Animal cell culture involves growing isolated animal cells or tissues in a controlled environment with nutrient medium. Different cell types can be cultured including fibroblasts, epithelial, and tumor cells. The history of animal cell culture began in the late 19th century with early experiments maintaining cells in vitro. Requirements for successful cell culture include sterile facilities, equipment for temperature control and media preparation, and single-use plasticware and glassware. While cell culture is useful for research and production, it cannot fully replicate the in vivo cellular environment.
Animal cell culture involves growing isolated animal cells or tissues in a controlled environment with nutrient medium. Different cell types can be cultured including fibroblasts, epithelial, and tumor cells. The history of animal cell culture began in the late 19th century with early experiments maintaining cells in vitro. Requirements for successful cell culture include sterile facilities, equipment for temperature control and media preparation, and single-use plasticware and glassware. While cell culture is useful for research and production, it cannot fully replicate the in vivo cellular environment.
Animal cell culture involves growing isolated animal cells or tissues in a controlled environment with nutrient medium. Different cell types can be cultured including fibroblasts, epithelial, and tumor cells. The history of animal cell culture began in the late 19th century with early experiments maintaining cells in vitro. Requirements for successful cell culture include sterile facilities, equipment for temperature control and media preparation, and single-use plasticware and glassware. While cell culture is useful for research and production, it cannot fully replicate the in vivo cellular environment.
Animal cell culture involves growing isolated animal cells or tissues in a controlled environment with nutrient medium. Different cell types can be cultured including fibroblasts, epithelial, and tumor cells. The history of animal cell culture began in the late 19th century with early experiments maintaining cells in vitro. Requirements for successful cell culture include sterile facilities, equipment for temperature control and media preparation, and single-use plasticware and glassware. While cell culture is useful for research and production, it cannot fully replicate the in vivo cellular environment.
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Animal Cell Culture
Cell culture • Animal cell culture refers to the rearing of isolated animal cells or animal tissues in suitable nutrient medium under controlled conditions. • In vitro cultivation of organs, tissues & cells at defined temperature using an incubator & supplemented with a medium containing cell nutrients & growth factors is collectively known as tissue culture. • Different types of cell grown in culture includes connective tissue elements such as fibroblasts, skeletal tissue, cardiac, epithelial tissue (liver, breast, skin, kidney) and many different types of tumor cells. History • 1885, Roux maintained the cells of medullary plate of chick embryo in warm saline. • 1903, Jolly observed cell division in Salamander leucocytes grown in vitro. • 1907, Ross Granville and Harrison established a methodology of animal tissue culture for the first time. • 1913, Rous and Jones used penicillin and streptomycin as antibiotics in animal cell cultures. History • 1916, Rous and Jones used laminar air-flow hoods for the aseptic transfer of cultures and media and for cultural operations. • 1940, Rous and Jones formulated a medium containing blood serum to culture animal cells and tissues. • Katherine prepared the clones of L-cells via animal cellculture. History • Enders, Weller and Robbin grew animal cell cultures to manufacture polio vaccine. • 1949, Alan Parks developed a protocol to freeze cells and kept them at -196°C • 1950, Rous and Jones evaluated necessary conditions such as temperature, pH, media components, inorganic ions, hormones and others for culture. History • 1951, famous Hela cell was derived from cancerous growth of Henrietta Lacks, who died of cervival cancer. • 1952, Harry Eagle developed a chemically defined culture medium. • 1954, Ender et al., employed human cell lines for the production of vaccines for humans and animals. History • 1955, Hayflick and Moorhead discovered the lifespan of normal human diploid cells in cell cultures. • 1960’s and 1970’s, several companies started developing disposable plastic and glass cell culture products, improved filtration products for cell culture works. • 1966, Alec Issacs demonstrated the presence of Interferon in animal cell cultures infected with some viruses. History • 1980, recombinant erythropoietin was prepared from Chinese hamster ovary (CHO) cell lines. • 1982, Thilly et al., established the scaling up process for the large scale culture of animal cells. • 2002, Human Genome society (France) funded for research leading to cloned human body. Why is cell culture used for? Areas where cell culture technology is currently playing a major role. • Model systems for Studying basic cell biology, interactions between disease causing agents and cells, effects of drugs on cells, process and triggering of aging & nutritional studies • Toxicity testing Study the effects of new drugs • Cancer research Study the function of various chemicals, virus & radiation to convert normal cultured cells to cancerous cells Why is cell culture used for? Contd., • Virology Cultivation of virus for vaccine production, also used to study there infectious cycle. • Genetic Engineering Production of commercial proteins, large scale production of viruses for use in vaccine production e.g. polio, rabies, chicken pox, hepatitis B & measles • Gene therapy Cells having a functional gene can be replaced to cells which are having non-functional gene It has many potential applications in production of vaccines, bioactive compounds, monoclonal antibodies and cell clones for gene therapy. Three categories (1) Explant culture Small pieces of tissues excised from the body of an animal, for animal tissue culture are called Explants.
Petriplates, conical flasks, roller bottles etc., are
used to culture the explants. Explant culture Three categories (2) Organ culture The culture of intact organ fragments or two or more cell types that make up the part of an organ in culture media is called organ culture. Organ Culture Three categories (3) Cell culture This is maintenance of individual cells in culture Media.
Unlike the explants and organ cultures, cell
Cultures are done in large flasks or bioreactors.
Cell cultures are done with a view of producing
tissue culture based products of commercial importance. Cell culture
Hela Cells Fibroblast Cells
Setup Requirements • Requires enough infrastructures such as equipments and chemicals to grow animal cells and tissues in a safe and efficient manner. • Single use facility: isolated work area for cell culture. • Work area should be free from contaminants. • Work surfaces should be thoroughly cleaned between each activities. Requirements • Materials used should be used after proper sterilization and should be free from bacteria, fungi and mycoplasma contaminants. Specific Requirements: (1) Laminar Flow Hoods. (2) Centrifuges. (3) Incubators. (4) Work area and flooring. (5) Plasticwares. (6) Gasswares. (7) Waterbath. (8) Inverted microscope. (9) Vaccum pump. (10)Liquid Nitrogen Container. Specific Requirements: Laminar Airflow • Laminar flow hoods are sterile air cabinets designed in such a way that sterile air passes across the work area continuously. • The air is pumped into the cabinet by an electric blower. • The air entering the work area is filtered by high efficiency particulate air supply filter (HEPA filter). • Contaminated air passes through HEPA filter. • If air filters are kept in vertical position, are called vertical laminar flow hoods and when kept in horizontal position, are called horizontal laminar flow hoods Vertical Laminar Flow Hood Horizontal Laminar Flow Hood Specific Requirements: Centrifuges
• Routinely used in tissue culture for the
concentration of cells for cryopreservation. • An ideal centrifuge should have a clear lid so that the condition of the load can be observed without opening the lid. • Care should be taken not to over fill the tubes and to balance them carefully. • These simple steps will reduce the risk of aerosols being generated. Specific Requirements: Incubators • They are constant temperature rooms used to grow cells and tissues. • Provides controlled temperature environment facilitating the growth of animal cells and tissues. • Degree of humidity and CO2 levels can be in a controlled manner. • Usually incubators are set to run at 28°C for insect cell lines and 37°C for mammalian cells. • Copper coated incubators reduces the risk of microbial contamination. • Water bath treatment trays will also reduce the risk of bacterial and fungal growth in the water trays. Specific Requirements: Incubators Specific Requirements: Work Area and Flooring • Work area and flooring must be smooth enough for easy cleaning. • They should be waterproof and resistant to a variety of chemicals such as acids, alkalis, solvents and disinfectants. • Cold storage rooms should be resistant to cracking. • Walls and windows should be covered with screens to avoid dusting. Specific Requirements: Plasticwares
• Cell culture vessels are commercially available
for single use as sterile packed, plasticwares. • Not effective when recycling. • They should be used only once (use and throw). • They provide a hydrophilic surface to facilitate the attachment of binding dependent cells. Specific Requirements: Plasticwares Specific Requirements: Glasswares • Flasks, roller bottle, hollow fibre catridge, petridishes, glass slides etc., are used. • Conical flasks are commonly used for formulation of medium. • Test tubes are used for trypsinization. • Pipettes are used for aseptic transfer of cell cultures and media b/w vessels. Water bath • To warm media, TRED and PBS before placing on cells • Can harbor fungi and bacteria, spray all items with 70% ethanol before placing in the hood. • Usually takes 10 -15 minutes for media to warm, 5-10 for TRED to thaw Vacuum pump • For permanent aspiration of liquids (media, PBS and TRED). • Use unplugged glass pasteur pipets, throw into sharps box when done. Inverted Phase Microscope • A phase contrast microscope with objectives below the specimen. • A phase plate with an annulus will aid in exploiting differences in refractive indices in different areas of the cells and surrounding areas, creating contrast Cell Culture • Pros • Use of animals reduced • Cells from one cell line are homogenous and have same growth requirements, optimizing growing patterns. • In vitro models allow for control of the extracellular environment • Able to monitor various elements and secretions without interference from other biological molecules that occurs in vivo • Cons • Removal of cells from their in vivo environment means removing the cells, hormones, support structures and various other chemicals that the cells interact with in vivo. • It is nearly impossible to recreate the in vivo environment. The artificial conditions could cause cells to de-differentiate which will cause them to behave differently and produce proteins other than it would in vivo. – Genotype: the genetic make-up of the cell – Phenotype: the appearance and behavior of a cell as a result of their genotype. Most often, scientists are looking at phenotypic changes in their analysis of cells in culture
