Suspension culture

All primary cells (cells derived directly from a subject) must first be removed from a subject, isolated (using digestion enzymes), and suspended in media before being cultured.[1] However, this does not mean that these cells are compatible with suspension culture, as most mammalian cells are adherent and need to attach to a surface to divide. White blood cells can be taken from a subject and cultured in suspension, since they naturally exist in suspension in blood.[12] Adhesion of white blood cells in vivo is typically the result of an inflammatory immune response and requires specific cell-cell interactions that should not occur in a suspension of a single type of white blood cell.[13]

Immortalized mammalian cell lines (cells that are able to replicate indefinitely), plant cells, and insect cells can be obtained cryopreserved from manufacturers and used to start a suspension culture.[14] To start a culture from cryopreserved cells, the cells must first be thawed and added to a flask or bioreactor containing media. Depending upon the cryoprotectant agent, the cells might need to be washed to avoid deleterious effects from the agent.[3]

Suspension cell cultures are similar to adherent cultures in a number of ways. Both require specialized nutrient containing media, containers that allow for gas transfer, aseptic conditions to avoid contamination, and frequent passaging to prevent overcrowding of cells. However, even within these similarities there are a few key differences between these culture methods. For example, though both adherent and suspension cell cultures can be maintained in standard flasks such as the T-75 tissue culture flask, suspension cultures need to be agitated to avoid settling to the bottom of the flask. While adherent cell cultures can be maintained in flat flasks with a lot of surface area (to promote cell adhesion), suspension cultures require agitation otherwise the cells will fall to the bottom of a flask, greatly impacting their access to nutrients and oxygen, eventually resulting in cell death.[4] For this reason, specialized flasks (including the spinner flask and shaker flask, discussed below) have been developed to agitate media and keep the cells in suspension. However, the agitation of media subjects the cells to shear forces which can stress the cells and negatively impact growth. Although both adherent and suspension cell cultures require media, media used in suspension culture may contain a surfactant to protect cells from shear forces in addition to the amino acids, vitamins and salt solution contained in culture media such as DMEM.[5]

Spinner flasks, which are used for suspension cultures, contain a magnetic spinner bar which circulates the media throughout the flask and keeps cells in suspension.[15] Spinner flasks contain one central capped opening flanked by two protruding arms which are also capped and allow for additional gas exchange. The magnetic spinner bar itself is typically suspended from a rod attached to the central cap so that it maximizes media circulation in the cell suspension. When culturing cells, the spinner flask containing cells is placed on a magnetic stir plate, inside of an incubator and the spinner parameters need to be adjusted carefully to avoid killing cells with shear forces.[16]

Orbital laboratory shaker.

Shaker flasks are also used for suspension cultures, and appear similar to typical Erlenmeyer flasks but have a semi-permeable lid to allow for gas exchange.[17] During suspension cell culturing, shaker flasks are loaded with cells and the appropriate media before they are placed on an orbital shaker. To optimize cell culture proliferation, the revolutions per minute of the orbital shaker must be adjusted within an acceptable range depending on the cells and media used. The media must be allowed to stir, but cannot disturb the cells too much causing them excessive stress. Shaker flasks are often used for fermentation cultures with microorganisms such as yeast.[18]

Passaging, or subculturing, suspension cell cultures is more straightforward than passaging adherent cells. While adherent cells require initial processing with a digestion enzyme, to remove them from the culture flask surface, suspension cells are floating freely in media.[19] A sample from the culture can then be taken and analyzed to determine the ratio of living to dead cells (using a stain such as trypan blue) and the total concentration of cells in the flask (using a hemocytometer). Using this information, a portion of the current suspension culture will be transferred to fresh flask and supplemented with media. The passage number should be recorded, particularly if the cells are primary and not immortalized as primary cell lines will eventually undergo senescence.[20] Suspension cells are often passaged outright without changing the media. In order to change the media for a suspension culture, all cells from the current container should be removed and centrifuged into a pellet. The excess media is then removed from the centrifuged sample, and the flask is refilled with fresh media before re-adding the cells to the flask. Media changes and subculturing are important to maintain cell lines, since cells will consume nutrients in media to expand. Cells will also grow exponentially until the environment becomes inhospitable due to lack of nutrients, extreme pH, or lack of space to grow.