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Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications (6th ed.) by R. Ian Freshney. Read online. Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications (7th ed.) by R. Ian Freshney. Read online, or download in secure PDF or. Culture of Animal Cells. SIXTH EDITION. A Manual of Basic Technique and Specialized Applications. R. IAN FRESHNEY. #WILEY-BLACKWELL. WWW.
The composition of each MCDB medium is optimized to promote the growth of a specific cell type. Instead of glucose, pyruvate and galactose is added at a high concentration in order to control pH drops due to the lactic acid that is produced during glucose metabolism and to promote the release of CO2 from the respiratory chain. Another major characteristic is that each of its constituent amino acids is added at approximately its respective maximal concentration. At present, formulations of popular media for cell culture are available commercially. Thus, there are investigators who are using culture media without understanding their details and background, particularly regarding the rationale for their development, the exact ingredients, as well as the cell types that these media are suitable for.
Lewis demonstrated that the Locke—Lewis solution—which is modified Locke's solution that additionally contains amino acids, bouillon, and glucose or maltose —is more effective for chick embryo cell cultivation than simple balanced salt solutions. Thus, their research did not lead to the molecular identification of any substance that is essential for cultured cells.
Joilannes P. They typically stop growing after a certain number of divisions ie, the Hayflick limit. In , Wilton R. Gey and his coworkers created an infinitely proliferating human cell line from a tissue of a patient with uterine cervical cancer HeLa cells. This state of affairs made it easier to examine and to precisely quantify the subtle differences in the effects of culture media on cells.
Thus, the development of culture media advanced rapidly as a result. In order to find the crucial components in those natural materials and to develop defined media that are comparably efficient in the cultivation of cells, relative to the media containing natural ingredients, two main strategies were undertaken. The first strategy was to use dialyzed serum for the support of cells at minimum levels and to add defined components to maximize the proliferation of cells.
The second strategy was not dependent on serum, or even proteins at all, and involved the formulation of media exclusively from definitive components. Fischer was a pioneer of the first strategy. Later, using dialyzed serum that had been prepared in accordance with Fischer's method, Thomas A. The result then was reported as the RPMI medium for use in lymphocyte cultivation.
He developed a chemically defined medium that was composed of glucose, inorganic salts, amino acids, iron, vitamins, and glutathione, with no protein at all. Serum and embryonic extract were still necessary for the primary culture. Other researchers also reported the necessity of a small amount of serum in Medium for cell proliferation. There was also a challenge: serum supplementation remained necessary for the cultivation of cell types other than CHO cells.
The reductive approach is used to analyze and identify the essential parts in the complex biological process. The synthetic approach is used to reconstruct the biological process by putting the known parts together.
Both approaches were instrumental in the development of synthetic culture media because these approaches had disadvantages relative to each other.
First, the reductive approach was undertaken strenuously by Carrel and others because the synthetic approach was almost impossible without any information about the active substances for the cultivation of cells; accordingly, the substances that were important for cell culture in natural media were lined up.
In contrast, the reductive approach could not be entirely successful at elucidating the complexities of the natural media because of the quantitative and qualitative limitations of the analytical method, complicated components of the natural media, and their variation from batch to batch.
Accordingly, the candidate substances based on the knowledge of the reductive approach were tested by means of the synthetic approach by Eagle and others and the substances that are essential for cell culture were eventually identified. It seems to be important when and how these approaches are used for the successful development of culture media. Evolution of Cell Lines, 21 2.
Senescence, 22 2. Transformation and the Development of Continuous Cell Lines, 22 3. Laboratory Design, Layout, and Equipment, 25 3. Layout, Furnishing, and Services, 25 3. Requirements, 25 3. Services, 28 3. Ventilation, 30 3. Layout, 30 3. Sterile Handling Area, 30 3. Laminar Flow, 30 3. Service Bench, 30 3. Incubation, 31 3. Preparation Area, 33 3. Storage, 34 4. Equipment and Materials, 37 4. Requirements of a Tissue Culture Laboratory, 37 4. Aseptic Area, 37 4.
Laminar-Flow Hood, 37 4. Service Carts, 41 4. Inverted Microscope, 45 4. CCD Camera and Monitor, 46 4. Dissecting Microscope, 46 4.
Centrifuge, 47 4. Cell Counting, 47 4.
Incubation and Culture, 47 4. Incubator, 47 4. Humid CO2 Incubator, 48 4. Temperature Recorder, 48 4. Roller Racks, 49 4. Magnetic Stirrer, 50 4. Culture Vessels, 50 4. Preparation and Sterilization, 50 4. Washup, 50 4.
Preparation of Media and Reagents, 51 4. Sterilization, 52 4. Storage, 53 4. Consumables, 53 4. Refrigerators and Freezers, 54 4. Cryostorage Containers, 55 4.
Controlled-Rate Freezer, 55 4. Supplementary Laboratory Equipment, 55 4. Computers and Networks, 55 4. Upright Microscope, 55 4. Low-Temperature Freezer, 56 4. Confocal Microscope, 56 4.
PCR Thermal Cycler, 56 4. Specialized Equipment, 56 4. Microinjection Facilities, 56 4. Colony Counter, 56 4. Centrifugal Elutriator, 56 4. Flow Cytometer, 56 5. Aseptic Technique, 57 5. Objectives of Aseptic Technique, 57 5.
Risk of Contamination, 57 5. Maintaining Sterility, 57 5. Elements of Aseptic Environment, 58 5. Laminar Flow, 58 5.
Quiet Area, 60 5. Work Surface, 61 5.
Personal Hygiene, 61 5. Reagents and Media, 61 5. Cultures, 61 5. Sterile Handling, 61 5. Swabbing, 61 5. Capping, 63 5. Flaming, 63 5. Handling Bottles and Flasks, 64 5. Pipetting, 64 5. Pouring, 65 5. Standard Procedure, 65 Protocol 5. Working on the Open Bench, 67 Protocol 5. Handling Dishes or Plates, 69 5. Apparatus and Equipment, 69 5.