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Laboratory Cell Disrupters
A review covering apparatus and techniques of cell disruption


Practical aspects of mechanical cell disruption are discussed. Sources and approximate prices of equipment are given. A more extensive review of the subject by the author, Tim Hopkins, is available in Purification and Analysis of Recombinant Proteins, Seetharam and Sharma, editors, published by Marcel Dekker, Inc (New York)1991.

BEAD MILL HOMOGENIZERS
In bead milling, a large number of minute glass beads are vigorously agitated by shaking or stirring. Disruption occurs by the crushing action of the glass beads as they collide with the cells. The method has been used for years to disrupt microorganisms and works successfully with tough-to-disrupt cells like cyanobacteria, yeast, spores and microalgae where other techniques have failed. It is considered the method of choice for disruption for yeast and fungi. More recently, bead mill homogenization has been applied to plant and animal tissue. The size of the glass beads is important. Optimal size for bacteria and spores is 0.1 mm , 0.5 mm for yeast, mycelia, microalgae, and unicellular animal cells such as leucocytes or trypsinized tissue culture cells and 1.0 or 2.5 mm for tissues such as brain, muscle, leaves and skin. The speed of disruption is increased about fifty percent by using like-sized ceramic beads made of zirconia-silica rather than glass (Hopkins, unpublished observations), presumably because of their greater density. The loading of the beads should be at least 50% of the total liquid-biomass volume but can be up to 90%, provided adequate agitation of the bead slurry is still possible. Generally, the higher the volume ratio of beads to cell suspension, the faster the rate of cell disruption. After treatment, the beads settle in seconds by gravity and the cell extract is easily removed.

Microorganisms can be disrupted manually by this technique. A cell suspension is added to the beads and the mixture is agitated at top speed on a vortex mixer for several minutes. The hand-held method is slow and tedious. Therefore, electromechanical bead mill agitators have been developed. Bead agitation by these devices is either by shaking or stirring.

Shaking-type Bead Mills. Most shaking-type bead mills are restricted to sample sizes of 3.0 mL or less. Heat generation is a concern in larger shaking bead mills and effective cooling is difficult to achieve. For example, bead milling of a 250 mL sample without external cooling increases the temperature l0 degrees per minute of homogenization. The German made Braun MSK (B. Braun Biotech, Bethlehem, PA) disrupts samples sizes up to 40 mL but relies on somewhat unreliable cooling with liquid CO2 to keep the sample at acceptable temperatures. Smaller volume shaking bead mills have smaller breakage chambers with surface area to volume ratios high enough to permit adequate heat dissipation without external cooling. Four such commercial devices are the Mini-BeadBeater (BioSpec Products, Bartlesville, OK), the Micro-Dismembrator II (B. Braun Biotech, Bethlehem, PA), Retsch Mixer (Brinkmann, Westbury, NY) and the FastPrep (Bio 101, Vista, CA). Disruption of microorganisms takes about 1-5 minutes. BioSpec Products manufactures two machines, one which holds a single 2 mL screw-cap microvial and the other capable of disrupting eight sample vials at a time. BioSpec Products has a machine capable of holding 96-well assay blocks under development. The price of shaking bead mills range from $600 to $5000.
Rotor-type Bead Mills. Larger capacity laboratory bead mill cell disrupters agitate the beads with a rotor rather than by shaking. Equipped with efficient cooling jackets, larger sample volumes can be processed without overheating. The most widely used is the Bead-Beater (BioSpec Products, Bartlesville, OK). This unit will disrupt about 250 mL or, with smaller chamber attachments, 50 or 15 mL batches of cell suspension in 3-5 minutes. Cell concentrations as high as forty percent (wet wt) can be used. VirTis Company (Gardiner, NY) offers an attachment for its line of high speed rotary homogenizers which efficiently agitate glass beads in a special test-tube or fluted flask. Complete homogenizer units cost about $500 and $800, respectively.
While the above small volume cell disrupters are used mostly for the disruption of microorganisms, they can also be used to homogenize and extract plant and animal tissue. This newer application is suitable for both soft tissue and tough or fibrous samples such as skin, tendon or leaves. Extraction yields of biomolecules such as nucleic acids, viruses, and receptor complexes are often superior to that of other methods. For nucleic acid isolation, consider disrupting the cells directly in the nucleic extraction solution (phenol, guanidinium SCN, etc). Nuclease concerns will be eliminated and yields enhanced. And, if PCR techniques are being used, shaking-type bead mills which use disposable micro-vials totally eliminate cross contamination between samples. Selective homogenization is sometimes possible using different bead sizes or speeds of bead agitation. For example, it is possible to selectively disrupt only the epidermal layer of whole leaves or to obtain intact subcellular organelles by using smaller charges of beads and/or shorter disruption times.
ROTOR-STATOR HOMOGENIZERS (also called colloid mills or Willems homogenizers) are well suited for plant and animal tissue and generally outperform cutting-blade type Benders. Compared to a blender, foaming, swirling and aeration are minimized and smaller sample volumes are accommodated. Most tissues are quickly and thoroughly homogenized with the apparatus. The cellular material is drawn into the apparatus by a rotor sited within a static tube or stator. The material is then centrifugally thrown outward to exit through slots or holes on the tip of the stator. Because the rotor is turning at very high speed, the tissue is rapidly reduced in size by a combination of turbulence and scissor-like mechanical shearing occurring within the gap of the rotor and stator. Since most rotor-stator homogenizers have an open configuration, the product is repeatedly recycled. The process is quite fast and, depending upon the toughness of the tissue sample, desired results are usually be obtained in 10-60 seconds. For the recovery of intracellular organelles or receptor site complexes, shorter times and/or reduced rotor speeds are used. The sample size prior to processing with the homogenizer must be small enough to be drawn inside the hole at the tip of the stator. Therefore, samples often must be pre-chopped or - fragmented with a scissors, single-edge razor blade or cryopulverizer (a device that quickly powders tissue at liquid nitrogen temperatures - see below). Unlike many other types of cell disrupters, rotor-stators homogenizers generate negligible heat during operation.
Most laboratory rotor-stator homogenizers are top driven with a compact, high speed electric motor which turns at 8,000 to 60,000 rpm. The size of the rotor-stator probe (also called the generator) can vary from the diameter of a drinking straw for 0.5-50 mL sample volumes to much larger units handling 10 liters or more. There is an important relationship between rotor speed and stator diameter. In principle, the top rotor speed of the homogenizer should double for each halving of the rotor diameter. It is not rpm per se but the tip velocity of the rotor that is the important operating parameter. Ten to twenty meters per second (2000 to 4000 fpm) are acceptable tip speeds for tissue disruption. Unfortunately, some of the smaller-sized commercial rotor-stator homogenizers do not meet this standard. Other factors such as rotor-stator design (there are many), materials used in its construction and ease of cleaning are also important factors to consider in selecting a rotor-stator homogenizer. Some manufactures are BioSpec Products (Bartlesville, OK), Brinkmann Instruments (Westbury, NY), Charles Ross & Son Company (Hauppauge, NY), Craven Laboratories (Austin, TX), IKA Works (Cincinnati, OH), Omni International (Gainsville, VA), Pro Scientific (Monroe, CT), Silverson Machines (Bay Village, OH), and VirTis Company (Gardiner, NY). The cost of complete units (motor plus rotor-stator head or generator) range from $600 to $5000.

Most laboratory sized homogenizers function properly only with liquid samples in the low to medium viscosity range (<10,000 cps). The speed and efficiency of homogenization is greatly degraded by using too small a unit, and the volume range over which a given homogenizer rotor-stator size will function efficiently is only about ten fold. Foaming and aerosols can be a problem with rotor-stator homogenizers. Keeping the tip of the homogenizer well submerged in the media and the use of properly sized vessels helps with the first problem. Square shaped homogenization vessels give better results than round vessels and it is also beneficial to hold the immersed tip off center. Aerosols can be minimized by using properly covered vessels (VirTis, Brinkmann and Omni). Even though a number of the laboratory rotor-stator homogenizers use fully enclosed motors, none of them are truly explosion-proof. Therefor, due caution should be followed when using flammable organic solvents by conducting the homogenization in a well ventilated hood.

Bottom-driven laboratory rotor-stator homogenizers are a new entry to the laboratory. The rotor-stator assembly is usually placed within a sealed chamber or container, fits blender motor bases and have working volumes of 100-1000 mL. They costs about $250 - $400 and are available from BioSpec Products (Bartlesville, OK) and Eberbach Corporation (Ann Arbor, MI).

Closely related homogenizers, called dispersers, are used for preparing large volumes of crude plant and animal aqueous extract. Operating like a household garbage disposal unit, the rotor-stator mechanism quickly homogenizes and liquefies kilogram quantities of biomass. The sample is suspended in one or more liters of media, loaded into a top reservoir and homogenized either in a continuous or batch mode. Costing $600 to $7000, two manufacturers are BioSpec Products and IKA Works.

BLADE HOMOGENIZERS. Although less efficient than rotor-stator homogenizers and aeration and foaming can be a problem, blade homogenizers (commonly called blenders) have been used for many years to produce fine brie and extracts from plant and animal tissue. Blenders are not suitable for disruption of microorganisms. In this class of homogenizer a set of stainless steel cutting blades rotate at speeds of 6,000-50,000 rpm inside a glass, plastic or stainless steel container. The blades are either bottom- or top-driven. Some of the higher speed homogenizers can reduce tissue samples to a consistent particulate size with distributions as small as 4 microns, as determined by flow cytometric analysis. After blending, some plant tissue homogenates undergo enzymatic browning - a oxidation and cross-linking process which can complicate subsequent separation procedures. Enzymatic browning is minimized by carrying out the extraction in the absence of oxygen or in the presence of oxygen scavenging thiol compounds such as mercaptoethanol. Sometimes, addition of polyethylene imine, metal chelators, or certain detergents such as Triton X-100 or Tween 80 also help.

When using a blender, use caution when blending with flammable solvents such as alcohol or acetone or when homogenizing diseased tissues. Blenders use brush motors to achieve their high speeds and, therefor, spark during operation. Also aerosols are readily formed while blending. Use a sealed blender container and operate it in a well ventilated hood. Blade homogenizers can process liquid sample sizes from 2 mL to one gallon. Accessories for blenders include cooling jackets for temperature control, closed containers to minimize aerosol formation and entrapment of air, special vessels made of stainless steel, semi-micro containers and even insulated vessels for use with cryogenic solvents (see Freeze fracturing). Manufactures of a scientific line of blenders include British Medical Enterprises (London, England), ESGE (Basel, Switzerland), Hamilton Beach Commercial (Washington, NC), Omni International (Waterbury, CT), Professional Diagnostic (Edmonton, Alberta Canada), The VirTis Company (Gardiner, NY) and Waring Products Division (New Hartford, CT). Accessory vessels for Hamilton-Beach brand blenders are manufactured by BioSpec Products (Bartlesville, OK) and for Waring brand blenders by Eberbach Corporation (Ann Arbor, MI). Prices for blade homogenizers range from about $100 to $2000.
FREEZE-FRACTURING. Both microbial pastes and plant and animal tissue can be frozen in liquid nitrogen and then ground with a common mortar and pestle at the same low temperature. Presumably the hard frozen cells are fractured under the mortar because of their brittle nature. Also, ice crystals at these low temperatures may act as an abrasive.

A freeze-fracturing device called the Bessman tissue pulverizer is useful for fragmenting 10 mg to 10 g quantities of fibrous tissue such as skin or cartilage to the size of grains of salt. This material is then easily homogenized by other methods. Looking somewhat like a tablet press, the pulverizer consists of a hole machined into a stainless steel base into which fits a piston. The base and piston are pre-cooled to liquid nitrogen temperatures. Ten mg to ten grams of hard frozen animal or plant tissue is placed in the hole. The piston is placed in the hole and given a sharp blow with a hammer. The resulting frozen, powder-like material can be further processed by Pestle and Tube, Bead Mill or Rotor-stator homogenizers. Manufactured by BioSpec Products (Bartlesville, OK) and by Spectrum Medical Industries (Carson, CA), the pulverizers come in several sizes and cost $200 to $400. In larger capacity models, BioSpec Products (Bartlesville, OK) has incorporated a built-in, spring loaded hammer looking much like a staple gun. This same company also makes a small screw press designed to pulverize 50-500 mg of hard frozen tissue. This pulverization tool works slower than the Bessman device (perhaps a minute rather than a few seconds per sample), but it is well suited for fresh bone and other hard tissue.

GRINDERS. Grinding biological material in a mortar or tube with fine sand, alumina or glass powder is roughly the equivalent of bead-milling (see Part 1). The method works reasonably well with all types of biomass but is strictly small scale and is labor intensive. Cell pastes or solid mass with a minimum volume of buffer are mixed with 0.5-1 volume of grinding media and ground with a mortar and pestle. Disruption efficiency is poor if lower cell densities or smaller charges of grinding media are used. Glass powder, having a high surface area, may adsorb significant amounts of charged biomolecules such as nucleic acids and proteins.

PESTLE AND TUBE HOMOGENIZERS (also called tissue grinders) are used to disrupt animal tissue. While variations of the pestle and tube homogenizer have names like Potter, Potter-Elvehjem, Dounce, and Ten Broeck, as a group they consist of test-tubes made of glass, inert plastic or stainless steel into which is inserted a tight-fitting pestle (clearance about 0.1-0.2 mm) made of like material. The walls of the test-tube and pestle can be smooth or have a ground finish. Most tissues must be cut or chopped into small pieces (1-5 mm) with scissors or a single-edge razor blade before being suspended in a 4-10 fold volume excess of medium in the test-tube. The pestle is manually worked to the bottom of the tube, thus tearing and fragmenting tissue as it is forced to pass between the sides of the pestle and the wall of the tube. The grinding action occurs again as the pestle is withdrawn. Five to thirty repetitions of this low shear method homogenizes the tissue. Rotation of the pestle at about 500-1000 rpm with an electric motor while the test-tube is manually raised and lowered speeds up the process. While pestle and tube homogenization is simple and the equipment used is usually inexpensive, it is both labor intensive and, in the case of fragile glass homogenizers, potentially dangerous. Even so, this homogenizer will continue to be popular because of its extremely gentle action. Often it is the method of choice for the preparation of small quantities of subcellular organelles from soft animal tissues such as brain or liver. Microorganisms cannot be disrupted with pestle homogenizers.

Commercially available glass or plastic pestle homogenizers with batch capacities of 0.1-50 mL generally cost $15-$100 and are available from many manufacturers including Ace Glass (Vineland, NJ), Bellco Glass (Vineland, NJ), BioSpec Products (Bartlesville, OK), Kontes (Vineland, NJ), Thomas Scientific (Swedesboro, NJ), Tri-R Instruments (Rockville Center, NY), Sage Products (Crystal Lake, IL) and Wheaton Industries (Milville, NJ). Disposable, plastic pestles which fit into microcentrifuge tubes are available from Kontes. They also offer a small, hand-held motor unit to drive the pestle. While precision stainless steel tissue grinders are more expensive ($200 - $250, BioSpec Products and Wheaton), they are efficiently cooled and tolerate vigorous homogenization without risk of breakage. A 'Rolls-Royce' homogenizer costing about $3000 has a variable speed motor, cooling jacket, and hand- operated lever to rise and lower the pestle (B. Braun Biotech Bethlehem, PA). A continuous pestle homogenizer is available from Yamato USA (Northbrook, IL). Grooves machined on the upper one-third of the pestle catch and guide tissue through the close tolerance region of the lower two-thirds of the cylinder pestle. The resultant homogenate exits from the bottom of the cylinder. Recycling is usually necessary. The machine comes in two sizes and costs $2000-$3000.

MEAT MINCER AND SOLIDS PRESS. The household meat grinder or mincer has been used for many years for the preparation of animal tissue extracts. Tissue is mechanically pressed through holes in a metal sieve plate while rotating blades slowly sweep across the face of the plate cutting the meat in 0.3-0.5 mm fragments. While it is not an effective way to disrupt cells per se , it is useful as a preliminary step for complete homogenization using other physical or chemical methods. Meat grinders cut flexible tissue like muscle better if the tissue is processed slightly frozen.
For small tissue samples, BioSpec Products (Bartlesville, OK) manufacture hand operated screw presses for the preparation of tissue extracts as does EDCO Scientific (Chapel Hill, NC). Capable of considerable force, sample sizes from 0.1 grams up to 50 grams of soft tissue are pushed through sieve plates having 0.5 to 3 mm holes, much like the action of a kitchen garlic press. Hard or fibrous tissues like tendon, skin, leaves and seeds will not pass through the press. The units cost from $25 to $400. Fred S. Carver (Wabash, IN) has a compact hydraulic laboratory tissue press for the extraction of intracellular liquids and oils for about $1600. Designed for plant leaves, Bioreba (Chapel Hill, NC) makes a hand-held grinder consisting of an circular array of steel balls which crush a few leaves inside a mylar plastic bag. The grinder and bags cost about $200.

ULTRASONIC DISINTEGRATORS are widely used to disrupt cells. These devises generate intense sonic pressure waves in liquid media. Under the right conditions, the pressure waves cause formation of microbubbles which grown and collapse violently. Called cavitation, the implosion generates a shock wave with enough energy to break cell membranes and even break covalent bonds.
Modern ultrasonic processors use piezoelectric generators made of lead zirconate titanate crystals. The vibrations are transmitted down a titanium metal horn or probe tuned to make the processor unit resonate at 15-25 kHz. The rated power output of ultrasonic processors vary from 10 to 375 Watts. What really counts is the power density at the probe tip. Higher output power is required to sustain good performance in large sized probes. For cell disruption, probe densities should be at least 100 W/cm2 and the larger the better for tip amplitude (typical range: 30-250 microns). Some manufacturers of ultrasonic disintegrators are Artek Systems (Farmington, NY), BioSpec Products (Bartlesville, OK), Branson Sonic Power Company (Danbury, CT), B. Braun Biotech (Bethlehem, PA), RIA Research Corp. (Hauppauge, NY), Sonic Systems (Newton, PA) and VirTis Company (Gardiner, NY). A new development is a cordless ultrasonic processor supplied by BioSpec Products (Bartlesville, OK). With a tip diameter of 1/8 inch, it easily fits into microtubes and 96 well titer plates.

Ultrasonic disintegrators generate considerable heat during processing. For this reason the sample should be kept ice cold if possible. For microorganisms the addition of 0.1 -0.5 mm diameter glass beads in a ratio of one volume beads to two volumes liquid is recommended. Tough tissues like skin or tendon should be macerated first in a tissue press or grinder or pulverized in liquid nitrogen (see above). Use small vessels during ultrasonic treatment and place the probe tip deep enough in the sample to avoid foaming. Finally, one should be aware that free radicals can be generated during sonication and that these radicals can react with most biomolecules. Damage by oxidative free radicals can be minimized by including scavengers like cysteine, dithiothreitol or other -SH compounds in the media.
Other cell disruption apparatus or techniques not covered in this review but discussed in detail by the author in the original book are High-Pressure Homogenizers, Autolysis, Enzymatic lysis, Dehydration, Chemical lysis, Solvent lysis and Programmed self-distruction.