Focus on Cell Disruption
Renewed interest in cell biology has created the need for techniques that efficiently disrupt cells’ outer membranes while sparing their contents.
Chemical and mechanical methods are the two general approaches to cell lysis, with numerous methods within those categories. “The choice of method depends on what you’re trying to get out of the cell,” says Tim Hopkins, PhD, CEO of BioSpec Products (Bartlesville, OK).
Bio-Rad Laboratories (Hercules, CA) classifies cell disruption methods as gentle or harsh. The former include osmotic lysis (suspending cells in high-salt solutions), freeze-thawing, detergent, and enzymatic methods. Because these techniques are slow and relatively inefficient, they provide some protection against overprocessing when analyzing for sensitive molecules or organelles. Harsher techniques include sonication, French press, grinding, glass-bead homogenization (beadbeating), and blending-type mechanical homogenization.
Chemical lysis, which is sometimes combined with mechanical disruption, employs chaotropic agents, detergents, and enzymes. Most are available as kits. Chemical lysis is gentle but unpredictable, as disruption conditions and durations may vary significantly among samples. Mechanical systems involve an up-front investment, while kits represent a recurring expense.
Literature from Hielscher USA (Ringwood, NJ), which specializes in ultrasonic homogenization, suggests that chemical lysis can alter protein structure and introduce purification problems, while enzymatic disruption requires long incubation times and is not reproducible.
Mechanical techniques
Manual cell disruption techniques have been used for decades. Although they are still preferred by some labs, they are low tech and mostly unautomated compared with more modern mechanical methods. One method involves freezing cell suspensions and grinding the mass with a mortar and pestle. Freeze-grinding is slow, messy, and difficult to scale. Other methods squeeze cells between a pestle and the sample vessel wall, or through tiny holes under high pressure (French press). These methods do not generate significant heat but are slow and laborintensive. All manual-mechanical techniques are unsuited to high-throughput operation.
Ultrasonic probes, rotor-stator homogenizers and beadbeaters are more modern—and popular— homogenization methods for cell work.
Ultrasonic probes operating at 20,000 cycles per second cause cavitation in aqueous solutions— microscopic areas of vacuum-like pressures and high temperatures that tear cells apart. Although temperatures may reach several thousand degrees Celsius, cavitation volumes are so small they do not heat the process significantly.
Assuming proper matching between sample and homogenizer, cell lysis by sonication takes between a few seconds and two minutes. Since sonication energy is user-defined on both handheld and benchtop sonicators, methods may be adjusted depending on the cell and end product. For example, DNA extraction requires “softer” disruption, while protein preparation from bacteria demands more rigorous sonication.
Hielscher suggests using short sonication burst cycles of up to 15 seconds on cooled samples to allow heat to dissipate without untoward effects on analytes.
Beadbeating, a technique perfected by Hopkins at BioSpec, shakes or vortexes cell samples sealed within microvials or microplate wells containing a large number of small spherical beads (0.1 to 6 mm in diameter) and usually, but not always, a lysis solution. The high-intensity wet-grinding process causes permeation of cell membranes or walls in less than three minutes. Beadbeating is well suited for high-throughput processing and also works well for small, intact tissue samples.
Cool operation
Laboratories using mechanical disruption and targeting proteins or intracellular organelles must pay close attention to operating temperature. “Ideally, you should keep samples ice cold during processing,” Hopkins advises, “but realistically you’ll probably be fine if temperatures do not rise above culture or tissue source temperature.”
Despite best efforts to keep samples cold, endogenous enzymes can destroy proteins, particularly but not limited to the time between lysis and downstream processing when cell homeostasis no longer exists but enzymatic activity persists. Bio-Rad provides the following advice on avoiding enzymatic degradation:
- Lyse cells in strongly denaturing buffers (urea, thiourea, detergent).
- Operate above pH 9, where protease activity is minimized, using sodium carbonate or Tris as the buffering agent.
- Consider adding a chemical protease inhibitor to the lysis buffer. For best results, use a combination of inhibitors in a protease inhibitor cocktail.
- When studying protein phosphorylation, include phosphatase inhibitors such as okadaic acid, calyculin A and vanadate.
- After cell disruption, check the efficiency of cell disruption by light microscopy and centrifuge all extracts (20,000 × g for 15 minutes at 15°C) to remove insoluble materials.
Tips for effective disruption
Another major mechanical disruption technique uses rotorstator homogenizers. The handheld instruments work by repeatedly forcing samples through open slits or holes on the distal end of a static tube by a rotor turning inside the tube at 30,000 rpm. Rotor-stator homogenizers work rapidly and generate very little heat. On the negative side, it is not a high-throughput method and may work poorly with certain monocellular organisms.
Sample disruption is a necessary early step in the isolation of RNA, DNA, proteins, and organelles from cells and tissue. Rotor-stator homogenizers are widely used for this purpose because they mechanically disrupt cells while sparing macromolecules from degradation.
“Homogenization methods should always be tailored to the cell or tissue type,” says Holly Yacko Archibald, director of sales at PRO Scientific (Oxford, CT). Animal and plant tissues require more rigorous initial disruption methods, which may be followed by less mechanically strenuous cell disruption. Many cultured cells, Archibald adds, are easily lysed by simply vortexing their suspension in the presence of a suitable homogenization reagent.
Archibald advises purchasers of homogenizers for cell disruption to consider vessel and probe size, variable speed control, and crosscontamination avoidance.
Probe/vessel dimensions determine whether the probe will fit into the sample container and/or supply sufficient energy for complete homogenization. Variable speed control permits operators to ramp up from low to high power and back again to avoid creating pockets of sample inhomogeneity, and it is especially suited to organelle preparation.
Cross-contamination is more difficult to control with rotorstator homogenizers than, say, with pipettors that universally use disposable tips. Cross-contamination results in the carryover of analytes from one sample to another; for example, the homogenate of treated versus untreated cells. Multiprobe homogenizers are one solution that provides multiplicity and automation but adds cost. The alternatives are single-use probes or the old-fashioned method of cleaning and sterilizing probes between runs.
For additional resources on homogenizers, including useful articles and a list of manufacturers, visit www.labmanager.com/homogenizers