Animal Cell Culture Book Pdf - From Theory to Practice in Regenerative Medicine

Animal cell culture is an important laboratory technique in the biological and medical sciences. It has become an essential tool for the study of most biochemical and physiological processes and the use of large-scale animal cell culture has become increasingly important to the commercial production of specific compounds for the pharmaceutical industry. This book describes the basic requirements for establishing and maintaining cell cultures both in the laboratory and in large-scale operations. Minimal background knowledge of the subject is assumed and therefore it will be a readable introduction to animal cell culture for undergraduates, graduates and experienced researchers. Reflecting the latest developments and trends in the field, the new topics include the latest theory of the biological clock of cell lines, the development of improved serum-free media formulations, the increased understanding of the importance and control of protein glycosylation, and the humanization of antibodies for therapeutic use.

The present work is directed primarily to the students of biotechnology, keeping in view the University Grants Commission Syllabus on animal cell culture prescribed for vocational course both for undergraduate and postgraduate students and also for the scientists in the medical, veterinary and biological sciences, whose curiosity and efforts are responding to the stimulus of current interest in biotechnology.

Animal Cell Culture Book Pdf -

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To ensure that the results can be extrapolated to the population of interest, there must also be replication relevant to the hypothesis being tested. For this, it helps to define the scientific or BU of interest, which is the entity (i.e., people, animals, and cells) that we would like to test a hypothesis or draw a conclusion about. For example, to conclude that a drug is better than a placebo, a large number of patients are required because the hypothesis is about patients. We cannot give Jim the drug and Bob the placebo, take a daily measurement for several weeks, and then make general conclusions about the drug's efficacy. At times, the BU may not be so apparent. Suppose we hypothesise that outbred mice are smarter than inbred mice. We cannot test this hypothesis with only 1 strain of outbred and 1 strain of inbred mice, even if we have many mice from each strain. There may be intelligence differences between these 2 strains that have nothing to do with their inbred/outbred status, much like there are differences between Bob and Jim that cannot be disentangled from the drug effect. The BU is the strain (because the hypothesis is about strains) and therefore we need multiple strains of both outbred and inbred mice.

Similar problems arise in cell culture studies. If we hypothesise that breast cancer cell lines proliferate at a faster rate than lung cancer cell lines, we need multiple breast and lung cell lines, as the hypothesis is about differences between the tissue of origin. If 1 breast and 1 lung cell line proliferate at different rates, we cannot attribute this to the tissue of origin as no 2 cell lines are expected to proliferate equally.

Although the EU often corresponds to a BU of interest (Fig 2A), it can also correspond to a collection or group of BUs, such as all mice in a litter if the treatment is applied to the pregnant dam (Fig 2B). In addition, the EU can correspond to parts of a BU; for example, individual eyes, patches of skin, or organotypic slice cultures from the same animal, as long as these parts can be randomly and independently assigned to different conditions (Fig 2C). Finally, an EU can correspond to a sequence of observations on a single BU. For example, the experiment is divided into time periods that are randomly assigned to different treatment conditions (e.g., on or off a drug), and a measurement is taken at each time period (Fig 2D). This last design is infrequent in biological experiments but forms the basis for N-of-1 and crossover designs (and differs from a longitudinal or repeated-measures design). Below we use concrete examples to illustrate these points.

We expect animals in a cage to influence each other on many relevant variables, from behaviour to microbiomes (and hence anything influenced by an animal's microbiome). Even if animals meet the first two criteria for genuine replication, mutual influence of animals in the same cage may render them unsuitable to be an EU. The solution is to house animals 1 per cage, or, if this is undesirable for ethical or experimental reasons, housing animals 2 per cage maximises the number of cages (which are now the EUs) for a fixed number of animals. The same idea applies to cells in a well or tissue, if there is mutual influence, or suspected influence, then N cannot refer to the number of cells. Next, we show how these ideas apply to different types of experiments.

Some ex vivo experiments are similar to the nonhuman primate example above, where, for example, an organotypic hippocampal culture has time periods randomised to the presence or absence of a serotonin receptor antagonist, and at each period electrophysiological recordings are made. Here we substitute the hippocampal culture for the subject, and the presence or absence of the antagonist for the pictures, and all previous points apply. In other ex vivo experiments, the treatment is applied to the animal, and multiple slice preparations are derived from each animal. Here, N is the number of animals, not the number of slice cultures.

In cell culture experiments cells are often both the OUs and BUs of interest, but rarely the EU. Suppose an aliquot of cells is thawed and the cell suspension is pipetted into different wells of a microtitre plate. Cells are randomised to wells, and then wells to treatments, so the first criterion is met. But treatments are applied simultaneously to all cells in a well, not independently to each cell, so the second criterion is not met. In addition, it is unreasonable to assume that cells in a well have no influence on each other; they form cell-to-cell connections, release signalling molecules, and compete for the same nutrients in the media. Hence, the third criterion is not met for using cells as the EU. Thus, a well, culture dish, or another plastic container is the appropriate EU for cell culture experiments.

But in vitro experiments are often finicky; the results depend on the unique conditions that vary each time the experiment is run. The experimental material (e.g., a cell line) is often artificially homogeneous and the conditions under which the experiment is run are so narrowly defined that it is hard to know what will happen if the experiment is run a second time. For this reason, in vitro experiments are usually repeated on multiple days, and the number of wells, aliquots, or culture dishes within a day are treated as subsamples. The aim is to establish that the phenomenon is robust enough to survive multiple replications of the entire experimental run or protocol in a highly artificial system. This situation has parallels to the nonhuman primate example above. We could do the experiment on 1 day and use the wells as the EU, and have a large sample size, but then we cannot comment on the generalisability of the results. If the experimental system is sensitive to the many details of how it is carried out, then repeating the whole procedure on multiple days provides further information in a way that using more wells on a single day does not. It provides an estimate of the consistency of the effects across the different experimental runs (days). The multiple wells on each day are then treated as subsamples and do not contribute to N (for example, by averaging values across wells in the same condition on each day). This is a scientific judgement about the relevant unit that we would like to make inferences about, and although opinions may differ, using more stringent criteria makes the results more believable.

This eagerly awaited edition reviews the increasing diversity of the applications of cell culture and the proliferation of specialized techniques, and provides an introduction to new subtopics in mini-reviews. New features also include a new chapter on cell line authentication with a review of the major issues and appropriate protocols including DNA profiling and barcoding, as well as some new specialized protocols. Because of the continuing expansion of cell culture, and to keep the bulk of the book to a reasonable size, some specialized protocols are presented as supplementary material online.

Animal cells are used in the manufacturing of complex biotherapeutic products since the 1980s. From its initial uses in biological research to its current importance in the biopharmaceutical industry, many types of culture media were developed: from serum-based media to serum-free to protein-free chemically defined media. The cultivation of animal cells economically has become the ultimate goal in the field of biomanufacturing. Serum serves as a source of amino acids, lipids, proteins and most importantly growth factors and hormones, which are essential for many cell types. However, the use of serum is unfavorable due to its high price tag, increased lot-to-lot variations and potential risk of microbial contamination. Efforts are progressively being made to replace serum with recombinant proteins such as growth factors, cytokines and hormones, as well as supplementation with lipids, vitamins, trace elements and hydrolysates. While hydrolysates are more complex, they provide a diverse source of nutrients to animal cells, with potential beneficial effects beyond the nutritional value. In this review, we discuss the use of hydrolysates in animal cell culture and briefly cover the composition of hydrolysates, mode of action and potential contaminants with some perspectives on its potential role in animal cell culture media formulations in the future.

Hydrolysates are the products of plant (soy, pea, rice, rapeseed, etc.) or animal (chicken, pork, fish, etc.) proteins after hydrolysis by acid, alkali, enzymes and fermentation processes. When a predominantly protein starting material is used to produce the hydrolysate, the product may be described as a protein hydrolysate, for example, rice protein hydrolysate and rapeseed protein hydrolysate. Hydrolysates often contain a mixture of peptides, amino acids, minerals, carbohydrates, lipids and proteins that are similar to the raw input material. Since the late 1970s, chicken and fish-derived hydrolysates, have been used as serum replacements for animal cell culture (Mizrahi 1977). However, as animal-derived hydrolysate would encounter the same issues as using animal serum, the use of plant-based hydrolysates such as those derived from soy, rice and cottonseed proteins have gained popularity. The relatively low cost of plant-based hydrolysates makes them attractive as serum replacement components for large-scale protein production. However, given hydrolysate products are not fully characterized, further understanding of their components and how these can influence cell growth and maintenance is key to their success as potential serum replacement components. In this review, we will describe the history of animal cell culture, including examples of animal cell cultures used for the production of biotherapeutics in the presence or absence of serum, the history of the use of hydrolysates in animal cell cultures for biotherapeutics and new modalities such as cultured meat production. The review will also describe the compositions of hydrolysate products, their modes of action and potential contaminants with some views on their future use in animal cell cultures. 2ff7e9595c