What Is Tissue Dissociation? Methods and Purpose

Tissue dissociation is the process of breaking down a solid piece of biological material, such as an organ or a tumor, to release the individual cells it contains. This procedure is a foundational step for many types of cellular research and therapeutic applications, with the goal of creating a suspension of single cells. The process can be compared to carefully separating individual bricks from a wall without causing them to break. The success of this procedure is measured by the ability to obtain a high number of healthy, functional cells from the original tissue.

The Purpose of Isolating Single Cells

Isolating single cells from a larger tissue mass enables a wide range of advanced biological analyses and medical applications. By studying cells one by one, researchers can uncover details about cellular diversity and function that are obscured when looking at the tissue as a whole. One major application is in single-cell analysis, which examines the unique properties of individual cells. This is useful in cancer research for identifying rare tumor cells within a complex tumor microenvironment, providing insights into disease progression and potential therapeutic targets.

The isolated cells are also frequently used for cell culture, which is the process of growing specific cell types in a laboratory setting. This allows scientists to expand a population of cells for further experiments, such as testing the effects of new drugs or developing cell-based therapies like stem cell treatments. Another common application is flow cytometry, a technique that requires a single-cell suspension to sort and analyze large numbers of cells based on their physical and chemical characteristics.

Enzymatic Dissociation Methods

Enzymatic dissociation uses specific proteins to break down the extracellular matrix (ECM), the scaffold holding cells together. The ECM’s composition varies between tissue types, so the choice of enzymes must be tailored to the specific tissue to ensure effective dissociation while keeping cells functional.

Several enzymes are commonly used, often in combination. Collagenase is frequently used as it breaks down collagen, the main structural protein in connective tissues. For tissues with strong cell-to-cell connections, a protease called trypsin is often employed to cleave the proteins that anchor cells to one another. Other enzymes like Dispase, a gentler protease, are effective at separating sheets of epithelial cells. Hyaluronidase is used to break down hyaluronic acid, a carbohydrate that helps fill the space between cells.

Mechanical and Chemical Dissociation Methods

In addition to enzymes, physical force and non-enzymatic chemicals are used to separate cells. Mechanical dissociation involves using physical methods to break the tissue into smaller pieces. This is often performed as an initial step to increase the surface area of the tissue, allowing enzymes to penetrate more effectively. Common techniques include mincing the tissue with scalpels, grinding it with a glass homogenizer, or forcing it through a fine screen. While mechanical force alone can be sufficient for loosely associated cells, it is frequently combined with other methods for more dense tissues.

Chemical methods involve the use of chelating agents, which are molecules that bind to specific ions. Cell adhesion proteins that hold cells together often require calcium ions to function properly. Chemicals like EDTA work by binding to these calcium ions, which weakens the cell-to-cell connections and makes it easier to separate the cells. This approach is often used with enzymatic or mechanical techniques to enhance the process.

Ensuring Cell Viability and Quality

Assessing the quality of the cell suspension is a standard part of the workflow, as the success of any downstream application depends on a high-quality sample. A primary step in quality control is to assess cell viability, which is the percentage of live cells in the suspension. A common method for this is the Trypan Blue exclusion assay. This technique uses a dye that can only pass through the compromised membranes of dead cells, staining them blue, while live cells with intact membranes exclude the dye.

In addition to viability, it is also necessary to determine the concentration of cells in the suspension. This is done using a specialized microscope slide called a hemocytometer, which has a grid of known dimensions etched onto its surface. By loading a sample onto the hemocytometer, scientists can count the cells within a specific area of the grid and calculate the overall cell concentration.