Cryopulverization, also called cryogrinding, is often the first step in homogenizing tough tissues (skin, cartilage), tissues containing heat- or shear-labile components (enzymes, RNA, DNA), or samples containing important volatile components or oils (cannabinoids, flavor extracts). Tissue samples frozen to cryogenic temperatures of -80ｰC or lower become hard and embrittled. In that physical state, an impact or grinding action shatters tissue much like it were a piece of glass. The end result is a granulated or powdered tissue ideally suited for cell disruption and extraction procedures. Typical product yields using this method are 10-20% higher than the direct extraction of fresh or air-dried biomaterial.
The classic cryogrinder, a ceramic mortar and pestle precooled to dry ice or liquid nitrogen temperatures, has been in use for generations. While uncomplicated and low in cost, mortar and pestles are not easy to clean between samples and total sample recovery is difficult.
Cryogrinders
Laboratories now have a choice of several different devices to cryopulverize or cryogrind small tissue samples. The most popular is a freeze-fracturing device called a BioPulverizer or a Bessman tissue pulverizer. This compact tool is manufactured by BioSpec Products and Spectrum Medical Industries, respectively. Looking somewhat like a tablet press and coming in a selection of tissue capacities, a pulverizer consists of a hole machined into a thick-walled, stainless steel base into which loosely fits a piston or rod. The base and piston are pre-cooled to liquid nitrogen (liq N2) temperatures ard pre-frozen animal or plant tissue is placed inside the chilled hole. The chilled piston is placed on top of the hard frozen tissue sample, and with a few sharp blows with a hand-held hammer, the piston fragments normally fibrous or rubbery tissue into particles the size of grains of salt or smaller. Still-frozen, the material is then transferred to cold cell lysis extraction media for final cell lysis using a mechanical homogenizer...for example, a high-speed bead-mill cell disrupter, rotor-stator homogenizer, ultrasonicator or a Dounce tube and pestle.
In addition to the BioPulverizer, three other hand-operated cryopulverizers are the MicroCryoCrusher, a hand-operated screw press especially suited for cryopulverizing small samples of bone or teeth; the BioCryoTissueGrinder, a high-speed blade mill that cryopulverizes 0.5 to 10 grams of plant or animal tissue in the presence of dry ice, and the Cryo-cup Grinder, an insulated mortar and pestle specifically designed for cryopulverization. All are manufactured by BioSpecProducts and their cost is about $100.
Snap Freezing
Also used in cryogenic applications, the BioSqueezer is used to "snap-freeze" (quick freeze) 10-100 mg of soft animal tissue such as liver, brain, or muscle. This tool is a redesign of the classic freeze-clamp tongs. Snap-freezing offers a "snapshot" of metabolic events in tissue cells. The device freezes a small sample in a few milliseconds.
Easier to use and delivering more reproachable results, the fresh tissue is inserted into a small poly-bag and compressed with a Liq N2-chilled stainless steel clamp head into a thin disc about the size of a coin. The hard frozen disc can be easily fragmented by hand or crushed in a chilled mortar and pestle for subsequent rapid extraction of labile nucleic acids, proteins, and other extracellular products.
Multi-Sample CryoGrinders
Several manufacturers make motor-driven beed, ball- or hammer-mills capable of cryopulverizing many tissue and microbial samples in vials or deep-well microplates. Companies manufacturing these shakers include BioSpec Products, Cole-Parmer, Fritsch, IKA, Retsch, and Spex. The price of these multi-sample grinding pulverizers range from $2000 to $7000, but their large throughput makes up for the expense. Although cryogrinding of biological samples with steel beads or balls generally takes only a few seconds, heat generated by the collision of the grinding media can quickly warm samples in the vial. Proactive maintenance of cryotemperatures during the grinding process is essential. These high throughput machines address this by using pre-chilled, thick-walled metal tube holders; pre-chilled metal sample tubes; or by bathing vials with a drizzle of liquid nitrogen or flow of chilled air flowing through pieces of dry ice.
The main application for many multi-sample bead-mill cell disrupters (aka, Beadbeaters) is “wet-milling” ... the grinding of a sample in an excess of liquid media. Wet milling is commonly done inside 2 ml screw-cap polypropylene microtubes using glass or ceramic beads. These shaking machines operate at very high frequencies (1500-4000 oscillations/min) with vial displacements greater than 3/4 inch. This can be a problem when cryogrinding (a type of “dry grinding”) using steel beads...a bead five times more dense than glass. Many common plastic microvials or plates containing steel beads chilled to cryo-temperatures will crack or be breached during the grinding process. This problem is overcome by using special 2 ml polypropylene screw cap vials having a heavier wall and cap thickness (called “XX Tuff” vials), or 2 ml stainless-steel vials having custom silicone stoppers. Both are available from BioSpecProducts.
Practical Tips
Sourcing liquid nitrogen or dry ice has become more convenient. Many welding supply stores now stock bulk tanks of liquid nitrogen and can half-fill a quart- or gallon-sized vacuum flask or a foam-insulated picnic juice container for a small fee. Use a loose-fitting styrofoam plug to cap the container. And for dry ice, it is often found in larger supermarkets.
For those exploring cryogrinding for the first time, handling a container of liquid N2, and its use to chill your sample is mostly a matter of common sense. Respectfully treat it as though it were a container of scalding hot water. Use a plastic cup or bowl to contain the liquid N2 while chilling the tool or sample. Wearing safety glasses, pour liquid N2 from its container slowly. As it comes in contact with the room-temperature tool or sample expect the liquid N2 to sizzle and boil like water droplets falling on a hot cooking pan. When the object you are cooling stops “boiling” away liquid N2, it has reached the temperature of Liq N2. Handle chilled vials and cryo-tools with insulated gloves and tongs. In most cases grinding and pulverizing tools are made of steel or ceramic and will maintain their low temperature long enough to complete the pulverizing and grinding procedure without the need for further re-chilling.