Cell culture techniques for viruses

When cultures are established initially from tissue taken directly from animals often from fetal organs or tissues , they contain several cell types, most of which are capable of only limited growth in vitro —perhaps 5 or 10 divisions at most. This restricts their value, whether for routine diagnostic work or vaccine production, because of the high cost and inconvenience of having to obtain fresh tissue each time, as well as lack of consistency from batch to batch.

Furthermore, the donor animals often harbor latent viruses which can confuse diagnosis or contaminate vaccines. Nevertheless, the presence of a diverse range of differentiated cell types in such primary cultures means that they tend to be very sensitive to many animal viruses. In veterinary diagnostic virology, it is common practice to inoculate samples suspected to contain virus into primary cultures derived from the same species of animal as that providing the samples.

These are cells that are capable of undergoing a number of divisions in culture that is roughly related to the life span of the species of animal—about 50 for fetal human cells and about 10 for fetal cells from horses and cows. They retain their original diploid chromosome number throughout.

Diploid strains of fibroblasts established from human fetuses or embryos are widely used in human diagnostic virology and vaccine production, but diploid strains have not been much used in veterinary vaccine production. These are cells of a single type that are capable of indefinite propagation in vitro.

Such immortal cell lines originate from cancers, or by spontaneous transformation of a diploid cell strain. Often they no longer bear close resemblance to their cell of origin, as they undergo many mutations during their prolonged culture. The usual indication of these changes is that the cells have lost the specialized morphology and biochemical abilities that they possessed as differentiated cells in vivo. For example, it is no longer possible to distinguish microscopically between the epithelial cell lines arising from various cells of ectodermal or endodermal origin, or between the fibroblastic cell lines arising from cells of mesodermal origin.

Cells of continuous cell lines are often aneuploid in chromosome number, especially if of malignant origin. Continuous cell lines derived from monkey e. The great advantage of continuous cell lines over primary cell cultures is that they can be propagated indefinitely by subculturing the cells at regular intervals.

Like other cells, they retain viability for many years when frozen in serum-containing medium with added dimethylsulfoxide and stored at very low temperature, e. Good laboratories follow the general microbiological precept that the surest way of faithfully maintaining the characteristics of a cultured cell line is to replace it periodically from frozen stocks.

Some continuous cell lines have been adapted to grow as suspensions of single cells. Such suspension cultures are particularly useful for biochemical studies of viral replication, because large numbers of identical cells are continuously available for regular sampling and processing.

Various methods have been devised to maximize the surface area to which cells can attach, while keeping the overall size of the vessel and the volume of medium within reasonable bounds. Round bottles can be continuously rolled, or may be filled with glass tubes, glass beads, or spiral plastic film.

Perhaps the most useful method for growing cells on a large scale for vaccine production is on plastic or Sephadex beads microcarriers maintained in suspension in large fermentation tanks. Of paramount inportance for virologists is the selection of cell lines that will allow optimal growth of the virus under study. Some viruses replicate in almost any cell line and some cell lines support the replication of many different types of viruses. On the other hand, many viruses are quite restricted in the kinds of cells in which they can be isolated from an infected animal.

On adaptation by serial passage, however, mutants with somewhat greater growth potential for a given cell line can be selected.

A useful general rule is to use a cell strain of low passage number from the same animal species for primary isolation of a virus. Once the virus has been isolated, alternative more convenient cell substrates may be sought. Cultured cells serve three main purposes: 1 isolation of viruses from clinical specimens see Chapter 13 , for which purpose a type of cell culture should be selected which is known for its high sensitivity and in which cell abnormality is readily recognized, 2 production of vaccines and antigens for serological diagnosis, for which the principal requirement is for a cell line giving a high yield of virus and free from contaminating agents see Chapter 14 , and 3 biochemical studies of viral replication see Chapter 4 , for which continuous cell lines, preferably growing as suspension cultures, are usually chosen.

The growth of viruses in cell culture can be monitored by a number of biochemical procedures indicative of the intracellular increase in viral macromolecules and virions see Chapter 4. In addition, there are simpler methods that are more commonly used for diagnostic work see Chapter 13 , as outlined below. Many viruses kill the cells in which they replicate, so that infected cell monolayers gradually develop visible evidence of cell damage, as newly formed virions spread to involve more and more cells in the culture.

These changes are known as cytopathic effects CPE , and the responsible virus is said to be cytopathogenic. Most cytopathic effects can be readily observed in unfixed, unstained cell cultures, under low power of the light microscope with the condenser racked down and the iris diaphragm partly closed to obtain the required contrast. A trained virologist can distinguish several types of cytopathic effects, even in unstained, living cultures Plate , Table Fixation and staining of the cell monolayer reveals further diagnostic details, notably inclusion bodies Plate , Fig.

These morphological consequences of viral infection are discussed in Chapter 6. Cytopathic effects produced by different viruses. A Enterovirus—rapid rounding of cells, progressing to complete cell destruction. B Herpesvirus—focal areas of enlarged, rounded cells.

C Paramyxovirus—focal areas of cells are fused to form syncytia or giant cells. D Hemadsorption. Erythrocytes adsorb to those cells in the monolayer that are infected. The technique is applicable to any virus that incorporated hemagglutinin into the plasma membrane. Most of the enveloped viruses that mature by budding from the cell membranes produce hemadsorption. Cultured cells infected with orthomyxoviruses, paramyxoviruses, or togaviruses, all of which bud from cytoplasmic membranes, acquire the ability to adsorb erythrocytes.

This phenomenon, know as hemadsorption, is due to the incorporation into the plasma membrane of newly synthesized viral protein that binds red blood cells Plate D. Hemadsorption can be used to demonstrate infection with noncytopathogenic as well as cytocidal viruses, and can be demonstrated very early, e.

Hemagglutination is a different, though related phenomenon, in which erythrocytes are agglutinated by free virus see below. The principle of cell culture was established when Roux, an embryologist used warm saline to maintain chicken embryo for several days, thereby, coming up with tissue culture principle [ 2 ].

Cell culture has therefore, been defined as the is removal of animal cells and its propagation and cultivation in vitro in an artificial environment that is suitable for its growth [ 3 , 4 ]. This usually begins with a primary culture aiming at achieving confluence, that is formation of monolayer of cell in a culture flask supplemented the required nutrients and growth factors.

With achievement of confluence, the cells are then passaged or sub cultured from the primary to secondary and subsequence to tertiary, until a continuous cell line is established. The isolation of virus in a cell culture is labour-intensive, and consumes time [ 5 , 6 ]. These might reduce the impact of tissue culture in clinical diagnosis, thereby making it less attractive in diagnosing human diseases [ 5 , 7 ] while, some scientist found tissue culture as a relatively unbiased, whose limitation is only by the ability of the virus to grow on the selected cell lines [ 8 , 9 ].

However, Vero E6 cells were considered as the most permissive of all cell lines by providing a versatile medium for recovery of unknown pathogens, together with Electron Microscopy EM to the detection and classification of unknown agent [ 10 , 11 ]. Observation of cell culture via EM can provide early clues on aetiologic agent and subsequently guide laboratory and epidemiologic investigations. This is of clinical important once most specially, during diseases outbreak since knowing the aetiologic agent will assist public health officials to institute a timely response and prevent or limit further spread of the causative agent [ 12 , 13 ].

Therefore, the use of classical techniques of viral isolation in tissue culture and examination under EM is said to be critical in detection of viruses that were previously unrecognized as such. Contrary to the earlier view, cell culture is a fundamental technique that can be accomplished in hospital diagnostics and microbiology laboratories if infectious viral agent is suspected.

This technique was used in discovering Ebola virus in a suspected yellow fever patient and vice versa in several studies [ 14 — 17 ]. Recent advances in metagenomics with deep sequencing techniques have made it possible to analyse the genome of microorganism without isolating the virus via cell culture.

This is done via high-throughput sequencing using random amplified DNA product and comparison of sequences with available extensive bank of sequences for the final identification of the detected agent.

This is possible because random primers can specifically amplify the template for sequencing without having a prior knowledge of the suspected agent [ 18 — 20 ]. This technique is readily advancing in the aspect of pathogen discovery. It has been used forever to discover viruses such as Lioviu virus [ 21 ], Schmallenberg virus [ 22 ] and Bas —Congo virus [ 23 ]. In the cases of severely ill patients or infectious diseases outbreak, it is important to identify the causative agent of infection.

As such this review is aimed at describing some of the events in which viruses are isolated for identifying the causative agent and recognition of emerging diseases, by additional laboratory diagnosis assay such as Electron Microscope EM , serological and molecular techniques. Inoculation of clinical specimens from a patient on to the culture cells enables biological amplification of the virus to the level at which it can be detected or viewed under EM and further confirmed by other techniques such as serology, immunohistochemistry as well as fluorescence antibody assays and molecular methods leading to further characterization of the species and strain of the virus [ 24 — 26 ].

However, the use and relative importance of virus culture has been on the decline due to development of rapid and accurate molecular techniques [ 28 — 30 ]. Therefore, the aim of this review is to critically summarize the views of researchers on the role of cell culture technology in diagnosis of human diseases.

All searches were limited to publication from to except were necessary an older publication might be consider. All publications were in English and duplicates were removed. The final articles searched were those published till 31 st May The online database serch resulted in articles which were screened base on the title and abstrcts relevance, exluding conference abstract, comments and short communications retaining for full text review studies.

Electron Microscopy EM and cell culture isolation are instrumental in finding the causative agent in an unusual clinical manifestation.

One of the studies reports the isolation of Bunya virus in patient with history of tick bites [ 31 ]. The cells were then processed for examination with EM after which a Bunya virus was observed rather than the bacteria that was suspected. The virus envelope is spherical with some projection on the surface of the virus particles and the virus has granular core.

The infected cells show visible granular particles and differentiated into macrophages with elongated pseudopodia. Cell culture and real time reverse transcription polymerase chain reaction qRT-PCR have been broadly used in clinical settings for identifying influenza viruses [ 32 , 33 ]. Although, time consuming and labor intensive and required high skill personnel with specific laboratory equipment and condition, making it not suitable for primary health care settings and low income countries.

Nevertheless, cell culture is still important in confirming causative agent of infection in an outbreak. Cell cultures metabolomics can be used for identifying biomarkers of a pathological condition as well as metabolic path ways that produce such biomarkers. Metabolites play an important role in cancer diagnosis, recurrences and prognosis by identifying novel cancer biomarkers.

A slight change in metabolism can be detected in products of cellular process leading to development of prognostic models that will be useful for early detection of cancer.

Several studies examined the ability of human cancer cells to secrete volatile organic compounds [ 35 , 36 ], some of which were able to detect acetaldehyde release from lung cancer cell lines CALU-1 and SK-MES [ 37 , 38 ]. With the existence of commercially produced, cultivated cell lines which are used for rapid detection of a variety of viruses like R-Mix Diagnostic hybrid, Inc which is a mixture of monolayers of cells which are selected based on their capability of isolating different viruses causing respiratory tract infection.

R-Mix contains tissue from the lungs MV1LU and A cells as fresh cells readily for use, or frozen cell suspension that can be aliquoted by the labouratory or as frozen monolayers in a shell vials ready to use. R mixed has therefore been reported to offer a fast and time sensitive technique of identifying viruses that are commonly involved in causing respiratory infection with no specialized skills required [ 39 , 40 ].

Transgenic technology in cell culture involves incorporation of stable genomic materials into the cell so that once a particular virus enters the cell, it triggeres, production of virus specific enzymes that is easily measurable [ 41 , 42 ]. The genetic materials can be of viral, bacterial or cellular origin and referred to as virus inducible reporter gene segment [ 43 , 44 ]. In diagnostic laboratory transgenic cells can only be useful if they have the desirable promoter which is quite in cells that are infected but significantly up regulated by means of viral trans-activator protein in a manner that is specific, but not allowing heterologous viral transactivation protein to stimulate the promoter.

For a transgenic system to work, the virus to be detected must be able to adhere to the cell wall and prime its replication cycle without reaching the finishing point but adequate to activate the gene through the promoter. This makes the use of genetically improved cell line in improving growth of viruses possible, thereby, facilitating the detection of cells infected with viruses thus, providing the detection system that is specific, sensitive and very simple to perform [ 45 , 46 ]. This technology was applied successfully in identifying the polio virus via the use of transforming cells susceptible HeLa cells [ 47 ].

However, its identification is by staining with monoclonal antibodies and it can be detected within 16 to 24 hours inoculation [ 48 , 49 ]. Conversely, a more rapid transgenic system capable of easily detecting HSV within 24 was developed in such a way that it does not require medical expertise or expensive monoclonal antibodies. It involves the use of UL39 derived HSV promoter which codes for large ribo nucleotide reeducates sub unit [ 50 , 51 ].

Recombinant protein technology is important in meeting the demand for easy to use, fast and reliable test in diagnostic laboratory and has been useful for serological survey of infection [ 52 ]. Recombinant protein can be expressed and used for the detection of influenza virus antibody. It was sequenced and confirmed to be in frame with the N-terminal, together with proper orientation.

The recombinant plasmids were then transformed into the host cell strain B12 DE3 pLysS for expression. Transformation process was achieved using heat shock method. This confirmed that the antigen could be used to detect specific antibodies against influenza viruses using ELISA, which has a considerable advantage over other techniques for detecting specific antibodies.

If the hemagglutinating virus is unknown, it can be identified by using a panel of known antibodies. A virus neutralization assay is used in conjunction with an infectivity assay, such as the plaque assay described above. This assay detects antibody that is capable of inhibiting virus replication or in other words, antibody that can neutralize virus infection. Virus neutralization is a specialized type of immunoassay because it does not detect all antigen—antibody reactions.

It only detects antibody that can block virus replication. This is important because related groups of viruses may share common antigens, but only a fraction of these antigens are targets of neutralizing antibody.

A virus serotype is usually based on virus neutralization although this is not always specified. For example, there are three major poliovirus serotypes neutralization serotypes. In order to protect against poliovirus infection, a successful vaccine must induce neutralizing antibodies to poliovirus Types 1, 2, and 3. The tag can be an enzyme that cleaves a substrate substrate turns color upon cleavage , a radioactive isotope, or a fluorescent molecule. Serologic assays are commonly used in research and diagnostic laboratories.

As proteins readily bind to many types of glass or plastic, the adsorption part of the assay is straightforward. These enzymes relatively stable, cheap, and easy to purify, and they can be chemically linked to an antibody to aid in detection of an immune complex.

There are inexpensive substrates that change from colorless to colored when cleaved by these enzymes. ELISAs can be used to detect either antigen a virus, for example or antibody from a potentially infected individual. Antigen detection ELISAs can be used to quantitate the amount of virus in a cell culture supernatant or patient sample. In this case the antigen is the standardized reagent.

The presence of antibody signifies that the patient has either been infected or vaccinated with the agent in question.

Primary antibodies i. These reagents facilitate detection of viral antigens in a sample. If a fluorescent molecule is attached to the primary antibody, the assay is a fluorescence-linked immunosorbent assay. The primary antibody is untagged, but a tagged secondary antibody is used to detect the presence of primary antibody. For example, if the primary antibody is developed in an immunized rabbit, the secondary antibody is anti-rabbit IgG perhaps obtained from mice immunized with rabbit IgG.

Indirect ELISAs are common because it is cheaper to tag one batch of antirabbit IgG than to tag the specific antibodies directed against hundreds of different viral or other proteins.

An antibody or a serum sample is used to coat the dish or tube. A sample containing an antigen is added and allowed to bind to the antibody. A second antibody is added to the wells. It will bind to the antigen if present. In this example the antigen must be able to interact with two antibodies.

Requiring that two different antibodies react with the same antigen can improve the specificity of the assay. The titer is expressed as the reciprocal of the highest dilution at produces a positive color. Highly specific antibodies tagged with fluorescent molecules can be used to detect the presence of viral antigens in cells.

Assays can be direct using labeled primary antibody or indirect using labeled secondary antibody Fig. The technique allows one to distinguish infected from uninfected cells, thus can be used to perform focus-forming assays. Cell-based IFAs use tagged antibodies to mark infected cells. Direct immunofluorescence used tagged primary antiviral antibodies.

Indirect immunofluorescence uses a secondary antibody that is tagged. Western blots are labor intensive and expensive, but provide a method of confirming the identity of a reactive antigen. Western blots are done by electrophoresis of antigen i. The proteins in a mixture are separated by size in the gel and are transferred from the gel to a solid paper-like substrate often called a membrane.

The membrane is incubated with patient sera and the presence of patient antibodies is detected by subsequently incubating the membrane with labeled secondary antibodies. The specificity of the western blot lies in its ability to demonstrate the molecular weights sizes of the proteins recognized by patient sera. False positive reactions can be identified when the immunoreactive protein band does not correspond in size to known viral proteins.

For diagnostic purposes, a positive western blot may require that more than one viral protein be bound by patient antibodies. The basis of immunohistochemistry is that a tissue section is incubated with enzyme-tagged antibodies. A colorless substrate is added to the sample. If enzyme-tagged antibodies are present, the substrate is cleaved to produce a colored precipitate. This is a powerful technique as it allows one to examine individual virus-infected cells in a tissue section.

Patient samples biopsies are often preserved in formaldehyde or are stored frozen at ultracold temperatures. If these samples are archived, they can be tested for the presence of viral antigen even after years or decades Fig. Immunohistochemistry is a technique used to detect viral antigens in tissue sections. In this example a brain section has been treated with antibodies to a bornavirus.

The antibodies are tagged with an enzyme, producing a brown precipitate that indicates bornavirus-infected cells. PCR is a very sensitive method and uses oligonucleotide primers designed to detect suspect viruses.

PCR assays are very sensitive, but sensitivity can be a disadvantage as well as an advantage. When performing PCR for diagnostic purposes, it is essential that every precaution be taken to avoid contaminating patient samples.

This often requires separate equipment and work areas. It is also important to test all purchased reagents for the presence of contaminating nucleic acids.

This requires multiple negative controls. For example, one might apply sterile buffer or water to a nucleic acid purification column to check the column for contamination with viruses that might have been introduced during the manufacturing process. While PCR amplification requires some prior knowledge of a viral sequence, it is now routine to sequence all nucleic acid DNA and RNA in a sample, using high throughput, unbiased sequencing techniques.

Powerful algorithms are used to analyze the data and compare it to information stored in public databases. Sequences of no interest human DNA in a patient sample can be ignored, allowing the investigator to quickly focus on any viral sequences that may be present. As in the case of PCR, the sensitivity of the assay is both a positive and a negative, and the most careful researchers will include multiple negative controls in their assays. Use of unbiased sequencing has resulted in an explosion of new viruses from humans, animals, and environmental samples.

The current challenge is to develop an understanding of which viruses might be threats and which are part of our normal viral flora. The purpose of diagnostic virology is to identify the agent most likely responsible for causing disease in a human or animal patient. Virus identification can be used to:. For the medical practitioner, methods for identifying the virus in an infected patient ideally should be sensitive, specific, and rapid, as once a patient has recovered or died diagnosis has less practical value although a diagnosis could benefit family and community members.

On the other hand, epidemiologic studies may include hundreds or thousands of samples requiring use of low cost, high throughput modalities. Some are designed to detect viral proteins, enzymes, or genomes directly from a patient sample blood, throat swab and these are useful in a clinical setting. One type of rapid diagnostic assay design lateral flow immunoassay uses the process of diffusion to move a sample across a test chamber.

The liquid sample contacts various dried reagents as it flows through the chamber. Tests can be designed to detect either antigen or antibody from a patient sample.

An example of an antigen capture assay is shown in Fig. The test strip contains three key assay reagents dried on the strip. Closest to the sample addition chamber is an antiviral antibody. In our example the antibody is conjugated to gold nanoparticles. When the liquid from the patient sample is applied, it flows, by capillary action, across the slide and will encounter the labeled antibody.

The labeled antibody is picked up in the flowing liquid and will bind to any viral antigen present in the sample. The liquid continues to move across the slide by capillary action until it hits the Test T strip.

In our example the T strip contains antibody to the virus in question. If the labeled antibody is bound to virus, it will be stopped captured at the test strip. If the labeled antibody is not bound to virus, it will move past the T strip and reach the control C strip. Anti-immunoglobulin antibodies are bound at the C strip and will capture any labeled immunoglobulin in the liquid.

Thus if the sample is positive for virus, a colored line should appear at the T strip and the C strip because there is excess labeled antibody. If the sample is negative for virus, the T strip will remain colorless but the C strip will be positive. An antigen capture, lateral flow immunoassay.

The test strip contains three key assay reagents. In this example a gold-conjugated antibody binds to virus in the test sample. The liquid moves across the slide by capillary action to the Test T strip. The T strip contains membrane bound antiviral antibody. It will bind to capture the virus particles and their associated gold tagged antibodies to generate a visible signal at the T strip. If no virus is present in the sample, the gold-labeled antibody travels past the T strip and binds to anti-immunoglobulin antibodies bound at the C strip.

A positive sample must show color reactions at both the T strip and the C strip. A negative sample must show a color reaction at the C strip to validate that the test worked correctly. Often there is not enough virus present in the infected host to allow for direct detection in a patient sample. In that case the sample may be sent to a diagnostic laboratory for inoculation into cultured cells or fertile eggs to generate higher concentrations of virus.

The infected cultures are closely observed for visible changes, such as cell killing cytopathic effects , changes in cell morphology, or formation of syncytia fused cells. Any of these changes in the infected cell cultures as compared to uninfected controls provides an indication that an agent is replicating, and that further testing is warranted to identify the agent.

Growing viruses is labor intensive, takes days to weeks, and presents biosafety issues. Patient samples are often inoculated into several different types of cells in the hopes that at least one type will be susceptible. Culturing hIPSCs to study inherited liver diseases: Small pieces of skin biopsies obtained from patients with an inherited liver disease can be cultured in a Petri dish with fibroblast growth medium. Primary fibroblast cultures will emerge from the skin tissue after 2—3 days.

This generates a pure population of fibroblasts, which can be scaled-up and virally transduced to express pluripotent genes e. Cells at this stage of differentiation will migrate from their colonies into a monolayer, downregulate their pluripotency genes and express markers of their endoderm fate e.

Further changes in the media composition and exogenous growth factors added to the definitive endoderm cells will direct them toward foregut endoderm and subsequent specification to hepatic endoderm. The final stage of the hepatic differentiation medium yields hepatocyte-like cells that secrete albumin and other serum proteins, take up l ow-density lipoprotein LDL , store glycogen, and metabolize drugs.

Importantly, these cells will also display the disease phenotypes observed in the patient from which the skin biopsy was initially obtained. While this process allows researchers to study and rescue disease mechanisms ex vivo [9] , it also sheds light on liver development and the emergence of disease in utero. One of the pillars of cell culture research is the design of a defined cellular environment in which single variables can be manipulated in order to monitor cellular responses.

To achieve this goal, the cellular environment in vitro is oftentimes oversimplified and relies, for example, on a single cell type cultured in a monolayer. However, data generated from such a cellular system does not truly phenocopy the intricate cellular interactions between different cell types and extracellular matrices of an in vivo environment. To address this drawback, there is currently significant research into the design of cell cocultures that allow paracrine signaling between cells that cohabitate space in vivo, as well as bioartifical matrices that facilitate cellular growth in their native 3D orientation.

The goal is the design of cellular systems that mimic the complexity of the multicellular in vivo niche, yet also allow standardization for cell culture assays. The oftentimes most relevant cell types for addressing translational research questions—primary cells—are in fact very difficult to isolate and culture in vitro due to their limited proliferation and functional capacity ex vivo. To delay senescence, viral transfection of primary cells can sequester tumor-suppressor proteins, thereby extending the number of possible passages and allowing the emergence of immortal cell lines.

Although this facilitates their culture ex vivo, this technique also introduces the expression of carcinogenic genes. In addition, immortalized cell lines can acquire mutations during subculturing that can further interfere with the cellular phenotype and create a nonphysiological cell culture system.

This chapter has described the vast possibilities to employ cell culture techniques to address basic and translational research questions and has explained the necessary considerations for setting up a cell culture lab.

It has also shown essential practices and techniques for successfully working with cell lines and explained the conditions required for creating a cellular environment that mimics their in vivo niche. National Center for Biotechnology Information , U. Basic Science Methods for Clinical Researchers. Published online Apr 7.

Segeritz 1 and Ludovic Vallier 1, 2. Guest Editor s : Francesca Y. Author information Copyright and License information Disclaimer. University of Liverpool, Liverpool, United Kingdom. All rights reserved. Elsevier hereby grants permission to make all its COVIDrelated research that is available on the COVID resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source.

Abstract Cell culture is a very versatile tool in the investigation of basic scientific and translation research questions. Keywords: Cell culture, asepsis, primary cell, contamination, medium, supplements, incubator, biosafety level, hazard group, maintenance.

Introduction Cell culture refers to laboratory methods that enable the growth of eukaryotic or prokaryotic cells in physiological conditions. In Principle The Cell Culture Laboratory Cell Culture Laboratory Safety The exciting application of cell culture techniques in biomedical research requires the management of potential hazards linked to infectious agents harbored by cultured cells e.

Open in a separate window. Figure 9. Routes of exposure to biohazards. Table 9. Upon handling hazardous agents, potentially contaminated gloves must be removed immediately and disposed of in the biohazard waste. Wash hands. Potentially infectious and hazardous material must be decontaminated and disposed of via their recommended route.

Safe Experimental Procedures in the Cell Culture Laboratory In order to ensure a safe working environment with cell lines and biohazardous agents, personal protective equipment PPE must be worn in the cell culture lab. Equipment for the cell culture laboratory Despite the various techniques and assays carried out in different cell culture labs, the common theme of cell culture work is asepsis—the creation of a microenvironment free of unwanted pathogenic microorganisms, including bacteria, viruses, fungi, and parasites.

Aseptic Cell Culture Practices While the previous section has explored methods aimed at decreasing the exposure of hazardous substances to the laboratory worker, this section will address the practices that should be put in place by laboratory workers to protect the cultured cells.

Creating an Aseptic Work Environment Given that atmospheric air is laden with microparticles of potentially infectious nature, the biosafety cabinet is the most crucial piece of equipment to restrict nonsterile aerosols and airborne components from contaminating cultured cells.

Using Aseptic Reagents and Media for Cell Culture The main sources of contamination are laboratory staff, the environment, and the culture medium. Contaminations Since contaminations can generally not be avoided altogether, it is important to train cell culture laboratory staff to recognize early signs in order to prevent the spread of contaminants to other cells or cell culture products.

Microbial contaminants in cell culture. Bacterial Contamination The bacteria kingdom includes highly ubiquitous, prokaryotic microorganisms characterized by the size of a few micrometers in diameter, wide diversity in their morphologies, and fast doubling times through asexual reproduction.

Fungal Contamination Yeasts are unicellular eukaryotes that form multicellular string-like structures during asexual reproduction. Viral Contamination Viruses are infectious agents that rely on host cells for their own replication.

Eliminating contaminations Regardless of the type of contamination identified, affected cell cultures should be removed from the cell culture room and discarded to prevent the spread of infectious agents to other cultures.

The Cell Line The choice of a cell line for cell culture depends heavily on the functional properties and specific readouts required of the cell model [4]. The Cell Culture Microenvironment Regardless of the cell line chosen, a common requirement will be the selection of suitable growth conditions. The Cell Culture Medium The goal to create an environment that allows for maximum cell propagation is achieved primarily through the incubator i.

Temperature, pH, CO 2 , and O 2 Levels The desired temperature for cell cultures depends on the body temperature of the species and the microenvironment from which the cultured cell types were isolated. Cell maintenance. In Practice This section explains the basic protocols required for the maintenance of cell cultures. Dissociating Adherent Cells from Culture Vessels for Subculturing Cells cultured in vitro over time will deplete nutrients supplied in the medium, release toxic metabolites and grow in number.

Subculturing of Suspension Cultures The subculturing of suspension cultures can be achieved by aseptically removing one-third of the cell suspension solution and replacing the volume with prewarmed complete medium. Quantification of Cells and Determining Cell Viability Cells can die in the process of culturing or during handling and passaging.

Cell quantification using Trypan Blue. Freezing Cells When a surplus of cells becomes available during subculturing, they can be preserved at that passage through freezing with cryoprotective agents e. Applications Model Systems in Health and Disease Cell culture is one of the most important techniques in cellular and molecular biology since it provides a platform to investigate the biology, biochemistry, physiology e.

Drug Development and Drug Testing Cell culture tools can also be applied to screen novel chemicals, cosmetics, and drug compounds for their efficacy and assess drug cytotoxicity in specific cell types. Virology and Vaccine Production Cell culture with mammalian cells offers a host for viruses to replicate, allowing researchers to study their growth rates, development, and conditions required for their infectious cycle.

Tissue Regeneration and Transplantation hIPSCs, embryonic stem cells, and adult stem cells have the capacity to regenerate and differentiate into specialized cell types that can be used as replacement tissues or organs.

Genetic Engineering and Gene Therapy The expression of specific genes and their impact on cells can be studied by the introduction of new genetic material e. Scenario Culturing hIPSCs to study inherited liver diseases: Small pieces of skin biopsies obtained from patients with an inherited liver disease can be cultured in a Petri dish with fibroblast growth medium. Key Limitations Discrepancies between Cellular Environments in vitro and in vivo One of the pillars of cell culture research is the design of a defined cellular environment in which single variables can be manipulated in order to monitor cellular responses.

Discrepancies between Gene Expression in Primary Cells and Immortal Cell Lines The oftentimes most relevant cell types for addressing translational research questions—primary cells—are in fact very difficult to isolate and culture in vitro due to their limited proliferation and functional capacity ex vivo.

Conclusion This chapter has described the vast possibilities to employ cell culture techniques to address basic and translational research questions and has explained the necessary considerations for setting up a cell culture lab.

References 1. Biological agents: managing the risks in laboratories and healthcare premises. Mycoplasma contamination of cell cultures: Incidence, sources, effects, detection, elimination, prevention.

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