Density gradient centrifugation

Reasons for cell separation

Cell separation or cell isolation is the process of segregating one or more specific living cell populations from a diverse mixture of cells. Experiments conducted on isolated cells allow researchers to gain crucial insight into the characteristics of certain cells. In cell therapy applications, specific cell types are separated and isolated from peripheral blood or other biological sources prior to cell expansion to increase their numbers. Common reasons for cell separation include:

• Molecular analysis of single cells, such as to evaluate RNA expression
• Genetic modification of particular cell types, as in chimeric antigen receptor (CAR T) cell therapy
• Adoptive cell transfer experiments in different animal models
• Improving sensitivity of analytical techniques like HLA and FISH analysis

Cell separation techniques

Several methods are used to isolate cells from complex biological samples. Different cell characteristics like density, size, morphology, and physiology dictate the separation method and reagents to use. Cell separation techniques come classified into the following broad categories:

• Density gradient centrifugation: Relies on the different densities of cells with a heterogeneous sample.
• Immunomagnetic cell separation: Magnetic particles with affinity for specific cell surface markers help isolate the target cells from heterogeneous blends.
• Fluorescence-activated cell sorting (FACS): This technique uses fluorescent probes to specific cell surface markers and flow cytometry to separate complex cell mixtures.
• Immunodensity cell separation: This negative selection method utilizes antibody-based labeling and density gradient centrifugation to remove unwanted cell types, such as red blood cells.
• Sedimentation: Gravity causes denser components to sediment quickly for easy separation. The supernatant undergoes successive centrifugations to enable cell isolation.

Density gradient centrifugation FAQs

Below are some answers to frequently asked questions regarding density gradient centrifugation.

What is density gradient centrifugation?

This is a procedure used to separate different cell types, suspended particles such as viruses, or molecules like DNA using medium of a specific density or a gradient of different densities, with the highest density medium at the bottom. Analytical centrifugation can be used to determine the density of different particles within a mixture.

What is density gradient centrifugation used for?

Common applications of density gradient centrifugation are to separate cells from animal, plant, or microorganism sources. This process allows the production of cell therapies, including CAR T and natural killer (NK cells).

In these applications leukocytes are typically separated from other components in peripheral blood, bone marrow, or cord blood using a sterile, high-quality medium tested to ensure low endotoxin levels. Polysucrose media at 1.077 g/mL density is most common. If there’s a need to isolate cells with specific cell surface markers, density gradient centrifugation may be combined with other isolation methods.