User:Aly Ross/Cell suspension

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A cell suspension or suspension culture is a type of cell culture in which single cells or small aggregates of cells are allowed to function and multiply in an agitated growth medium, thus forming a suspension. Suspension cultures are used in addition to adherent cultures. The cells themselves can either be derived from homogenized tissue or from another type of culture. Suspension cell culture is commonly used to culture nonadhesive cell lines (like hematopoietic cells), plant cells, and insect cells. While some mammalian cell lines are cultured in suspension, the majority of commercially available cell lines are adherent.



History
The history of suspension cell culture aligns closely with the overall history of cell and tissue culture. Wilhelm Roux laid the groundwork for future tissue culture, by developing a saline buffer that was used to maintain living cells (chick embryos) for a few days. Ross Granville Harrison then developed in vitro cell culture techniques, including modifying the hanging drop technique for nerve cells and introducing aseptic technique to the culture process. Later, Montrose Thomas Burrows adapted Harrison's technique and collaborated with Alexis Carrel to establish multiple tissue cultures that could be maintained in vitro using fresh plasma combined with saline solutions. Carrel went on to develop the first known cell line, a line derived from chicken embryo heart which was maintained continuously for 34 years. Though the "immortality" of the cell line was later challenged by Leonard Hayflick, this was a major breakthrough and inspired others to pursue creating other cell lines. Notably, George Otto Gay and his assistant Mary Kubicek cultured the first human derived immortalized cell line - HeLa. While the other cell lines were adherent, HeLa cells were able to be maintained in suspension.

(GENERAL TIMELINE)

Isolating cells and starting a culture
Main article: Cell isolation

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. 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. 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.

Immortalized cell lines (cells that are able to replicate indefinitely), (plant cells/insect) can be obtained cryopreserved from manufacturers and used to start a suspension culture. 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.

Suspension cell culture maintenance
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. For this reason, specialized flasks (including the spinner flask and shaker flask) 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.

Spinner flasks
The major aspect of a spinner flask differentiating it from a normal flask is the magnetic spinner bar which circulates the media throughout the flask and keeps cells in suspension. A spinner flask looks like a large, cylindrical flask with one central capped opening flanked by two protruding arms which are also capped. These arms are either capped loosely or with semi-permeable caps and allow for additional gas exchange. The magnetic spinner bar itself is typically suspended from a rod attached to the central cap, keeping it off the bottom of the flask. Spinner settings need to be tuned to avoid killing cells due to shear forces. When culturing cells, the spinner flack containing cells is placed on a magnetic stir plate and both are kept inside of an incubator. (PICTURE, also bioreactor?)

Shaker flasks
Shaker flasks appear similar to typical Erlenmeyer flasks, but have a semi-permeable lid to allow for gas exchange. Similar to spinner flasks, shaker flasks require additional equipment, in this instance a orbital shaker, to function. During cell culture, shaker flasks filled with cells an media are placed on orbital shakers inside of incubators. Adjusting the revolutions per minute of the orbital shaker is necessary to tune the appropriate speed. The media must be allowed to stir, but cannot disturb the cells too much and cause them excessive stress. Shaker flasks are often used for fermentation cultures with microorganisms such as yeast.

Passaging (subculturing) cells
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 should be floating freely in media. A sample from the culture can be taken and analyzed to determine the ratio of live 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. Suspension cells are often passed outright instead of having their media changed, since it is more straightforward to passage the cells than to change the media. If one wants to change the media for a suspension culture, they must first remove all the cells from the current container and centrifuge them down into a pellet. Then, the excess media is removed from the centrifuged sample. The flask is refilled with fresh media and the cells are then added back into 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, high pH, or lack of space to grow.

Commercial applications of suspension cell culture
Unlike adherent cultures, which are limited by the surface area provided for them to expand on, suspension cultures are limited by the volume of their container. Meaning, suspension cells can exist in much larger quantities in a given flask and are preferred when using cells to make products including proteins, antibodies, metabolites or just to produce a high volume of cells. However, there are far fewer mammalian suspension cell lines than mammalian adhesive cell lines, limiting the scope of research involving suspension cell lines.

Some examples of suspension cell culture:


 * Antibody production by hybridomas
 * Fermentation cultures for beer
 * Therapeutic protein production by CHO cells
 * Secondary metabolite production for drugs in plant cells
 * Recombinate protein production in insect cells