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A cell suspension or suspension culture is a type of
cell culture Cell culture or tissue culture is the process by which cells are grown under controlled conditions, generally outside of their natural environment. The term "tissue culture" was coined by American pathologist Montrose Thomas Burrows. This te ...
in which single
cells Cell most often refers to: * Cell (biology), the functional basic unit of life Cell may also refer to: Locations * Monastic cell, a small room, hut, or cave in which a religious recluse lives, alternatively the small precursor of a monastery w ...
or small aggregates of cells are allowed to
function Function or functionality may refer to: Computing * Function key, a type of key on computer keyboards * Function model, a structured representation of processes in a system * Function object or functor or functionoid, a concept of object-oriente ...
and
multiply Multiplication (often denoted by the cross symbol , by the mid-line dot operator , by juxtaposition, or, on computers, by an asterisk ) is one of the four elementary mathematical operations of arithmetic, with the other ones being additi ...
in an agitated
growth medium A growth medium or culture medium is a solid, liquid, or semi-solid designed to support the growth of a population of microorganisms or cells via the process of cell proliferation or small plants like the moss ''Physcomitrella patens''. Differen ...
, thus forming a
suspension Suspension or suspended may refer to: Science and engineering * Suspension (topology), in mathematics * Suspension (dynamical systems), in mathematics * Suspension of a ring, in mathematics * Suspension (chemistry), small solid particles suspend ...
. Suspension culture is one of the two classical types of cell culture, the other being adherent culture. The history of suspension cell culture closely aligns with the history of cell culture overall, but differs in maintenance methods and commercial applications. The cells themselves can either be derived from homogenized tissue or from heterogenous cell solutions. Suspension cell culture is commonly used to culture nonadhesive cell lines like
hematopoietic Haematopoiesis (, from Greek , 'blood' and 'to make'; also hematopoiesis in American English; sometimes also h(a)emopoiesis) is the formation of blood cellular components. All cellular blood components are derived from haematopoietic stem cells. ...
cells,
plant cell Plant cells are the cells present in green plants, photosynthetic eukaryotes of the kingdom Plantae. Their distinctive features include primary cell walls containing cellulose, hemicelluloses and pectin, the presence of plastids with the capabi ...
s, and insect cells. While some cell lines are cultured in suspension, the majority of commercially available mammalian cell lines are adherent. Suspension cell cultures must be agitated to maintain cells in suspension, and may require specialized equipment (e.g. magnetic stir plate, orbital shakers, incubators) and flasks (e.g. culture flasks, spinner flasks, shaker flasks). These cultures need to be maintained with nutrient containing media and cultured in a specific cell density range to avoid cell death.


History

The history of suspension cell culture is closely tied to the overall history of cell and tissue culture. In 1885,
Wilhelm Roux Wilhelm Roux (9 June 1850 – 15 September 1924) was a German zoologist and pioneer of experimental embryology. Early life Roux was born and educated in Jena, Germany where he attended university and studied under Ernst Haeckel. He also attended ...
laid the groundwork for future tissue culture, by developing a saline buffer that was used to maintain living cells (chicken embryos) for a few days.
Ross Granville Harrison Ross Granville Harrison (January 13, 1870 – September 30, 1959) was an American biologist and anatomist credited for his pioneering work on animal tissue culture. His work also contributed to the understanding of embryonic development. Harrison ...
in 1907 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 in 1910,
Montrose Thomas Burrows Montrose Thomas Burrows (1884 – 1947) was a US surgeon and pathologist specializing in cancer research and surgery. He was born into a Scots-Irish Presbyterian family in Halstead, Kansas. (includes photo). Along with Dr. Alexis Carrel, a ...
adapted Harrison's technique and collaborated with
Alexis Carrel Alexis Carrel (; 28 June 1873 – 5 November 1944) was a French surgeon and biologist who was awarded the Nobel Prize in Physiology or Medicine in 1912 for pioneering vascular suturing techniques. He invented the first perfusion pump with Charle ...
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 Leonard Hayflick (born 20 May 1928) is a Professor of Anatomy at the UCSF School of Medicine, and was Professor of Medical Microbiology at Stanford University School of Medicine. He is a past president of the Gerontological Society of America and ...
, this was a major breakthrough and inspired others to pursue creating other cell lines. Notably in 1952, George Otto Gay and his assistant Mary Kubicek cultured the first human derived immortalized cell line -
HeLa HeLa (; also Hela or hela) is an immortalized cell line used in scientific research. It is the oldest and most commonly used human cell line. The line is derived from cervical cancer cells taken on February 8, 1951, named after Henrietta La ...
. While the other cell lines were adherent, HeLa cells were able to be maintained in suspension.


Methods and maintenance


Isolating cells and starting a 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. 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 cell White blood cells, also called leukocytes or leucocytes, are the cell (biology), cells of the immune system that are involved in protecting the body against both infectious disease and foreign invaders. All white blood cells are produced and de ...
s 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 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. 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 for laboratories

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, discussed below) have been developed to agitate media and keep the cells in suspension. However, the agitation of media subjects the cells to
shear force In solid mechanics, shearing forces are unaligned forces acting on one part of a body in a specific direction, and another part of the body in the opposite direction. When the forces are collinear (aligned with each other), they are called t ...
s 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 Surfactants are chemical compounds that decrease the surface tension between two liquids, between a gas and a liquid, or interfacial tension between a liquid and a solid. Surfactants may act as detergents, wetting agents, emulsifiers, foaming ...
to protect cells from shear forces in addition to the amino acids, vitamins and salt solution contained in culture media such as
DMEM Minimal Essential Medium (MEM) is a synthetic cell culture medium developed by Harry Eagle first published in 1959 in ''Science'' that can be used to maintain cells in tissue culture. It is based on 6 salts and glucose described in Earle's salts ...
.


Spinner flasks

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. 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.


Shaker flasks

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. 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.


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 are be floating freely in media. 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 Trypan blue is an azo dye. It is a direct dye for cotton textiles. In biosciences, it is used as a vital stain to selectively colour dead tissues or cells blue. Live cells or tissues with intact cell membranes are not coloured. Since cells a ...
) and the total concentration of cells in the flask (using a
hemocytometer The hemocytometer (or haemocytometer) is a counting-chamber device originally designed and usually used for counting blood cells. The hemocytometer was invented by Louis-Charles Malassez and consists of a thick glass microscope slide with a ...
). 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 Senescence () or biological aging is the gradual deterioration of functional characteristics in living organisms. The word ''senescence'' can refer to either cellular senescence or to senescence of the whole organism. Organismal senescence inv ...
. 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.


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. Most large scale suspension culture involves non-mammalian cells and takes place in bioreactors. 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 * Recombinant protein production in insect cells * Bulk protein production for enzyme and vaccine research * Producing cell suspension cultures to support oncolytic adenovirus used in cancer immunotherapy


List of suspension cell lines


See also

*
Cell culture Cell culture or tissue culture is the process by which cells are grown under controlled conditions, generally outside of their natural environment. The term "tissue culture" was coined by American pathologist Montrose Thomas Burrows. This te ...
* Adherent culture *
Bio-MEMS Bio-MEMS is an abbreviation for biomedical (or biological) microelectromechanical systems. Bio-MEMS have considerable overlap, and is sometimes considered synonymous, with lab-on-a-chip (LOC) and micro total analysis systems (μTAS). Bio-MEMS is ...
*
Cell adhesion Cell adhesion is the process by which cells interact and attach to neighbouring cells through specialised molecules of the cell surface. This process can occur either through direct contact between cell surfaces such as cell junctions or indir ...
**
Cell adhesion molecule Cell adhesion molecules (CAMs) are a subset of cell surface proteins that are involved in the binding of cells with other cells or with the extracellular matrix (ECM), in a process called cell adhesion. In essence, CAMs help cells stick to each ...


References

{{reflist Cell biology Cell culture techniques