Culture Of Animal Cells By R Ian Freshney Pdf Download

The goal of Animal Cell Culture Techniques is for students to acquire the necessary practical skills for the isolation of animals cells for in vitro studies, maintenance of animal cells in vitro, manipulation of animal cells in vitro, and application of molecular techniques to in vitro situations.

Cell-line misidentification and contamination with microorganisms, such as mycoplasma, together with instability, both genetic and phenotypic, are among the problems that continue to affect cell culture. Many of these problems are avoidable with the necessary foresight, and these Guidelines have been prepared to provide those new to the field and others engaged in teaching and instruction with the information necessary to increase their awareness of the problems and to enable them to deal with them effectively. The Guidelines cover areas such as development, acquisition, authentication, cryopreservation, transfer of cell lines between laboratories, microbial contamination, characterisation, instability and misidentification. Advice is also given on complying with current legal and ethical requirements when deriving cell lines from human and animal tissues, the selection and maintenance of equipment and how to deal with problems that may arise.

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Histopathological confirmation. A small portion of the sample being used to originate the culture should be fixed in formalin and used for histopathological assessment, ideally by the same histopathologist reporting the surgical specimen if this is from a patient. This step is particularly important if a patient sample is supplied to the laboratory directly by a clinician, because it may not be representative of the surgical specimen sent to the histopathologist. For instance, it may be taken at some distance from a tumour and consequently lack cancer cells, or it may be from a region that is unaffected by a specific pathology caused by a genetic or epigenetic defect.

Although it may be necessary to use antibiotics in the primary culture, they should be removed as soon as possible and the cells tested for mycoplasma (see Section 4.2.4 and Table 3). The type of assay used for mycoplasma detection should be stated, as should the frequency and date of the last test.

If a cell line is genetically modified (including methods used to achieve immortality when relevant), it is essential to describe the process used, including details of sequences, mode of insertion and antibiotic resistance markers. Additional tests may be necessary to demonstrate lack of infectivity, for example, following transduction using lentiviral or retroviral vectors. For hybridomas, details of the sources of both sets of cells are needed. Where animal tissue is used to originate a culture, it is important to record the species and strain, age, sex and genetic status.

The first publication should include the information described in the previous sections and subsequent publications should cite the first publication. Every publication should confirm that the cultures have been tested for mycoplasma (see Table 3 and Section 4.2.4) and that the test is negative. It is possible to eliminate at least some types of mycoplasma from cell lines, although this would only be worth attempting for particularly valuable or unique cells. The first publication should also provide evidence that the cells have been derived from the individual claimed to be the source, with subsequent publications comparing stocks of that cell line to the STR profile or other evidence cited within the first publication. Some journals insist on cell lines being made available as a condition of publication, so that other laboratories can repeat the work. Some funding agencies and institutions also encourage or insist that cell lines derived with their support are made available to others, free or at cost, even if they also require an MTA. Information on deposits in cell banks or whom to contact to obtain cells is helpful in this regard. Publication of work with the cell line implies its entry into the public domain and the right of others to acquire the cell line from the originator or the nominated cell bank.

Human cell lines may carry pathogens, including viral contamination, representing a potential health hazard to laboratory workers (see Section 3.1). They may also become contaminated with bacteria, fungi, mycoplasma or viruses, which may spread to other cell lines. These contaminants may also be potential pathogens. If the cells are to be used in animals, whether as grafts of normal tissue or to derive tumours, or to make chimeras, it is also critical that they are tested and shown to be free of relevant pathogens, which might otherwise harm the animal colony or those who care for the animals. The cells or their derivatives may also be re-isolated from the animals for further study in vitro, in which case they need to be treated as a new sub-line and subject to further characterisation for genetic status as well as mycoplasma and other pathogens. Human cells passaged through animals could in theory have acquired replication-competent retroviruses from the animal host that could subsequently infect human cells, although the risk of this and of them being pathogenic to humans is very low.

The user should also confirm that the cell line they obtain is fit for their own particular purpose. Even if a cell line is shown to be authentic, it may have lost a particular key characteristic with prolonged passaging. Karyotyping is a simple test that can reveal changes in a cell line. Indeed, it is routine to show that a line of ES cells or iPSCs has a normal karyotype if they are to be used for experiments involving production of chimeras and germ line transmission. Molecular assays to look for CNV or RNA profiling will also be indicative of changes, but are more costly. Nevertheless, a great deal of time and effort can be saved by confirming the appropriate characteristics before commencing work. It is also advisable to capture an image of the cell line in culture at different cell population densities and perform basic characterisation (e.g., calculating the population doubling time for that cell line) soon after arrival.

Misidentification occurs as a result of cross-contamination, poorly controlled manipulation or clerical error and implies a failure in good cell culture practice (GCCP); for example, accidental transfer of cells to a stock bottle of medium, having two cell lines in an MSC at the same time, mislabelling a flask or ampoule, or thawing the wrong ampoule. Other sources of cross-contamination are if feeder cells (e.g., as often used in ES cell culture) are still mitotically active due to inadequate irradiation or treatment with mitomycin C, or if conditioned media is prepared without adequate filtration to remove cells. Whenever a rapidly growing, continuous cell line is maintained in a laboratory there is a risk that it may cross-contaminate (i.e., replace) other, more slowly growing lines. There is a long history of this problem, highlighted in the 1960s and 1970s (Gartler, 1967; Nelson-Rees and Flandermeyer, 1976; Nelson-Rees and Flandermeyer, 1977), but now often ignored. Few authors using cell lines such as KB, Int-407, WISH, Chang liver or Hep-2 acknowledge that they are in reality working with HeLa cells. Similarly, some cell lines with a variety of names and claimed tissues of origin are in fact MCF-7 (breast cancer) or T24 (bladder cancer) cells. Whatever the purpose of the experiments, it is essential to know the derivation of the cells. Even if the process being studied is not cell type-specific, others may cite the work in a context where it is.

Transferring a cell line between laboratories may involve transport within a city, country or between continents. Therefore, consideration will have to be given to the condition of the cells, the means of transport and the legal requirements (see Section 2.1.7). Cell lines may be transported either as growing cultures or as vials of frozen cells.

While there are few restrictions on the movement of cell cultures within the European Union, importation of certain animal cells from other countries into the UK requires a permit from the Department for Environment, Food and Rural Affairs (DEFRA, 2013). This is particularly important for cells from agricultural species, including poultry, where there is a serious risk of importing non-endemic viruses.

The use of animals in experiments and testing is regulated under the Animals (Scientific Procedures) Act 1986 (ASPA). ASPA has now been revised to transpose European Directive 2010/63/EU on the protection of animals used for scientific purposes (EU Directives, 2010) and the revised legislation came into force on 1 January 2013. ASPA is not directly relevant to the derivation of a cell line from an animal that has been killed (by a schedule 1 method). However, it is relevant if any regulated procedure is required, such as tissue biopsy of a live animal, administration of substances, or derivation of a genetically altered animal. It is also relevant if cells are to be introduced into a live-born animal or animal embryo. While for most experiments it will make little difference with respect to regulation under ASPA whether the cells are of animal or human origin, some involving the latter may be considered contentious, especially if they concern the reproductive system or have the potential to lead to human characteristics developing in an animal (see Academy of Medical Sciences Report on Animals Containing Human Material (www.acmedsci.ac.uk)). New regulations and guidance on this type of research are being introduced and such experiments will be considered by the new Animals in Science Committee of the Home Office.

Therefore primary human tissue and cells (i.e., those removed directly from a person) are defined as relevant material under the HT Act. Cell lines derived from expansion of primary cell cultures in vitro are not relevant material, as all of the original cells will have divided and so the cell line has been created outside of the human body. The storage of cell lines created from primary human tissue, for research purposes, does not require an HTA licence and the use of such cell lines is not covered by the HT Act or regulated by the HTA. However obtaining, retention and storage of any of the primary material from which the cell line was derived will be subject to the HT Act and HTA regulation, as will any cell lines derived with the intention of use in human therapy under the HTA (2007). 17dc91bb1f