The Basics of Cell Separation Methods: A Deep Dive on Density

Cellular density, defined here as the mass of a cell per unit volume and typically expressed in units of grams per mL or grams per cubic centimeter, 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 & Regulation of Cell Density). Cellular composition can change, leading to overall changes in cellular density by way of physiological changes such as stages of differentiation, malignant transformation, and entry into senescence. Thus, cell density can be used both as a tool to measure a population of cells as well as to separate distinct populations of cells. This blog focuses on some frequently asked questions around the importance of cell density and the role density plays in cell separation.

How is cellular density related to cell health & viability?
Cell health is frequently measured with a variety of stains that interact with cells in different physiological states. For example, trypan blue is frequently used to distinguish live cells from dead. Trypan blue interacts with intracellular proteins in the cytoplasm of dead cells, giving them a dark blue appearance. However, trypan blue is excluded from live cells, which are observed to have a clear cytoplasm readily distinguishable from dead cells.

Cells that are viable can be separated from those that have been damaged and are undergoing apoptosis, though not yet dead, through use of markers. Scientists can assess the state of individual cells using a protein (Annexin V) that binds cell-surface exposed phosphatidylserine 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, whose ratio makes up cell density?
Cell mass is the amount of matter that makes up a cell. Water constitutes the majority of a cell’s mass at about 70%. Cells are usually classified based on their organic macromolecule content (lipid, carbohydrate, protein, nucleic acids), which constitute the majority of a cell’s weight. The organic macromolecule composition of a cell determines its basic chemistry, ultimately defining its structure and function and how it interacts with the external world. While inorganic ions of a cell are the least abundant at 1% or less of the cell mass, they play a significant 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 a cell’s volume which is controlled by water influx/efflux for homeostatic function. Cell volume can define not only cell shape, but also modulate other cellular functions such as cell proliferation, migration, and death. Apoptosis is related to volume shrinkage and changes in cell deformability.

How does cellular osmotic stress impact cell density and thus cell function?
Regulation of cell volume is a critical function of cells. When cells are exposed to osmotically active environments, normal cellular function is to maintain equilibrium through regulation of osmotic stress. Movement of water via osmosis (influx or efflux) alters intracellular macromolecule concentrations. 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 like cell size, density, and cell surface markers. Cellular density is a specific physical property that allows cell populations to be isolated from each other. Use of cell surface markers enable identification and specific isolation of subpopulations. Cell separation methods, also referred to as cell isolation, provide avenues of cellular enrichment for scientists. 

Cell isolations can be performed using
density gradient centrifugation, separating cells based on their buoyant density in solutions such as sucrose. Specific cell types, such as blood cells that are already in single-cell suspensions, can be easily separated in density gradient mediums. Altering the concentration of the density gradient media or layering several different gradients on top of each other can influence where the cells eventually settle after centrifugation.

Cells that are separated based on properties such as protein expression are often isolated with analytical techniques such as flow cytometry or
Fluorescence-activated cell sorting (FACS). This is typically done by flowing the cells under pressure in a stream, hydrodynamically focusing cells to partition single cells into individual droplets. Single cells in droplets are then electrostatically deflected into tubes to sort their respective populations based on user defined gating strategies.

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

Levitation technology
is a unique enrichment method that is label-free. Cells are mixed with an inert, paramagnetic compound then exposed to an externally applied magnetic field via a specifically designed cartridge. The cells levitate in solution to specific heights determined by the cells’ intrinsic properties, including density and magnetic susceptibility. Viable cells levitate higher than dead cells or debris, due to permeability of the membranes of dead cells, which allows for reproducible separation.

Single-cell suspensions from both blood and tissue samples are often used to determine cell function, cell state (I.e. disease), or even how cells respond to treatments and drugs. Cellular enrichment and purification improve downstream single-cell molecular analysis on desired cell types such as single-cell RNA sequencing (scRNA-seq), immunocytochemistry (ICC), or single-cell protein analysis

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