Methods for Disrupting Cells and Preparing Their Contents for Analysis

Numerous methods exist for cell disruption and preparing their contents for downstream analysis. The choice of method typically depends on the nature of the cells or tissue being processed. For easily lysed cells like cultured or blood cells, gentler techniques are preferred, while more aggressive methods are necessary for robust bacterial or plant cells, or mammalian cells within connective tissue.

Detergent-based Lysis

Detergent-based lysis is a gentle method suitable for mammalian cells, bacteria, yeast, and plants. In this approach, cell suspensions are gently centrifuged and resuspended in a lysis buffer containing detergents that disrupt the cell membranes. These detergents solubilize the membranes, leading to cell lysis and the release of intracellular contents. Depending on the analysis, the detergents may need to be removed in later stages if they interfere with the intended assay or production process.

Freeze-thaw Lysis

Freeze-thaw lysis is applicable for mammalian or bacterial cell suspensions. In this method, the cell suspension is rapidly frozen, typically with liquid nitrogen. After freezing, the sample is thawed and resuspended by pipetting or vortexing gently in a lysis buffer. This freezing and thawing cycle is repeated multiple times to ensure thorough disruption. After each cycle, the sample is centrifuged, and the supernatant, which contains the soluble protein, is retained.

Osmotic Shock

Osmotic shock is a very gentle lysis technique used primarily for suspensions of mammalian or bacterial cells. It works by altering the osmotic environment, typically from high to low osmotic pressure. This method is often combined with mechanical disruption and is ideal when the lysate is to be fractionated into subcellular components. It is a mild technique that avoids the need for detergents.

Ultrasonication

Ultrasonication involves the use of high-frequency sound waves to disrupt cells. A probe is inserted into the sample, and the sound waves create regions of low pressure, causing cell membrane disruption. This method is commonly applied to cell suspensions for protein extraction and is effective for breaking down both soft and tough cell walls.

Mechanical Methods

Mechanical methods, such as crushing and grinding, are often used for protein extraction. These methods include techniques like Dounce or Potter-Elvehjem homogenization, where cell membranes are disrupted by liquid shear forces. For tissues, homogenization can be done by chopping or mincing the sample in a chilled buffer using a Waring blender or Polytron® homogenizer. Other methods include freezing tissues in liquid nitrogen and grinding them to a fine powder with a mortar and pestle, often using alumina or sand for added disruption. Rapid agitation of cells with fine glass beads can also break open cells, which is particularly useful for Gram-positive and Gram-negative bacteria.

Enzymatic Digestion

Enzymatic digestion is frequently used when extracting proteins from bacteria, yeast, or eukaryotic cells embedded in fibrous tissues. In this method, enzymes such as Lysozyme, Mutanolysin, MetaPolyzyme, Lysonase, and Pronase are used to dissolve or disrupt cell walls, capsules, and other protective structures. Tissue digestion enzymes like Collagenase, Chondroitinase, and Hyaluronidase are also employed to break down tough tissues. This enzymatic digestion can be followed by homogenization, sonication, or vigorous vortexing in a lysis buffer to further facilitate cell disruption.

To prevent the degradation of the target molecule by proteases and phosphatases released during cell lysis, it is crucial to add inhibitors to the lysis buffer to protect the sample during disruption and subsequent purification.