A single cell suspension is a laboratory preparation where individual cells, whether from a solid tissue or an attached culture, are separated from each other and suspended in a liquid medium. This preparation is fundamental in biological research, allowing scientists to study cells one by one rather than as a bulk group. The process aims to isolate cells while preserving their integrity, which is a delicate balance to achieve.
Applications of Single Cell Suspensions
Single cell suspensions enable various research possibilities by allowing analysis of individual cellular components. Flow cytometry, for example, relies on individual cells passing single-file through a laser beam to detect and quantify specific cellular properties, such as size, granularity, and protein expression. This technique allows for the sorting of different cell types based on their unique characteristics, which is not possible with clumped cells. Clumps can block the instrument or cause inaccurate measurements.
Single-cell sequencing is another application that investigates the genetic or protein information of individual cells. By analyzing each cell separately, researchers can uncover subtle differences in gene expression or mutations within a seemingly uniform tissue, providing a high-resolution view of cellular heterogeneity. Single cell suspensions are also used to initiate new cell cultures, particularly primary cell cultures derived directly from living tissues. This allows researchers to grow and study specific cell types in a controlled laboratory environment for various downstream experiments, including drug testing or disease modeling.
Generating Suspensions from Tissue Samples
Transforming a solid tissue into a single cell suspension involves a combination of mechanical and enzymatic methods to break down its structure. The initial step involves mechanical dissociation, where the tissue is physically disrupted to increase its surface area and separate cells. This can be achieved by mincing the tissue into small pieces using a scalpel or sharp dissection scissors. The minced tissue may then be further processed by forcing it through a mesh screen or using a mechanical dissociator, which applies physical force to release individual cells.
Following mechanical disruption, enzymatic digestion is employed to dissolve the extracellular matrix, the “cellular glue” holding cells together. Enzymes like collagenase are used to cleave peptide bonds in collagen, a major component of the extracellular matrix. Other proteases, such as dispase, also break down matrix components. These enzymes liberate cells into a suspension while minimizing damage to the cell membrane and surface proteins, which is important for maintaining cell viability and functionality for downstream analyses.
Creating Suspensions from Cultured Cells
Preparing single cell suspensions from cultured cells differs depending on whether the cells grow attached to a surface or are already floating in their medium. Suspension cultures, where cells naturally grow unattached, require a simpler collection process. These cells are already free-floating, so they are collected, centrifuged to form a pellet, and then resuspended in fresh medium to achieve a desired concentration. This process is faster and less disruptive to the cells compared to detaching adherent cultures.
Adherent cultures, however, grow attached to the surface of a flask or plate and require specific steps to detach them into a suspension. The procedure begins by washing the cells with a balanced salt solution to remove residual growth medium. Next, an enzyme solution, most frequently Trypsin-EDTA, is added. Trypsin is a proteolytic enzyme that cleaves protein bonds responsible for cell adhesion, while EDTA, a chelating agent, binds calcium and magnesium ions that contribute to cell-cell adhesion, enhancing the enzyme’s effectiveness. After a brief incubation, the cells detach and can be gently collected and resuspended in fresh medium for further use.
Assessing Suspension Quality
After generating a single cell suspension, verifying its quality is an important step to ensure reliable experimental results. Cell viability is a primary concern, determining the percentage of live cells within the suspension. The Trypan Blue dye exclusion method is widely used for this purpose. Live cells have intact cell membranes that exclude the dye, appearing clear under a microscope. Dead cells, with compromised membranes, take up the Trypan Blue dye and appear blue, allowing for their distinction and quantification.
Cell counting is another quality check, determining the concentration of cells in the suspension. This is performed using a hemocytometer, a specialized glass slide with a grid etched onto its surface. A small volume of the cell suspension is loaded onto the hemocytometer, and cells within specific squares of the grid are manually counted under a light microscope. This count, along with the known volume of the grid squares and any dilution factors, allows for the calculation of the total cell concentration and the number of viable cells per milliliter.