Isolation of cells Cells can be
isolated from tissues for
ex vivo culture in several ways. Cells can be easily purified from blood; however, only the
white cells are capable of growth in culture. Cells can be isolated from solid tissues by digesting the extracellular matrix using
enzymes such as
collagenase,
trypsin, or
pronase, before agitating the tissue to release the cells into suspension. Alternatively, pieces of tissue can be placed in
growth media, and the cells that grow out are available for culture. This method is known as
explant culture. Cells that are cultured directly from a subject are known as primary cells. With the exception of some derived from tumors, most
primary cell cultures have limited lifespan. An established or
immortalized cell line has acquired the ability to proliferate indefinitely either through random mutation or deliberate modification, such as artificial
expression of the
telomerase gene. Numerous cell lines are well established as representative of particular
cell types.
Maintaining cells in culture For the majority of isolated primary cells, they undergo the process of
senescence and stop dividing after a certain number of population doublings while generally retaining their viability (described as the
Hayflick limit). Aside from temperature and gas mixture, the most commonly varied factor in culture systems is the cell
growth medium. Recipes for growth media can vary in
pH, glucose concentration,
growth factors, and the presence of other nutrients. The growth factors used to supplement media are often derived from the serum of animal blood, such as
fetal bovine serum (FBS), bovine calf serum, equine serum, and porcine serum. One complication of these blood-derived ingredients is the potential for contamination of the culture with viruses or
prions, particularly in medical
biotechnology applications. Current practice is to minimize or eliminate the use of these ingredients wherever possible and use human
platelet lysate (hPL). This eliminates the worry of cross-species contamination when using FBS with human cells. hPL has emerged as a safe and reliable alternative as a direct replacement for FBS or other animal serum. In addition,
chemically defined media can be used to eliminate any serum trace (human or animal), but this cannot always be accomplished with different cell types. Alternative strategies involve sourcing the animal blood from countries with minimum
BSE/
TSE risk, such as The United States, Australia and New Zealand, and using purified nutrient concentrates derived from serum in place of whole animal serum for cell culture. Plating density (number of cells per volume of culture medium) plays a critical role for some cell types. For example, a lower plating density makes
granulosa cells exhibit estrogen production, while a higher plating density makes them appear as
progesterone-producing
theca lutein cells. Cells can be grown either in
suspension or
adherent cultures. Some cells naturally live in suspension, without being attached to a surface, such as cells that exist in the bloodstream. There are also cell lines that have been modified to be able to survive in suspension cultures so they can be grown to a higher density than adherent conditions would allow. Adherent cells require a surface, such as tissue culture plastic or
microcarrier, which may be coated with extracellular matrix (such as collagen and laminin) components to increase adhesion properties and provide other signals needed for growth and differentiation. Most cells derived from solid tissues are adherent. Another type of adherent culture is
organotypic culture, which involves growing cells in a three-dimensional (3-D) environment as opposed to two-dimensional culture dishes. This 3D culture system is biochemically and physiologically more similar to
in vivo tissue, but is technically challenging to maintain because of many factors (e.g. diffusion).
Cell culture basal media There are different kinds of cell culture media which being used routinely in life science including the following: •
MEM •
DMEM •
RPMI 1640 •
Ham's f-12 •
IMDM • Leibovitz L-15 • DMEM/F-12 • GMEM
Components of cell culture media Typical Growth conditions Cell line cross-contamination Cell line cross-contamination can be a problem for scientists working with cultured cells. Studies suggest anywhere from 15 to 20% of the time, cells used in experiments have been misidentified or contaminated with another cell line. Problems with cell line cross-contamination have even been detected in lines from the
NCI-60 panel, which are used routinely for drug-screening studies. Major cell line repositories, including the
American Type Culture Collection (ATCC), the European Collection of Cell Cultures (ECACC) and the German Collection of Microorganisms and Cell Cultures (DSMZ), have received cell line submissions from researchers that were misidentified by them. Such contamination poses a problem for the quality of research produced using cell culture lines, and the major repositories are now authenticating all cell line submissions. ATCC uses
short tandem repeat (STR)
DNA fingerprinting to authenticate its cell lines. To address this problem of cell line cross-contamination, researchers are encouraged to authenticate their cell lines at an early passage to establish the identity of the cell line. Authentication should be repeated before freezing cell line stocks, every two months during active culturing and before any publication of research data generated using the cell lines. Many methods are used to identify cell lines, including
isoenzyme analysis,
human lymphocyte antigen (HLA) typing, chromosomal analysis, karyotyping, morphology and
STR analysis.
Other technical issues As cells generally continue to divide in culture, they generally grow to fill the available area or volume. This can generate several issues: • Nutrient depletion in the growth media • Changes in pH of the growth media • Accumulation of
apoptotic/
necrotic (dead) cells • Cell-to-cell contact can stimulate cell cycle arrest, causing cells to stop dividing, known as
contact inhibition. • Cell-to-cell contact can stimulate
cellular differentiation. •
Genetic and
epigenetic alterations, with a
natural selection of the altered cells potentially leading to overgrowth of abnormal, culture-adapted cells with decreased differentiation and increased proliferative capacity. The choice of
culture medium might affect the
physiological relevance of findings from cell culture experiments due to the differences in the nutrient composition and concentrations. A systematic bias in generated datasets was recently shown for
CRISPR and
RNAi gene silencing screens, and for metabolic profiling of cancer
cell lines. and Human Plasma Like Medium (HPLM), were developed.
Manipulation of cultured cells Among the common manipulations carried out on culture cells are media changes, passaging cells, and transfecting cells. These are generally performed using tissue culture methods that rely on
aseptic technique. Aseptic technique aims to avoid contamination with bacteria, yeast, or other cell lines. Manipulations are typically carried out in a
biosafety cabinet or
laminar flow cabinet to exclude contaminating micro-organisms.
Antibiotics (e.g.
penicillin and
streptomycin) and antifungals (e.g.
amphotericin B and
Antibiotic-Antimycotic solution) can also be added to the growth media. As cells undergo metabolic processes, acid is produced and the pH decreases. Often, a
pH indicator is added to the medium to measure nutrient depletion.
Media changes In the case of adherent cultures, the media can be removed directly by aspiration, and then is replaced. Media changes in non-adherent cultures involve centrifuging the culture and resuspending the cells in fresh media.
Passaging cells Passaging (also known as subculture or splitting cells) involves transferring a small number of cells into a new vessel. Cells can be cultured for a longer time if they are split regularly, as it avoids the senescence associated with prolonged high cell density. Suspension cultures are easily passaged with a small amount of culture containing a few cells diluted in a larger volume of fresh media. For adherent cultures, cells first need to be detached; this is commonly done with a mixture of
trypsin-
EDTA; however, other enzyme mixes are now available for this purpose. A small number of detached cells can then be used to seed a new culture. Some cell cultures, such as
RAW cells are mechanically scraped from the surface of their vessel with rubber scrapers.
Transfection and transduction Another common method for manipulating cells involves the introduction of foreign DNA by
transfection. This is often performed to cause cells to
express a gene of interest. More recently, the transfection of
RNAi constructs have been realized as a convenient mechanism for suppressing the expression of a particular gene/protein. DNA can also be inserted into cells using viruses, in methods referred to as
transduction,
infection or
transformation. Viruses, as parasitic agents, are well suited to introducing DNA into cells, as this is a part of their normal course of reproduction.
Established human cell lines cells have been stained with
Hoechst turning their
nuclei blue, and are one of the earliest human cell lines descended from
Henrietta Lacks, who died of cervical cancer from which these cells originated. Cell lines that originate with humans have been somewhat controversial in
bioethics, as they may outlive their parent organism and later be used in the discovery of lucrative medical treatments. In the pioneering decision in this area, the
Supreme Court of California held in
Moore v. Regents of the University of California that human patients have no property rights in cell lines derived from organs removed with their consent. It is possible to fuse normal cells with an
immortalised cell line. This method is used to produce
monoclonal antibodies. In brief, lymphocytes isolated from the
spleen (or possibly blood) of an
immunised animal are combined with an immortal myeloma cell line (B cell lineage) to produce a
hybridoma which has the antibody specificity of the primary lymphocyte and the immortality of the myeloma.
Selective growth medium (HA or HAT) is used to select against unfused myeloma cells; primary lymphoctyes die quickly in culture and only the fused cells survive. These are screened for production of the required antibody, generally in pools to start with and then after single cloning.
Cell strains A cell strain is derived either from a primary culture or a cell line by the selection or cloning of cells having specific properties or characteristics which must be defined. Cell strains are cells that have been adapted to culture but, unlike cell lines, have a finite division potential. Non-immortalized cells stop dividing after 40 to 60 population doublings and, after this, they lose their ability to proliferate (a genetically determined event known as senescence). ==Applications of cell culture==