Density Gradient Medium for Cell Separation

Cell density, defined here as the mass of a cell per unit volume and usually expressed in units of grams per mL or grams per cubic centimetre, is an intrinsic property of a cell. It is generally thought to be a highly regulated property of a given cell type, as cells maintain tight control over the concentration of cellular components (Relevance and regulation of cell density). The cellular composition can change, leading to overall changes in cell density through physiological changes such as stages of differentiation, malignant transformation, and senescence. Therefore, cell density can be used both as a tool to measure a population of cells and to separate different populations of cells. This blog focuses on some frequently asked questions about the importance of cell density and the role that density plays in cell separation.

How is cell density related to cell health and viability?

Cellular health is frequently measured with a variety of stains that interact with cells in different physiological states. For example, trypan blue is often used to distinguish living cells from dead cells. Trypan blue interacts with intracellular proteins in the cytoplasm of dead cells, giving them a dark blue appearance. However, trypan blue is excluded from living cells, which are observed to have clear cytoplasm easily distinguishable from dead cells.

Cells that are viable can be separated from those that have been damaged and are undergoing apoptosis, although not yet dead, by using markers. Scientists can assess the status of individual cells using a protein (Annexin V) that binds to exposed phosphatidylserine on the cell surface for apoptosis or dyes for DNA replication (ethidium monoazide or propidium iodide (PI)). which are indicative of cell division/proliferation.

What is the difference between cell mass and cell volume, the ratio of which makes up cell density?

Cell mass is the amount of matter that makes up a cell. Water makes up the bulk of a cell’s mass at around 70%. Cells are generally classified based on their content of organic macromolecules (lipids, carbohydrates, proteins, nucleic acids), which make up the majority of a cell’s weight. The organic macromolecule composition of a cell determines its basic chemistry and ultimately defines its structure and function and how it interacts with the outside world. While the inorganic ions of a cell are the least abundant in the 1% or less of the cell mass, they play an important role in cell metabolism and overall cell function.

Cell volume is a cellular characteristic defined as the amount of space a cell occupies. A balance between intracellular osmolarity and extracellular tonicity determines the volume of a cell that is controlled by the inflow/outflow of water for homeostatic function. Cell volume can define not only cell shape, but also modulate other cell functions such as cell proliferation, migration, and death. Apoptosis is related to volume contraction and changes in cell deformability.

How does cellular osmotic stress affect cell density and therefore cell function?

Regulation of cell volume is a critical function of cells. When cells are exposed to osmotically active environments, the normal cellular function is to maintain equilibrium through the regulation of osmotic stress. The movement of water by osmosis (in or out) alters the concentrations of intracellular macromolecules. These changes in extracellular osmolarity alter cell volume and therefore cell density. The inability to respond to an osmotic challenge can result in impaired function of a cell.

What are some common cell separation methods? What are the benefits of cell separation?

Cells can be separated by physical properties such as cell size, density, and cell surface markers. Cell density is a specific physical property that allows cell populations to be isolated from each other. The use of cell surface markers allows for the identification and specific isolation of subpopulations. Cell separation methods, also known as cell isolation, provide avenues for cell enrichment for scientists.

Cell isolations can be performed by density gradient centrifugation, separating cells based on their buoyant density in solutions such as sucrose. Specific cell types, such as blood cells already in single-cell suspensions, can be easily separated on density gradient media. Altering the concentration of density gradient media or overlapping several different gradients can influence where cells ultimately settle after centrifugation.

Cells that are separated based on properties such as protein expression are often isolated using analytical techniques such as flow cytometry or fluorescence-activated cell sorting (FACS). This is generally done by flowing the cells under pressure in a stream, hydrodynamically focusing the cells to split individual cells into individual droplets. Individual cells in droplets are then electrostatically shunted into tubes to sort their respective populations based on user-defined activation strategies.

Magnetic cell isolation is used via magnetic beads attached to target antibodies that bind to cellular proteins to identify cells of interest. Magnetic forces attract magnetically labelled cells toward a columnar bead array or to the side of the tube within the magnetic field. Both labelled and unlabeled fractions can be collected. Using this technique, cells of interest can be separated from unwanted cells.

Levitation technology is a unique enrichment method that has no labels. The cells are mixed with an inert paramagnetic compound and then exposed to an externally applied magnetic field through a specifically designed cartridge. Cells levitate in solution at specific heights determined by the cells’ intrinsic properties, including density and magnetic susceptibility. Viable cells levitate higher than dead cells or debris, due to the permeability of the dead cell membranes, allowing reproducible separation.

Single-cell suspensions of blood and tissue samples are often used to determine cell function, cell status (ie, disease), or even how cells respond to treatments and drugs. Cell enrichment and purification enhance subsequent single-cell molecular analysis in desired cell types, such as single-cell RNA sequencing (scRNA-seq), immunocytochemistry (ICC), or single-cell protein analysis.

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