Cell Disruption by Nitrogen Decompression A rapid and effective way to: of heating it. In addition, the entire cycle can be conducted at low • Homogenize cells and tissues temperature by pre-chilling or by operating the vessel in an ice • Release intact organelles bath. The vessel can also be filled with ice to keep the sample • Prepare cell membranes cool during the processing period. • Release labile biochemicals There is no oxidation. The blanket of inert nitrogen gas • Produce uniform and repeatable homogenates which saturates the cell suspension and the homogenate offers excellent protection against oxidation of any labile cell compo- without subjecting the sample to extreme nents. Although other gases: carbon dioxide, nitrous oxide, chemical or physical stress. carbon monoxide and compressed air have been used in this technique, nitrogen is preferred because of its non-reactive A Widely Accepted Method nature and because it does not alter the pH of the suspending Cell disruption by rapid decompression from a pressure vessel medium. In addition, nitrogen is preferred because it is generally has been used for many years by investigators who wanted available at low cost and at pressures suitable for this procedure. to overcome the limitations imposed by other cell disruption Any suspending medium can be used. The suspending procedures. Although the technique is not new, interest in the medium can be chosen for its compatibility with the end use of decompression method and many new applications for it have the homogenate and without regard for its adaptability to the grown rapidly following the introduction of convenient pressure disruptive process. This offers great flexibility in the preparation equipment such as the Parr Cell Disruption Vessel. of cell suspensions and produces a clean homogenate which does not require intermediate treatment to remove contaminates Many Applications which might be introduced when using other disruption methods. The nitrogen decompression method is particularly well suited Each cell is exposed only once. Once released, subcellular for treating mammalian and other membrane-bound cells. It has substances are not exposed to continued attrition which might also been used successfully for treating plant cells, for releasing denature the sample or produce unwanted damage. There is no virus from fertilized eggs and for treating fragile bacteria. It is not need to watch for a peak between enzyme activity and percent recommended for untreated bacterial cells, but this restriction can disruption. be eliminated by using various pretreatment procedures (p. 7) to The product is uniform. Since nitrogen bubbles are weaken the cell wall. Yeast, fungus, spores and other materials generated within each cell, the same disruptive force is applied with tough walls do not respond well to this method. uniformly throughout the sample, thus ensuring unusual uni- formity in the product. Cell-free homogenates can be produced. How It Works The principle of the method is quite simple. Large quantities It’s Easy To Apply of nitrogen are first dissolved in the cell under high pressure Use any sample size. Cell disruption by this method is inde- within a suitable pressure vessel. Then, when the gas pressure pendent of sample size or concentration. Any size sample from a is suddenly released, the nitrogen comes out of the solution as few cc’s to five hundred can be treated equally well in a Parr Cell expanding bubbles which stretch the membranes of each cell Disruption Vessel with excellent recovery of the starting material. until they rupture and release the contents of the cell. In addition, a wide variety of materials can be treated with the opportunity for scale-up work where labile cell components or Why It Is So Effective organelles are involved. It’s a gentle method. Although sometimes referred to as Easy to control. The degree of cell fractionization is easily “explosive decompression,” nitrogen decompression is actually controlled by adjusting the nitrogen pressure. High pressures a gentle method for homogenizing or fractionating cells since which dissolve large quantities of nitrogen within the cell usually the chemical and physical stresses which it imposes upon the produce total homogenization. Or, moderate pressures can sub-cellular components are held to an absolute minimum. It is be employed to reduce the disruptive forces and thus release much more protective of delicate enzymes and organelles than nuclei, active mitochondria and other organelles intact. Operating ultrasonic and mechanical homogenizing methods. In fact, it conditions can also be adjusted to homogenize suspensions of compares favorably to the controlled disruptive action obtained subcellular components such as nuclei and mitochondria that are in a Teflon and glass mortar and pestle homogenizer, but it does normally difficult to disrupt because of their small size. the job faster and more uniformly, with the added ability to treat Special skills are not required. The few simple steps large samples quickly and conveniently. required to operate the Parr vessel are easily learned. After oper- There is no heat damage. While other disruptive methods ating conditions have been established, uniform and repeatable depend upon friction or a mechanical shearing action which results can be obtained from run to run, usually within less than generates heat, the nitrogen decompression procedure is accom- twenty minutes – even with large samples. panied by an adiabatic expansion which cools the sample instead www.parrinst.com 2 Parr Instrument Company Cell Disruption Vessels Cell Disruption Vessels Cell Disruption Vessels in Five Convenient Sizes arr stainless steel vessels for processing cell suspensions by the nitrogen decompression Pmethod are made in several sizes with full opening heads and self-sealing closures which can be handled easily on any laboratory bench without special tools or fixtures. Each vessel has two valves and a pressure gage: one valve for charging with nitrogen and the other for withdrawing the homogenate and discharging it through an attached delivery tube. All of the fittings as well as the vessel itself are made of stainless steel with polished surfaces for good corrosion resistance and freedom from contamination. The individual parts are easily cleaned and can be thoroughly sterilized. Designs For Large and Small Samples These vessels are made in five different sizes to accommodate samples ranging from 0.5 mL to 5 liters, as listed in the table on page 4. The maximum charging capacity for each vessel is limited to two-thirds of the internal volume of the vessel, but smaller samples can be treated in any of these vessels by simply placing the sample in a beaker or test tube and positioning it under the dip tube within the vessel. The 4635, 920 mL vessel is Model 4635 and Model 4639 Parr Cell Disruption Vessels the general purpose model with a capacity for treating samples ranging in size from a few milli- liters up to 600 mL. The 4639, 45 mL vessel is designed specifically for small samples from 30 mL down to less than one milliliter. It has a tapered bottom which drains directly into the discharge valve, ensuring complete recovery of all of the sample. The larger vessels, 4636, 4637, and 4638, are intended for large volume applications. Users are urged to run preliminary experiments in the 4635 Vessel to confirm the suitability of the procedure before scaling up to these larger sizes. Model 4635 Model 4639 1-800-872-7720 Parr Instrument Company Bulletin 4635 3 Cell Disruption Vessels Easy Access To Vessel Chamber Custom Modifications Each of these vessels, except the small 45 mL size, has a split- Most cell disruption procedures can be handled readily in the ring closure – an exclusive Parr design which allows the vessel to 920 mL, 4635 Vessel with its customary fittings and attachments, be opened or closed easily without disturbing any of the fittings yet applications may arise in which it will be desirable to modify or connecting lines attached to the head. In this closure two ring a vessel to meet special requirements. If initial studies show a sections slide into place from the sides of the vessel to lock the need for modifications, the user can return any vessel to the head in position while a self-sealing O-ring maintains a tight seal factory for such changes or additions as may be needed. at all pressures. The ring sections are secured by a latch mech- anism that is connected to the split rings. Changes sometimes requested include: On the small, 45 mL vessel, a firm closure is obtained by Multiple sample capacity. Up to three additional dip tubes simply turning down a knurled cap until it is hand tight. No and discharge valves can be installed in any vessel (except the wrenches or fixtures are required. 45 mL size). With these additional outlets and appropriate sample holders, as many as four samples can be processed indepen- Safety Protection dently and simultaneously in a single unit. All Parr Cell Disruption Vessels are designed to accommodate Special valves. Valves with either larger or smaller orifices a commercial nitrogen cylinder. The 920 mL and 1850 mL vessels can be provided. Users processing large quantities of materials are protected by a safety rupture disc which will burst at approxi- that have a tendency to plug the orifice of the standard throttling mately 3000 psig, well below the pressure at which any parts valve may want to substitute either a larger needle valve or of the vessel would fail. The 1 and 2 Gallon O-ring vessels are a ball valve to permit faster flow rates or to avoid stoppage. protected by 2200 psig rupture disc to protect the assembly as Fine metering valves with smaller orifices and very precise this is the maximum allowable working pressure for its compo- control capabilities may be helpful when treating samples that nents.