In vitro cell culture is a method used for studying the behavior of animal cells in a controlled environment, free of systemic variations. Currently, different types of cell cultures have been adapted and developed. Animal cell cultures have been applied for studying basic cell biology, interactions of drugs and other chemicals with cells, production of vaccines and proteins, etc. This chapter covers a brief summary of the main features, types, requirements and applications of cell culture methodology. Also, we describe applications and principles for cell separation techniques, ranging from basic to more advanced applications.
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Cell culture was developed in the early twentieth century as a method to study the behavior of animal cells in an environment free of the systemic changes that can be found in an animal during the normal homeostasis and stress of an experiment. The first model chosen for cell culture were amphibian cells, presumably for being exothermic animals and given that their cells would not require successive incubations. Later, medical science breakthroughs led to an interest in endothermic animals, where the normal and pathological development is similar to that of humans. The advent of mouse strains genetically pure brought mammals to the research laboratories. While the embryos provided a wide range of cell types in primary culture, mouse models had the advantage of continuous cell lines and a substantial tumor cell repertoire.
The progression of cell culture as a modern and sophisticated technique is based mostly on the needs of two major medical branches: virology and oncology. Cell culture has also been welcomed in many medicine and manufacturing routine applications. Cytogenetically analysis of amniocentesis derived-cells has the ability to disclose genetic disorders in the fetus. Likewise, viral infections can be evaluated quantitatively and qualitatively in host-cultured cells. Toxic effects of potential pharmaceutical compounds and contaminants can be evaluated also by using cell cultures.
The main uses of cell culture systems include:
Experimental model systems in basic and medical sciences. Cell culture offers certain advantages over the environmental and biological variability of other models. In addition, the use of genetically defined and characterized cell lines can simplify the analysis of experimental data. On the other hand, results obtained with specific cellular systems may not be representative of a wide range of other types of cells.
Study of physiological requirements for certain cell types. These include studies on positive effects of growth factors, growth-promoting substances, negative effects of cytotoxic compounds or xenobiotics and events related to programmed cell death (apoptosis), as well as cell proliferation, cell activation, cell signaling or any other cellular process.
Studies of cell development and differentiation. these include aspects of cell cycle and gene expression.
Pathological studies. these are enclosed in the characterization of cells using karyotyping to determine their genetic status.
Genetic Manipulation. cell culture techniques have played an essential role in the development of molecular biology, through the development of methods such as transfection.
Biotechnology. based on the manufacturing and industrial production of therapeutic proteins, vaccines and monoclonal antibodies.
Cellular systems can be established from whole organisms (e.g., chicken embryo), discrete organs (e.g., mouse liver), or blood cells (e.g., lymphocytes). While, in theory, it is possible to grow nucleated cells from any source, in practice, the highest probability of success is achieved with active young cells. The main considerations for the development of cell cultures are:
Biosafety. It is important to be aware of the potential infection risks when culturing animal cells. Although animal cells have a reduced risk of disease transmission compared to human cells, cultures should always be handled as a potential source of pathogenic microorganisms.
Using primary culture or continuous cell lines. freshly isolated cells more easily reflect the biochemical dynamics of the cells in vivo, although having a limited life span. Continuous cell lines are easy to use and offer the advantage of a priori knowledge of their specific growth requirements.
Culture media requirements. These include the provision of inorganic ions (such as a balanced salt solution), a carbon source, organic nutrients and other supplements that include antimicrobial agents to counteract the risk of contamination. For growth support, usually a basal medium supplemented with serum (e.g., fetal bovine serum) or a homologous serum supplement with a set mixture composition of proteins, polypeptides, hormones, lipids, and trace elements. Levels of CO2 and O2 should be considered; many cultures are buffered with bicarbonate and should be kept in a CO2 rich atmosphere.
Clearly, the study of cellular activity in vitro has several advantages and disadvantages. The main advantage is the consistency and reproducibility of results that can be obtained from using a batch of clonal cells. Cell cultures have a highly control of the physicochemical environment (i.e., pH, temperature, osmotic pressure, oxygen, and carbon dioxide tension) which can be controlled very accurately, and the control of physiological conditions, which can be constantly examined.
The disadvantages of cell culture are: highly skilled personnel, techniques must be performed using strict asepsis techniques because animal cells grow slower than many of the common contaminants (e.g., bacteria, viruses and fungi). Additionally, animal cells may not survive when isolated and therefore are not capable of an independent sustainable existence without providing a complex environment. One of the main limitations of cell culture is the expense and effort that has to be applied to obtain a relatively low amount of cells.
In addition, tissue composition is variable and heterogeneous. Replicas from the same sample have various constituents. To replicate an experimental result, cell lines must be manipulated many times in serial passages. For instance, every culture is going to be different from the original and less uniform in its constitution. In order to resolve this issue, the replicas are randomly mixed in each passage and the selective pressure of growing conditions tends to produce an optimal prevalent phenotype.
Cell culture is a collection of techniques and resources in which cells that were part of an organism are growth in an artificial controlled environment. Usually, tissue must be previously treated to disrupt it by mechanical or enzymatic processes depending on the origin of the tissue and the purpose of the cell culture. However, some cells can be culture without this treatment such as in the case of liquid samples.
During tissue culture, tissue characteristics and architecture must be retained, at least partially. For instance, growth is slow and restricted to the use of embryonic tissue or 3D cell culture procedures. The 3D cell culture mimics the microenvironment, architectural design and functionality of a normal tissue under control conditions. In some cases, cell migration is observed through the solid phase when a piece of tissue is placed on a solid liquid-interface. Thus generating primary explants that grow outside the original tissue
The Primary Culture is the first culture that grows successfully after the cell isolation from a tissue. As mentioned above, cell should be subculture in a series of passages in order to keep the best condition for cell growth. As result, these cell lines go into senescence after the thirtieth division cycle. For this reason, cells are storage in a cell bank system to maintain them for long periods of time. In some cases, the cells can be immortalized, e.g., B cell lymphocyte can be immortalized with the Epstein- Barr virus to confer them the ability to proliferate indefinitely. These transformed cells have the advantage of unlimited availability, but they have the disadvantage of losing its initial characteristics. shows the main ways to start cell cultures (for further information please see recommended readings).
Main type of sources for cell cultures. Adapted from (1).
Cell culture refers to the removal of cells from an animal or plant and their subsequent growth in a favorable artificial environment. The cells may be removed from the tissue directly and disaggregated by enzymatic or mechanical means before cultivation, or they may be derived from a cell line or cell strain that has already been established.
Primary CulturePrimary culture refers to the stage of the culture after the cells are isolated from the tissue and proliferated under the appropriate conditions until they occupy all of the available substrate (i.e., reach confluence). At this stage, the cells have to be subcultured (i.e., passaged) by transferring them to a new vessel with fresh growth medium to provide more room for continued growth.
Cell LinesAfter the first subculture, the primary culture becomes known as a cell line or subclone. Cell lines derived from primary cultures have a limited life span (i.e., they are finite; see below), and as they are passaged, cells with the highest growth capacity predominate, resulting in a degree of genotypic and phenotypic uniformity in the population.
Cell StrainIf a subpopulation of a cell line is positively selected from the culture by cloning or some other method, this cell line becomes a cell strain. A cell strain often acquires additional genetic changes subsequent to the initiation of the parent line.
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Normal cells usually divide only a limited number of times before losing their ability to proliferate, which is a genetically determined event known as senescence; these cell lines are known as finite. However, some cell lines become immortal through a process called transformation, which can occur spontaneously or can be chemically or virally induced. When a finite cell line undergoes transformation and acquires the ability to divide indefinitely, it becomes a continuous cell line.
Culture conditions vary widely for each cell type, but the artificial environment in which the cells are cultured invariably consists of a suitable vessel containing the following:
Most cells are anchorage-dependent and must be cultured while attached to a solid or semi-solid substrate (adherent or monolayer culture), while others can be grown floating in the culture medium (suspension culture).
If a surplus of cells are available from subculturing, they should be treated with the appropriate protective agent (e.g., DMSO or glycerol) and stored at temperatures below '130°C (cryopreservation) until they are needed. For more information on subculturing and cryopreserving cells, refer to the Guidelines for Maintaining Cultured Cells.
Cells in culture can be divided into three basic categories based on their shape and appearance (i.e., morphology).
Fibroblastic (or fibroblast-like) cells are bipolar or multipolar, have elongated shapes, and grow attached to a substrate.
Epithelial-like cells are polygonal in shape with more regular dimensions, and grow attached to a substrate in discrete patches.
Lymphoblast-like cells are spherical in shape and usually grown in suspension without attaching to a surface.
Cell culture is one of the major tools used in cellular and molecular biology, providing excellent model systems for studying the normal physiology and biochemistry of cells (e.g., metabolic studies, aging), the effects of drugs and toxic compounds on the cells, and mutagenesis and carcinogenesis. It is also used in drug screening and development, and large scale manufacturing of biological compounds (e.g., vaccines, therapeutic proteins). The major advantage of using cell culture for any of these applications is the consistency and reproducibility of results that can be obtained from using a batch of clonal cells.
This video provides an overview of the basic equipment used in cell culture and proper laboratory set-up. Guidance on how to work safely and aseptically in a cell culture hood is introduced and demonstrated.
This video is focused on the steps you should take to prevent contamination of your cell culture. All of the basic actions required to perform cell culture using best practice sterile techniques are demonstrated.
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