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Homogenization Technology Overview

 

Manual Methods - Mortar & Pestle and Dounce homogenizers:

Manual methods of homogenization use mechanical force applied by hand in order to crush tissue and cells. The most common form of homogenizers are mortar and pestle homogenizers, in which a hard, blunt object (the pestle) is pressed against the container holding the sample (the mortar). In most cases, the pestle is a small plastic object and the microcentrifuge tube or other tube containing the sample acts as the mortar. Plastic mortars are very inexpensive, however because the entire homogenization is performed by hand, human error is high, reproducibility is low, homogenization effectiveness is very poor, and repeated homogenization becomes very tedious and is very slow. The use of battery-operated pestle holders, which spin the pestle to produce additional shear force on the samples and assist in homogenization, make the method slightly less tedious and slightly more effective.A slight improvement on this method is the dounce homogenizer. In a dounce system, the mortar and pestle come bundled and are specially crafted for use with each other. This ensures a tighter fit and improves homogenization efficiency, however cross-contamination becomes an issue since the dounce must be thoroughly washed between each sample. Buying multiple dounces is an option, however quickly becomes expensive. Furthermore, a dounce system is still slow, tedious, and highly irreproducible due to human error.

Rotor-Stator homogenizers:

Also known as mechanical shear homogenizers, these units use shear force, usually produced by spinning blades, to homogenize samples. Laboratory blenders that are similar in design to the blenders one would have in their kitchen do exist, however they are not practical for most applications and represent a small minority of rotor-stator homogenizers. The vast majority of rotor-stator homogenizers are probe-style instruments which contain smaller motor-driven blades (the "knife") at the end of a rotor shaft. Liquid enters the rotor-stator and the knife creates a shear force to disrupt the samples. These types of instruments are far more powerful than manual homogenization, and can be used with cells in suspension. Various models of varying probe size and power are designed to handle different sizes and toughness of samples. Metal probes require cleaning; however some units have plastic probes which are disposable. Some are hand-held, but many can be mounted for hands-free use, helping to reduce human error somewhat. Fully automated systems are available which largely eliminate human error but these are often an expensive solution to single-sample homogenization, costing upwards of $5000 or more. Automated systems which can homogenize multiple samples also exist, however these are very expensive, often $10000 or more. Within a reasonable budget, one is usually limited to single-sample systems. Due to the rapid mechanical shearing action inside the samples, a significant amount of heat can build up with extended use. Since this heat is generated within the sample itself, it is not well controlled by cooling methods. Lastly, because the sample container needs to be open and the rotor stator creates rapid blending action, operation of a rotor-stator can become messy and aerosols may be a problem if working with hazardous substances.

Ultrasonic homogenizers:

Ultrasonic homogenizers, or sonicators, use an electronic generator to send waves of high-frequency mechanical energy through a transducer to a "horn", which then rapidly vibrates. This vibration creates a rapid forming and collapsing of small bubbles within the system, a process known as cavitation. Similar to rotor-stators, most sonicators are designed to homogenize one sample at a time, and are available at a wide range of powers and sizes for different sized samples. Automated systems are also available for sonication, with some high-end high-throughput systems reaching well into the tens of thousands of dollars. Simple, lower-powered systems are usually available for under $1000, however these systems are not suitable for rapid homogenization of multiple samples. Most often used for cell culture, sonicators are also not well suited for tearing through tough tissue, as the rapid cavitation simple cannot provide enough force, nor is it optimal for large amounts of any tissue. Sonicators are, however, the best homogenization devices for breaking apart small subcellular organelles. The main drawback of sonication is the thorough denaturation of protein due to cavitation, and the addition of a large amount of heat, making high-power sonicators a poor choice for temperature-sensitive assays including most RNA work. Sonicators also use expensive probes that must be polished and carefully cared for to prevent erosion or pitting, which can greatly decrease the effectiveness of the sonicator.

High-pressure homogenizers:

High-pressure homogenization, where a press is used to lyse cells, is an old method applied mostly to single-celled, non-filamentous microorganisms. In pressure homogenizers, such as the once popular but discontinued French Pressure Cell ("French Press"), cell suspensions are forced through small channels under high pressure. This creates a large pressure drop and strong shearing forces to lyse cells. Lysis is effected by the pressure, flow rate, and temperature of the system, and a recycling flow is often implemented to subject the suspension to the pressure drop and shear force multiple times. Steel plates or rings placed near the low-pressure exit of the channels, upon which the cells collide, is sometimes implemented and also aids in disruption. High-pressure homogenizers exist which can process many thousands of liters of cell suspensions per hour, and their throughput for the homogenization of single-celled organisms is unmatched. These systems are generally unable to homogenize tissue without prior dissociation, and attempting to do so will merely clog the device. High-pressure homogenization also generates a significant amount of heat, usually requiring pre-cooling of both the machine and the sample to mitigate. Most systems have also been frequently criticized for being messy to use, and all require thorough cleaning after use both to keep the unit sterile and to prevent cross-contamination. The systems are expensive, usually well over $10,000, and are also large and extremely heavy. For these reasons, researchers are moving away from pressure cell homogenization in favor of other methods. Some, however, remain loyal due to the excellent cell lysis capabilities.

Bead Mill homogenizers:

Bead mill homogenizers use the rapid agitation of beads to tear through tissue and lyse cells. The homogenization beads tear through tissue to dissociate it and collide to create high-energy areas which lyse cells. Many bead-mill systems are high-throughput and can handle tough tissue about as well as a rotor-stator. Bead-mill systems are hands-free and automated, and most can homogenize a large amount of samples in a few minutes or less. A distinct advantage of a bead-mill system is afforded by the use of different sizes of beads. Larger beads are less efficient at disrupting small particles, while smaller beads are very efficient. By varying the bead size and thoroughness of the homogenization (via alteration of the time and speed settings), one can homogenize small cells, nuclei and other subcellular organelles, and other small particles, or leave organelles or even whole cells intact. This makes bead mills far more versatile than other systems. Because the systems are fully autoamted and the samples remain contained, there is extremely high reproducibility and virtually no risk of cross-contamination.The drawback to most bead mill systems is the tradeoff between power and price - most cheaper systems cannot handle tougher samples, and most systems capable of homogenizing tough tissue are prohibitively expensive. Furthermore, most bead mill systems require a lot of power and therefore generate a lot of heat, requiring liquid or dry ice cooling. However, all of these drawbacks are addressed in the Bullet Blender®.

What sets the Bullet Blender® apart:

The Bullet Blender® uses a patented, simple yet powerful striking technology in which "bullets" spin around on spokes and rapidly strike the tubes to agitate the homogenization beads. This hub-and-spoke system is far more efficient than the traditional shaking system, allowing us to use a smaller motor and therefore generate far less heat while still being able to homogenize tough samples, such as muscle, carcinoma, and roots. The heat generated is so small that even with repeated operation our simple Air Cooling™ system is enough to keep the samples within a just few degrees of the ambient temperature (and the Bullet Blender can be used in a 4°C room). Also, because the device has just one moving system it is far more reliable than other devices - the less components a system has, the less of a chance there is that something will break. Furthermore, because using a Bullet Blender allows you to homogenize in standard tubes, the cost of operating the Bullet Blender is uniquely low. This, combined with a price 3-4x lower than other high-performance, high-throughput bead mills, creates a unique value that allows researchers access to top-quality performance at a reasonable price.

 

 

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