Adherent cell cultures are a type of

cell culture Cell culture or tissue culture is the process by which cell (biology), cells are grown under controlled conditions, generally outside of their natural environment. After cells of interest have been Cell isolation, isolated from living tissue, ...

that requires

cells Cell most often refers to: * Cell (biology), the functional basic unit of life * Cellphone, a phone connected to a cellular network * Clandestine cell, a penetration-resistant form of a secret or outlawed organization * Electrochemical cell, a d ...

to be attached to a surface in order for growth to occur. Most vertebrate-derived cells (with the exception of

hematopoietic cell A blood cell (also called a hematopoietic cell, hemocyte, or hematocyte) is a cell produced through hematopoiesis and found mainly in the blood. Major types of blood cells include red blood cells (erythrocytes), white blood cells (leukocytes) ...

s) are anchorage dependent and require a two-dimensional monolayer to facilitate cell adhesion, spreading and replication. Cell samples can be taken from tissue explants or cell suspension cultures. Adherent cell cultures with an excess of nutrient-containing

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''. Differe ...

will continue to grow until they cover the available surface area.

Protease A protease (also called a peptidase, proteinase, or proteolytic enzyme) is an enzyme that catalysis, catalyzes proteolysis, breaking down proteins into smaller polypeptides or single amino acids, and spurring the formation of new protein products ...

s 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 In biology, a subculture is either a new cell culture or a microbiological culture made by transferring some or all cell (biology), cells from a previous culture to fresh growth medium. This action is called subculturing or passaging the cells. Su ...

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.

Methods and maintenance

Isolating cells

Primary cells used for adherent cultures must be isolated and treated from a subject, or may be transferred from pre-existing cell lines. Adherent cells must first be transferred to a

monolayer A monolayer is a single, closely packed layer of entities, commonly atoms or molecules. Monolayers can also be made out of cells. ''Self-assembled monolayers'' form spontaneously on surfaces. Monolayers of layered crystals like graphene and molyb ...

attached to a surface, and are categorized by their morphological differences. Fibroblast-like adherent cells have a linear and stretched shape, and

migrate Migration, migratory, or migrate may refer to: Human migration * Human migration, physical movement by humans from one region to another ** International migration, when peoples cross state boundaries and stay in the host state for some minimum le ...

when attached to the monolayer.

Epithelial Epithelium or epithelial tissue is a thin, continuous, protective layer of cells with little extracellular matrix. An example is the epidermis, the outermost layer of the skin. Epithelial ( mesothelial) tissues line the outer surfaces of man ...

-like adherent cells have a wider and polygonal shape, and do not migrate when attached to the monolayer. Once cells are properly isolated from their source and are transferred to the media, cell

can be conducted.

Adherent cell culture maintenance for laboratories

While

adherent cell cultures, spent media must be repeatedly pipetted out and replaced with fresh media. The culture vessel can also be repeatedly tapped, which should be combined with either mechanical or enzymatic methods to facilitate cell detachment. The culture vessel can also be centrifuged, forming a

supernatant In an aqueous solution, precipitation is the "sedimentation of a solid material (a precipitate) from a liquid solution". The solid formed is called the precipitate. In case of an inorganic chemical reaction leading to precipitation, the chemic ...

that can be extracted using a pipette. Cells must be fed 2 to 3 times per week, and must be cultured at an appropriate temperature, humidity, light, and pH in order to ensure optimal

cell proliferation Cell proliferation is the process by which ''a cell grows and divides to produce two daughter cells''. Cell proliferation leads to an exponential increase in cell number and is therefore a rapid mechanism of tissue growth. Cell proliferation ...

Passaging (subculturing) cells

While adherent cultures share similarities with suspension cultures, there are many key differences in how they are cultured and passaged. For adherent culture passaging, the spent media is first pipetted out of the flask containing cells as a waste product. Cells are adhered to the media that was not removed in a culture vessel, and a series of wash and incubation steps are then necessary to detach the cells. For the wash steps, a balanced salt solution is poured to the side opposite the cell culture, and the culture vessel is then shaken before draining the balanced salt solution. Heat is applied to the culture vessel for the incubation steps, causing protein denaturation and the gradual separation of the cells from the media. Similarly to suspension cultures, the total number of cells can then be calculated using a

hemocytometer The hemocytometer (or haemocytometer, or Burker's chamber) 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 mi ...

and trypan blue. Epithelial cells that reach confluence normally swiftly stop dividing due to

contact inhibition In cell biology, contact inhibition refers to two different but closely related phenomena: contact inhibition of locomotion (CIL) and contact inhibition of proliferation (CIP). CIL refers to the avoidance behavior exhibited by fibroblast-like cell ...

of growth related to the formation of intercellular

adherens junctions In cell biology, adherens junctions (or zonula adherens, intermediate junction, or "belt desmosome") are protein complexes that occur at cell–cell junctions and cell–matrix junctions in epithelial and endothelial tissues, usually more basal ...

Commercial applications and limitations

Adherent cultures are most commonly used for

cytology Cell biology (also cellular biology or cytology) is a branch of biology that studies the structure, function, and behavior of cells. All living organisms are made of cells. A cell is the basic unit of life that is responsible for the living an ...

and for harvesting cellular products on a small scale. Since their growth is limited to 2D, it is difficult to use adherent cultures to study in-vivo cell structure and function. Research is being done to grow adherent cell cultures using 3D microcarriers in order to avoid this limitation and to use adherent cell cultures for drug testing. Commercial applications of adherent cultures include: * Producing adherent cells that create proteins of interest used for vaccine development. * Adherent cells used in conjunction with viral vectors for cell and gene therapy. * Delivering micro and nanotechnology to adherent cells in vitro. * Adjusting adherent cell morphology for cancer cell screening.

References

{{Reflist Cell culture