<h5 id="81-historical-perspective">8.1 Historical Perspective</h5> <ul> <li> <p>In the 1950s, George Gey established the first human cell line (HeLa) from a patient’s cervical cancer, leading to significant discoveries in medical sciences.</p> </li> <li> <p>The need for large-scale cell culture became apparent with the demand for viral vaccines, and cell culture technologies have since been used in various areas, including drug efficacy and toxicity assessment, vaccine, and biopharmaceutical manufacture.</p> </li> <li> <p>A variety of culture media have been developed to support cell survival, proliferation, and functionality; the addition of growth factors such as nerve growth factor, epidermal growth factor, insulin-like growth factor, fibroblast growth factor, platelet-derived growth factor, and transforming growth factor has increased cellular proliferation.</p> </li> <li> <p>In 1976, the development of serum-free media was accelerated.</p> </li> <li> <p>The timeline for the historical perspective of animal cell culture is presented in Table 8.1.</p> </li> </ul> <p>======</p> <h5 id="82-culture-media">8.2 Culture Media</h5> <ul> <li>Natural media consists of biological substances, such as plasma, serum, and embryo extract. The disadvantage is poor reproducibility and reduced uniformity due to the exact composition being unknown.</li> <li>Synthetic media, also known as artificial media, is composed of a basal medium and supplements like serum, growth factors, and hormones.</li> <li>Serum-containing media uses human, bovine, equine, or other serum as a supplement. However, the quality of serum varies from batch to batch and deteriorates within one year, requiring fresh testing for every batch.</li> <li>Serum-free media uses crude protein fractions, such as bovine serum albumin or a- or β-globulin, as supplements. This can make a medium selective for a particular cell type.</li> <li>Protein-free media uses undefined components, such as peptide fractions, as supplements. Chemically defined media uses basal media like Phosphate buffered saline (PBS) and complex media like Eagle’s Minimum Essential Medium (EMEM).</li> <li>Antibiotics like penicillin and streptomycin are commonly used in culture medium to control the growth of contaminants, although they are not essential for cell growth.</li> </ul> <p>======</p> <h5 id="83-physical-environment-for-culturing-animal-cells">8.3 Physical Environment for Culturing Animal Cells</h5> <ul> <li>Animal cell culturing in vitro requires simulating micro-environments, nutritional, physical, and hormonal conditions for optimal cell growth.</li> <li>Controlling temperature, osmolality, pH, gaseous requirements, a supporting surface, and protecting cells from physical, chemical, and mechanical stresses are essential.</li> <li>Temperature is maintained at 37°C using CO2 incubators to simulate the body temperature of Homosapiens, as most cells are derived from warm-blooded animals.</li> <li>Osmolality, affected by glucose, salt, and amino acids, should be approximately 300 mOsmol for optimal cell growth, and can be measured by an osmometer.</li> <li>Two buffering systems are used: natural (with CO2) and chemical (HEPES-based), with phenol red as a pH indicator, changing color according to pH levels.</li> </ul> <p>======</p> <h5 id="84-equipment-used-for-cell-culture">8.4 Equipment Used for Cell Culture</h5> <ul> <li>Cell culture maintenance requires culture flasks, Petri dishes, or multi-well plates, maintained at an appropriate temperature, humidity, and gas mixture ($37^{\circ} \mathrm{C}, 95 %, 5 % \mathrm{CO}_{2}$ for mammalian cells) in an incubator.</li> <li>Essential equipment for an animal cell culture laboratory includes laminar flow hoods, $\mathrm{CO}_{2}$ incubators, an inverted microscope, autoclave, and centrifuge.</li> <li>Laminar flow hoods provide a sterile environment, free from particulates and microbes, and are of two types: horizontal and vertical flow hoods.</li> <li>$\mathrm{CO}<em>{2}$ incubators maintain sterility, constant temperature, $\mathrm{CO}</em>{2}$ level (5-10%), and high relative humidity (95%) for cell culture.</li> <li>An inverted microscope with phase-contrast optics and preferably a photographic facility is required for monitoring cell morphology and detecting problems at an early stage.</li> </ul> <p>======</p> <h5 id="85-types-of-animal-cell-cultures-and-cell-lines">8.5 Types of Animal Cell Cultures and Cell Lines</h5> <ul> <li>Primary cell cultures are obtained directly from the host tissue and contain heterogeneous cells similar to their parents. They can be adherent or suspension cells.</li> <li>Adherent cells are anchorage-dependent, proliferate as a monolayer, and require attachment to a solid or semi-solid substrate for proliferation. Examples include fibroblasts and epithelial cell types.</li> <li>Suspension cells, also called anchorage-independent or non-adherent cells, are not attached to the surface of the culture vessels and float in the culture medium. Examples include hematopoietic stem cells and tumor cells.</li> <li>Secondary cell cultures are obtained by sub-culturing or passaging primary cell cultures. They can be finite or continuous cell lines, depending on the culture life span.</li> <li>Finite cell lines have a limited number of cell divisions and life span, while continuous cell lines can divide indefinitely and are immortal and tumorigenic.</li> <li>Aseptic techniques are essential in cell culture to prevent bacterial and fungal infections. Subculturing or passaging is the process of using enzymes to enable the cells to detach from the old medium and transfer them to a fresh flask after centrifugation and addition of fresh media.</li> </ul> <p>======</p> <h5 id="86-cell-viability-determination">8.6 Cell Viability Determination</h5> <ul> <li> <p>Cell viability determination is crucial in cell culture to evaluate the live status of culture cells.</p> </li> <li> <p>There are two main types of viability assays: dye exclusion and metabolic viability assays.</p> </li> <li> <p>Dye exclusion viability assays use a dye or stain that enters the cell, indicating loss of cell membrane integrity and cell death. Examples of dyes include trypan blue, propidium iodide, and 7-AAD.</p> </li> <li> <p>Metabolic viability assays rely on the ability of cells to execute a specific biochemical reaction, such as the reduction of a tetrazolium compound (e.g., MTT assay). These methods estimate the cell number based on the cellular content of enzyme or substrate and subsequent extraction of the dye.</p> </li> <li> <p>The MTT assay measures the conversion of a yellow-coloured water-soluble salt to purple-coloured insoluble formazan crystals, which are proportional to the cell number.</p> </li> <li> <p>The conversion of non-fluorescent acetomethoxy derivative of calcein to a fluorescence emitter component after ester hydrolysis by intracellular esterase is also used in metabolic viability assays.</p> </li> </ul> <p>======</p> <h5 id="87-scale-up-of-animal-cell-culture-process">8.7 Scale-up of Animal Cell Culture Process</h5> <ul> <li>Spinner flasks are used for scaling up suspension cell cultures, consisting of a flat surface glass flask with a suspended central teflon paddle for agitation and improved gas exchange.</li> <li>Roller bottles are used for adherent cell cultures, with cells adhering to microcarrier beads that increase available growth space; these bottles can be rotated in specialized CO2 incubators for even exposure to medium.</li> <li>Microcarrier beads of various diameters and materials can effectively grow monolayer cells, with a maximum surface area to volume ratio of nearly 90,000 cm2 l-1.</li> <li>Culturing monolayer cells on microbeads allows for cells to be treated as a suspension and efficiently stirred without damage.</li> <li>Under optimal conditions, adherent cells can be grown to high densities, but cells at high densities may exhaust the medium quickly, requiring frequent replenishing.</li> </ul> <p>======</p> <h5 id="88-advantages-of-animal-cell-culture">8.8 Advantages of Animal Cell Culture</h5> <ul> <li>Animal cell culture allows growth in a controlled physico-chemical environment.</li> <li>It can proliferate into a homogenous genetic population.</li> <li>The process facilitates easy gene addition (transfection) or regulation of protein levels (RNAi).</li> <li>Adequate numbers of cells are available for chemical studies and biopharmaceutical production.</li> <li>However, it is a sensitive technique with productivity potentially reduced by small changes.</li> <li>Scale-up is possible but can be challenging, and it might not fully represent in vivo phenotype/genotype.</li> </ul> <p>======</p> <h5 id="89-applications-of-animal-cell-culture">8.9 Applications of Animal Cell Culture</h5> <ul> <li>Follicle Stimulating Hormone (FSH) is a glycoprotein hormone that stimulates the bone marrow to produce more red cells and is produced in CHO cells. It is used for treating certain types of anemia caused by chemotherapy, AIDS, and chronic renal failure.</li> <li>Erythropoietin (EPO) is a glycoprotein hormone that is secreted by kidney under hypoxic or anoxia conditions and is used to treat anemia and chronic renal failure. Recombinant human EPO (r-HuEPO) is produced using CHO cell lines.</li> <li>Factor VIII and Factor IX are glycoproteins produced in CHO cells used to treat Haemophilia A and B respectively. Factor VIII is required for blood clotting and is not produced in Haemophilia A patients while Factor IX is deficient in Haemophilia B patients.</li> <li>Tissue Plasminogen Activator (tPA) is a serine protease produced in CHO cells that catalyzes the conversion of plasminogen to plasmin which is responsible for dissolving blood clots. It is approved for use in certain patients having a heart attack or stroke.</li> <li>Monoclonal antibodies (mAbs) are produced by hybridising antigen-activated B lymphocytes with a myeloma cell using polyethylene glycol. This technology has revolutionized the area of diagnostics and antibody-based therapies.</li> </ul> <p>======</p>