Suspension culture

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CHO cells in suspension Cells.jpg
CHO cells in suspension

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 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 cells, plant cells, and insect cells. [1] While some cell lines are cultured in suspension, the majority of commercially available mammalian cell lines are adherent. [2] [3] 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). [4] These cultures need to be maintained with nutrient containing media and cultured in a specific cell density range to avoid cell death. [5]

Contents

History

SH-SY5Y cells adhered to a surface SH-SY5Y cells, transmitted light phase gradient contrast microscopy with ZEISS Celldiscoverer 7 (30614936722).jpg
SH-SY5Y cells adhered to a surface

The history of suspension cell culture is closely tied to the overall history of cell and tissue culture. In 1885, Wilhelm Roux 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. [6] Ross Granville 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. [7] Later in 1910, 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. [8] Carrel went on to develop the first known cell line, a line derived from chicken embryo heart which was maintained continuously for 34 years. [9] 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. [10] Notably in 1952, 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. [11]

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. [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 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. [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

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. 201107 shaker.png
Orbital laboratory shaker.

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. [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 (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 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.

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:

List of suspension cell lines

Cell lineMeaningOrganismOrigin tissue Morphology Links
CHO Chinese Hamster OvaryHamsterOvaryEpithelial ECACC Cellosaurus
HeLa "Henrietta Lacks"HumanCervix epitheliumCervical carcinoma ECACC Cellosaurus
H-9HumanEmbryonic stem cellsLymphoblast Cellosaurus
Jurkat HumanWhite blood cellsLymphoblast ECACC Cellosaurus
C6/36Insect - Asian tiger mosquito Larval tissue ECACC Cellosaurus
High Five Insect (moth) - Trichoplusia ni Ovary Cellosaurus
S2 Schneider 2 Insect - Drosophila melanogaster Late stage (20–24 hours old) embryos ATCC Cellosaurus
Sf21 Spodoptera frugiperda 21Insect (moth) - Spodoptera frugiperda Ovary ECACC Cellosaurus
Sf9 Spodoptera frugiperda 9Insect (moth) - Spodoptera frugiperda OvaryEpithelial ECACC Cellosaurus
SH-SY5YHumanBone marrowEpithelial Cellosaurus
PC-3HumanProstateEpithelial Cellosaurus

See also

Related Research Articles

<span class="mw-page-title-main">Tissue culture</span> Growth of tissues or cells in an artificial medium separate from the parent organism

Tissue culture is the growth of tissues or cells in an artificial medium separate from the parent organism. This technique is also called micropropagation. This is typically facilitated via use of a liquid, semi-solid, or solid growth medium, such as broth or agar. Tissue culture commonly refers to the culture of animal cells and tissues, with the more specific term plant tissue culture being used for plants. The term "tissue culture" was coined by American pathologist Montrose Thomas Burrows. This is possible only in certain conditions. It also requires more attention. It can be done only in genetic labs with various chemicals.

Organ culture is the cultivation of either whole organs or parts of organs in vitro. It is a development from tissue culture methods of research, as the use of the actual in vitro organ itself allows for more accurate modelling of the functions of an organ in various states and conditions.

<span class="mw-page-title-main">Cultured meat</span> Animal flesh product created outside of a living animal

Cultured meat is a form of cellular agriculture where meat is produced by culturing animal cells in vitro.

<span class="mw-page-title-main">Chinese hamster ovary cell</span>

Chinese hamster ovary (CHO) cells are an epithelial cell line derived from the ovary of the Chinese hamster, often used in biological and medical research and commercially in the production of recombinant therapeutic proteins. They have found wide use in studies of genetics, toxicity screening, nutrition and gene expression, particularly to express recombinant proteins. CHO cells are the most commonly used mammalian hosts for industrial production of recombinant protein therapeutics.

<span class="mw-page-title-main">Bioreactor</span> System that supports a biologically active environment

A bioreactor refers to any manufactured device or system that supports a biologically active environment. In one case, a bioreactor is a vessel in which a chemical process is carried out which involves organisms or biochemically active substances derived from such organisms. This process can either be aerobic or anaerobic. These bioreactors are commonly cylindrical, ranging in size from litres to cubic metres, and are often made of stainless steel. It may also refer to a device or system designed to grow cells or tissues in the context of cell culture. These devices are being developed for use in tissue engineering or biochemical/bioprocess engineering.

<span class="mw-page-title-main">Cell culture</span> Process by which cells are grown under controlled conditions

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 technique is also called micropropagation. After the cells of interest have been isolated from living tissue, they can subsequently be maintained under carefully controlled conditions. They need to be kept at body temperature (37 °C) in an incubator. These conditions vary for each cell type, but generally consist of a suitable vessel with a substrate or rich medium that supplies the essential nutrients (amino acids, carbohydrates, vitamins, minerals), growth factors, hormones, and gases (CO2, O2), and regulates the physio-chemical environment (pH buffer, osmotic pressure, temperature). Most cells require a surface or an artificial substrate to form an adherent culture as a monolayer (one single-cell thick), whereas others can be grown free floating in a medium as a suspension culture. This is typically facilitated via use of a liquid, semi-solid, or solid growth medium, such as broth or agar. Tissue culture commonly refers to the culture of animal cells and tissues, with the more specific term plant tissue culture being used for plants. The lifespan of most cells is genetically determined, but some cell-culturing cells have been “transformed” into immortal cells which will reproduce indefinitely if the optimal conditions are provided.

<span class="mw-page-title-main">Embryoid body</span> Three-dimensional aggregate of pluripotent stem cells

Embryoid bodies (EBs) are three-dimensional aggregates of pluripotent stem cells.

In biology, a subculture is either a new cell culture or a microbiological culture made by transferring some or all cells from a previous culture to fresh growth medium. This action is called subculturing or passaging the cells. Subculturing is used to prolong the lifespan and/or increase the number of cells or microorganisms in the culture.

Trypsinization is the process of cell dissociation using trypsin, a proteolytic enzyme which breaks down proteins, to dissociate adherent cells from the vessel in which they are being cultured. When added to cell culture, trypsin breaks down the proteins that enable the cells to adhere to the vessel. Trypsinization is often used to pass cells to a new vessel. When the trypsinization process is complete the cells will be in suspension and appear rounded.

<span class="mw-page-title-main">Immunocytochemistry</span>

Immunocytochemistry (ICC) is a common laboratory technique that is used to anatomically visualize the localization of a specific protein or antigen in cells by use of a specific primary antibody that binds to it. The primary antibody allows visualization of the protein under a fluorescence microscope when it is bound by a secondary antibody that has a conjugated fluorophore. ICC allows researchers to evaluate whether or not cells in a particular sample express the antigen in question. In cases where an immunopositive signal is found, ICC also allows researchers to determine which sub-cellular compartments are expressing the antigen.

<span class="mw-page-title-main">Microcarrier</span>

A microcarrier is a support matrix that allows for the growth of adherent cells in bioreactors. Instead of on a flat surface, cells are cultured on the surface of spherical microcarriers so that each particle carries several hundred cells, and therefore expansion capacity can be multiplied several times over. It provides a straightforward way to scale up culture systems for industrial production of cell or protein-based therapies, or for research purposes.

<span class="mw-page-title-main">Plant tissue culture</span> Growing plant cells under known conditions

Plant tissue culture is a collection of techniques used to maintain or grow plant cells, tissues, or organs under sterile conditions on a nutrient culture medium of known composition. It is widely used, to produce clones of a plant in a method known as micropropagation. Different techniques in plant tissue culture may offer certain advantages over traditional methods of propagation, including:

Embryo rescue is one of the earliest and successful forms of in-vitro culture techniques that is used to assist in the development of plant embryos that might not survive to become viable plants. Embryo rescue plays an important role in modern plant breeding, allowing the development of many interspecific and intergeneric food and ornamental plant crop hybrids. This technique nurtures the immature or weak embryo, thus allowing it the chance to survive. Plant embryos are multicellular structures that have the potential to develop into a new plant. The most widely used embryo rescue procedure is referred to as embryo culture, and involves excising plant embryos and placing them onto media culture. Embryo rescue is most often used to create interspecific and intergeneric crosses that would normally produce seeds which are aborted. Interspecific incompatibility in plants can occur for many reasons, but most often embryo abortion occurs In plant breeding, wide hybridization crosses can result in small shrunken seeds which indicate that fertilization has occurred, however the seed fails to develop. Many times, remote hybridizations will fail to undergo normal sexual reproduction, thus embryo rescue can assist in circumventing this problem.

<span class="mw-page-title-main">Moss bioreactor</span>

A moss bioreactor is a photobioreactor used for the cultivation and propagation of mosses. It is usually used in molecular farming for the production of recombinant protein using transgenic moss. In environmental science moss bioreactors are used to multiply peat mosses e.g. by the Mossclone consortium to monitor air pollution.

Schneider 2 cells, usually abbreviated as S2 cells, are one of the most commonly used Drosophila melanogaster cell lines. S2 cells were derived from a primary culture of late stage Drosophila melanogaster embryos by Dr. Imogene Schneider, likely from a macrophage-like lineage.

A 3D cell culture is an artificially created environment in which biological cells are permitted to grow or interact with their surroundings in all three dimensions. Unlike 2D environments, a 3D cell culture allows cells in vitro to grow in all directions, similar to how they would in vivo. These three-dimensional cultures are usually grown in bioreactors, small capsules in which the cells can grow into spheroids, or 3D cell colonies. Approximately 300 spheroids are usually cultured per bioreactor.

A Hollow fiber bioreactor is a 3 dimensional cell culturing system based on hollow fibers, which are small, semi-permeable capillary membranes arranged in parallel array with a typical molecular weight cut-off (MWCO) range of 10-30 kDa. These hollow fiber membranes are often bundled and housed within tubular polycarbonate shells to create hollow fiber bioreactor cartridges. Within the cartridges, which are also fitted with inlet and outlet ports, are two compartments: the intracapillary (IC) space within the hollow fibers, and the extracapillary (EC) space surrounding the hollow fibers.

<span class="mw-page-title-main">Cellular agriculture</span> Production of agriculture products from cell cultures

Cellular agriculture focuses on the production of agricultural products from cell cultures using a combination of biotechnology, tissue engineering, molecular biology, and synthetic biology to create and design new methods of producing proteins, fats, and tissues that would otherwise come from traditional agriculture. Most of the industry is focused on animal products such as meat, milk, and eggs, produced in cell culture rather than raising and slaughtering farmed livestock which is associated with substantial global problems of detrimental environmental impacts, animal welfare, food security and human health. Cellular agriculture is a field of the biobased economy. The most well known cellular agriculture concept is cultured meat.

Entomoculture is the subfield of cellular agriculture which specifically deals with the production of insect tissue in vitro. It draws on principles more generally used in tissue engineering and has scientific similarities to Baculovirus Expression Vectors or soft robotics. The field has mainly been proposed because of its potential technical advantages over mammalian cells in generating cultivated meat. The name of the field was coined by Natalie Rubio at Tufts University.

<span class="mw-page-title-main">Adherent culture</span>

Adherent cell cultures are a type of cell culture that requires cells to be attached to a surface in order for growth to occur. Most vertebrate derived cells can be cultured and require a 2 dimensional monolayer that to facilitate cell adhesion and spreading. Cell samples can be taken from tissue explants or cell suspension cultures. Adherent cell cultures with an excess of nutrient-containing growth medium will continue to grow until they cover the available surface area. Proteases like trypsin are most commonly used to break the adhesion from the cells to the flask. Alternatively, cell scrapers can be used to mechanically break the adhesion if introducing proteases could damage the cell cultures. Unlike suspension cultures, the other main type of cell culture, adherent cultures require regular passaging performed using mechanical or enzymatic dissociation. The culture can be visualized using an inverted microscope, however the growth of adherent cultures is dependent on the available surface area. For this reason, adherent cell cultures are not commonly used to obtain a high yield of cells, instead the use of suspension cultures is preferred.

References

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