Various methods have been devised for disrupting tissues and suspended cells, but the method of choice is usually the procedure that causes minimal damage to the released cell constituents.
Most physical procedures are based on the effects of shearing forces, and because the released cell parts undergo rapid deterioration at room temperature, these procedures are usually carried out at low temperature and in cold buffer solution.
Among the older methods used is grinding the sample with a mortar and pestle, often with the aid of abrasive materials such as sand, alumina, or ground glass.
This procedure has several disadvantages, including the loss of some cell constituents by adsorption to the abrasive and the necessity of removing the abrasive material either before or during the fractionation procedure.’
Shearing forces adequate to disrupt most cells and tissues may also be obtained using a blender in which steel blades rapidly rotate through the cell or tissue suspension. The product of this and similar techniques is called a homogenate (e.g., “cell homogenate” or “tissue homogenate”).
Tissues may also be homogenized by placing them, along with cold buffer solution, in a cylindrical glass tube fitted with a glass or Teflon plunger. As the plunger is driven down the tube (generally by hand), the tissue and buffer are forced upward through the narrow annular space between the wall of the tube and the plunger.
The shearing forces that are generated are usually sufficient to disperse the tissue after several up-and-down strokes. This is the method of choice for the disruption of soft tissues, such as liver, brain, and kidney one of the more rigorous methods for disrupting cells is by the use of a pressure cell, which consists of a steel cylinder and close-fitting steel piston.
The piston is pushed into one end of the cylinder using a press or hydraulic jack, and the sample is forced out of the cylinder through a narrow opening at the other end. The size of this opening may be controlled by a needle valve. Bacteria and other microorganisms enclosed within a tough cell wall are frequently disrupted using this approach.
Cells may also be disrupted by insonation (ultrasound) using a sonifier. In this procedure, the probe of the sonifier is immersed in the cell suspension and caused to vibrate in the fluid (usually at about 20,000 cycles per second). These ultrasonic vibrations produce a number of effects in the fluid that act collectively to cause the disruption of the cells. Shock waves (alternate compressions and rarefactions) arising from the tip of the probe create turbulent flow of the fluid in which the cells are suspended and may disrupt the cells.
The shock waves also cause cavitation of the fluid, that is, microscopic bubbles are formed in the fluid near the tip of the probe and these rapidly stream away from the probe along with some of the fluid. The friction created between this stream and the suspended cells also contributes to their disruption. Some of these bubbles disintegrate into still smaller bubbles that travel away from the probe like miniature projectiles; when these impinge upon the cells, the cells may be disrupted.
Cells can also be disrupted by chemical means. For example, enzymes that specifically degrade the components of the cell wall or plasma membrane may be added to the tissue or cell suspension. Alternatively, proteolytic or lipolytic agents (such as certain detergents) that dissolve the membrane may be used.
Some cells are sufficiently fragile that they may be disrupted by successive freezing and thawing. Erythrocytes (red blood cells) and certain other cells can be broken by the osmotic pressure created within them when they are placed in a hypotonic salt solution or distilled water.
After the cells have been disrupted, the goal is to separate and isolate the structures that have been released from them. Because cellular organelles and other constituents vary in size, shape, and density, they settle through the liquid in which they are suspended at different rates.
Consequently, disrupted cells are more often fractionated by some form of centrifugation than by any other method. Indeed, centrifugation has become one of the most widely employed procedures in cellular research and one of the most important tools of the cell biologist.