
"In a research environment, purchase of an expensive cell disrupter which processes a wide variety of cell types may be easier to justify than a specialized disrupter," he adds. "And if the long-term goal is to scale up, the choice of disruption methods narrows considerably."
Some techniques that can easily process a few milliliters of cell sample in a researcher's lab are of no use when the sample is scaled up to several liters. Another key consideration in selecting a cell disrupter is cell type.
"Some cells are more difficult to rupture than others," says Carol Ostrom, marketing manager at Microfluidics Corp. in Newton, Mass. "For example, mammalian cells, in many methods, are easier. Yeast are really hard to penetrate."
In her company's Cell Disruption Microfluidizer, cells caught at the point where two high-speed and highly pressurized streams of proprietary liquids meet are ruptured. Mammalian cells, relatively delicate because they lack cell walls, rupture with one pass at a pressure of 2,000 psi, while insect blood cells require three passes at 5,000 psi or one pass at 15,000 psi.
The notoriously tough yeast cells can require two passes at 20,000 psi, Ostrom says. The advantage of the device, according to Ostrom, is that "the whole product is treated in a uniform manner, making it easy to scale up from research and development to production."
While Microfluidics emphasizes the ability of its device to rupture the toughest cells, a Cell Disruption Bomb from Parr Instruments Co. in Moline, Ill., targets delicate mammalian cells and works on the principle of nitro- gen decompression. This is the same phenomenon feared by deep- sea divers and known as "the bends," in which nitrogen in the blood bubbles out of solution as the divers ascend from great depths.
With the Cell Disruption Bomb, nitrogen is dissolved in cells in a high-pressure vessel. A sudden release of the pressure sends the nitrogen into bubbles, and the cells burst. The technique is fast and uniform, and handles large samples without generating heat.
"It is physically and chemically quite gentle and can be used to recover delicate biochemicals with high metabolic activities," says Sherman Hamel, vice president of sales and marketing at Parr. "The disruptive action can also be closely controlled to release intact nuclei and functional mitochondria from most mammalian cells."
Bead milling is another effective way to disrupt the tough- to-crack yeast cells, other fungi, cyanobacteria, microalgae, and spores. In a shaking-type bead mill, electromechanical forces agitate glass beads in a container of cell-rich fluid. Speed, duration, and bead size are chosen to suit the particular cell type and the type of material the researcher wants to collect. It takes one to five minutes to disrupt bacterial cells, for example, says Hopkins. After movement stops, the beads settle immediately to the bottom of the container and the broken cells can be removed from the material on top. The shaking type bead mill can handle samples up to 3 ml. A rotor-type bead mill, in which a moving rotor provides the shearing force, can process samples up to 250 ml.
Another common cell-disrupting tool, the rotor-stator homogenizer, tears cells by applying turbulence and shearing generated by forces between a stationary component, the stator, and a moving component, the rotor.
"The stator is a hollow tube, and the rotor is a blade inside it that turns swiftly," says Alison Lippincott, marketing coordinator at Omni International Inc. in Gainesville, Va. "The stator has slots," Lippincott says. "When the rotor spins, it sucks the sample up and cuts it while it is rotating. When the sample is pushed out of the windows [slots], it is cut further. Outside [of the slots], the sample meets a pressure differential, which shears it even further."
Ultrasound is the basis for another widely used cell- disruption technology. Devices based on ultrasound are called sonicators and work on the principle of cavitation. An electrical current is converted to mechanical vibrations, which traverse a device called a horn, which intensifies the vibrations. This establishes pressure waves.
When the horn contacts a liquid, millions of microscopic bubbles, or cavities, form in the presence of the resulting positive and negative forces generated by the pressure waves.
As the bubbles expand under negative pressure and implode, or collapse, under positive pressure, they send a powerful shock wave through a probe tip, which shears cells it contacts. The cavitation actually occurs just in front of the probe's tip. Vendors supply ultrasound devices and a variety of horns, probe tips, cooling jackets, and sound- proofing modifications.
"Every unit does the exact same thing," says Anthony Borrelli, assistant marketing manager at Sonics and Materials Inc., Danbury, Conn. "With higher wattages, you can go with a higher volume. A couple of models have more whistles and bells."
While ultrasound manufacturers highlight the efficiency and speed of sonicators, many life scientists are wary of the devices.
"Ultrasound generates a tremendous amount of heat, which is a great disadvantage in biology," says Stefan Surzycki, a professor in the Institute for Molecular and Cell Biology at Indiana University, Bloomington. Heat, he explains, can destroy organelles and unravel biological molecules.
Birth Of BioNeb Surzycki's dissatisfaction with cell disrupters on the market led him to invent a new entrant into this rather classical field.
"Ultrasound leads to heat, which is uncontrollable," he says. "Systems that use a blade shatter everything. With many methods, you just smash everything and hope you don't smash what you want."
Surzycki, along with fellow Indiana biology professor Robert Togasaki and associate Masahiko Kitayama, embarked on building a better cell disrupter. The technique they settled on takes advantage of a natural phenomenon called nebulization--basically, the formation of droplets. The aspect of nebulization that is important to the process is similar to what happens in a capillary tube, Surzycki explains.
"If you have a very small capillary tube, flow is in layers," Surzycki says. "The center flows faster and layers towards the side of the tube are much slower. If you put a cell in the area of differential speed, it is broken because one end of the cell is flowing faster than the other. The cell stretches and breaks."
Engineering a capillary tube small enough to hold a cell and facilitate this process proved a daunting challenge. Enter nebulization.
Nebulization "occurs when you blow a gas over a surface of a liquid, following the same principles as a perfume sprayer or air-painting device," Surzycki says. The gas flow causes droplets to form--but not instantaneously, he explains. "For a moment they are connected to the liquid, with the neck size about half the diameter of the droplet," he adds. For a millisecond, that "neck" between liquid surface and emerging droplet resembles a tiny capillary tube. In Surzycki's device, called BioNeb, cells are sheared within that neck because of the differential flow.
BioNeb works well on a variety of cell types and sources, including cyanobacteria, E. coli, yeast, algae, and plant and mammalian cells, its manufacturer says. One convert to BioNeb from sonicators and a high-pressure device called a French press is David W. Krogmann, a professor of biochemistry at Purdue University, Lafayette, Ind. "We have used the BioNeb to break open cells of the cyanobacterium Synechocystis, an especially difficult cyanobacterium to break," he says. He adds that the two methods he had been using broke only 40 percent of the cells in his samples over repeated cycles, compared with 60 percent or more in a single pass with BioNeb.
And Wendy Boss, a professor of botany at North Carolina State University in Raleigh, uses BioNeb to open carrot cells gently enough so that she can ease out and collect their nuclei.
Surzycki points out another major advantage of this gentle approach--it is time-independent. If a user runs it a bit too long, the sample isn't ruined. With other systems, the longer you operate, the more you destroy. The device is also cool and very fast, handling a liter of cells in two to three minutes.
"It uses low pressure, so hazards are much less," says Raymond Rickert, president of Glas-Col Apparatus Co. in Terre Haute, Ind., which is entering the biotechnology market with the BioNeb Cell Disruption System. "You can turn it over to an assistant without worrying about blowing up the lab."
Ricki Lewis is a freelance science writer based in Scotia, N.Y.
Ricki Lewis is a freelance science writer based in Scotia, N.Y.