32.5:
Tissue Homogenization and Cell Lysis
Tissue homogenization techniques disrupt tissue architecture and lyse cells to isolate subcellular organelles or macromolecules, such as nucleic acids and proteins.
Depending on the sample and the intended downstream application, homogenization can be performed using mechanical or non-mechanical methods.
Solid-based mechanical methods such as a tissue grinder or bead beater are used for hard or fibrous tissue samples like bone and muscle.
While a tissue grinder uses a manually or automatically operated mortar and pestle to grind a sample, the bead beater uses collision between the sample and the beads to break apart the tissue.
Ultrasonication is a liquid-based mechanical homogenization method that can shear cells in suspension using high-frequency sound waves generated by a vibrating probe.
Non-mechanical techniques include enzymatic, biological, or chemical homogenization methods.
These comprise the use of enzyme cocktails containing collagenase or trypsin or chemical agents like detergents or chaotropes, freeze-thaw cycles, or osmosis to breach the cell wall and plasma membrane and release the cell's contents into the solution.
Non – mechanical methods are often combined with mechanical lysis to obtain optimal results
32.5:
Tissue Homogenization and Cell Lysis
Tissue homogenization involves disintegrating tissue architecture and lysing cells, and is an early step in isolating and analyzing cellular components. The method used for homogenization depends on the sample type, the amount of sample available, the analyte to be obtained, and the sensitivity of the method. These methods are broadly classified as mechanical and non-mechanical methods.
Mechanical methods of tissue homogenization
These methods rely on applying external physical force to disrupt tissues and cells. They make use of specialized tools and instruments for homogenization. These instruments use grinding, shearing, blending, beating, or shock to disintegrate the sample. For example, in a French press, the sample is pushed through a small opening under pressure which causes the cells to disrupt. Other homogenizers such as Waring blenders and rotor-stators cut and shear the tissues into significantly smaller sizes.
Non-mechanical methods of tissue homogenization
Non-mechanical lysis methods involve chemical disruptions rather than physical forces to lyse the cells. The tissue is homogenized in a lysis buffer that regulates pH, ionic strength, osmotic strength, and enzymatic activity. The lysis buffer thus aids cell lysis and protects the cell components from damage.
While the enzymes of the lysis buffer help degrade the extracellular matrix of tissues to release individual cells, the surfactant or detergent helps disrupt cell membranes and denature proteins. Sodium dodecyl sulfate (SDS) and Triton-X 100 are two popularly used detergents in these buffers. Another component, the chaotropes, disrupt weak interactions between molecules, thus denaturing the proteins and keeping nucleic acids intact during isolation.
Other non-mechanical physical methods involve using temperature cycles in which the sample is frozen on dry ice or in an ethanol bath and then thawed at room temperature or 37℃. These repeated cycles cause the cell membranes to weaken and rupture. Cell membranes can also be ruptured by osmotic imbalance by placing them in a hypotonic or hypertonic solution. The inward or outward movement of water due to the osmotic gradient causes the cells to swell and burst, or shrink and collapse, releasing their internal contents.
While numerous methods and tools are available for homogenization, each has pros and cons that must be evaluated based on the specific requirements. Often, mechanical methods alone may not wholly or efficiently homogenize a sample. In such cases, mechanical methods may be combined with non-mechanical methods for complete homogenization.
While numerous methods and tools are available for homogenization, each has pros and cons that must be evaluated based on the specific requirements. Often, mechanical methods alone may not wholly or efficiently homogenize a sample. In such cases, mechanical methods may be combined with non-mechanical methods for complete homogenization.
Suggested Reading
- Takach E. Tissue homogenization. Tissue analysis for drug development. Unitec House, 2 Albert Place, London N3 1QB, UK: Future Science Ltd; 2013. p. 22–35.
- Goldberg S. Mechanical/physical methods of cell disruption and tissue homogenization. Methods Mol Biol. 2008;424:3–22.