Stem Cell Purification

Isopycnic Centrifugation Sorting

Isopycnic centrifugation sorting is utilized in primary cell recovery by separating cells on the basis of size density and adhesiveness; as the name implies, centrifugal force is regularly applied in the protocol. The sorting is either based on density gradient or cell culture.

The density gradient method entails the loading of a mixture of cells into a solution with differing densities. This mixture is then spun in a centrifuge, the process allowing cells to move through the gradient until they arrive at an isopycnic location, where the cells and the solution have the same density. Commercially available density gradient tools include Peroll, Ficoll, Lymphoprep, and Dextran. Percoll consists of microscopic silica particles, whereas the other density gradients tools primarily contain polysaccharides (chains of glucose molecules). The density gradient method is typically used for enrichment purposes and is followed by other, more refined, purification methods. Some of the advantages of density gradient are short process times, reproducibility, and high cell viability. The limitations are low resolution, low purity, labor intensiveness, and nonspecificity, among others.

Cell culture purification utilizes the adhesiveness of cells. In this method, the rapid adherence of stem cells relative to that of other cells allows for nonadherent cells to be washed out; the stem cells adhere to the vessel in which the cells are cultured. The adhered cells are subsequently recovered by trypsinization, in which the enzyme trypsin breaks down the proteins that enable the cells to adhere to the vessel. The cell culture method can achieve high levels of purity and high levels of cell viability. However, it is time consuming and labor intensive and requires specialized equipment and reagents.

Fluorescence-Activated Cell Sorting

Fluorescence-activated cell sorting (FACS) is a type of flow cytometry. In flow cytometry, cells are suspended in a stream of fluid and travel in single file past a detection point that measures the fluorescence and light from the cells. This technology is used in cell counting, cell sorting, and biomarker detection. In FACS, a laser of a specific wavelength is aimed at a flowing stream of cells, and the light-scattering properties of each cell are analyzed by a computer system to determine the size of the cell, granularity, or DNA content.

...