BIO62004 Instrumentation in Medical Diagnostic, Laboratory and Blood Banking PDF

BIO62004 Instrumentation in Medical Diagnostic, Laboratory and Blood Banking Topic 2: Maintenance of Mammalian Cell Culture 2.1 (L4): Aseptic Techniques in maintenance of cell culture in vitro ...

BIO62004 Instrumentation in Medical Diagnostic, Laboratory and Blood Banking Topic 2: Maintenance of Mammalian Cell Culture 2.1 (L4): Aseptic Techniques in maintenance of cell culture in vitro  Learning objectives Students should be able to: 1. Discuss the principles of animal cell culture 2. Demonstrate animal cell culture techniques 3. Relate animal cell culture to specialized applications 4. Understand the importance of aseptic technique (Sterile work area, Personal hygiene, Sterile reagents and media) 5. Disuses the principles of cryopreservation and cell banking  Animal Cell Culture Cell culture refers to the process by which cells are grown in a controlled artificial environment. Cells can be maintained in vitro outside of their original body by this process which is quite simple compared to organ and tissue culture. https://microbeonline.com/animal-cell-culture-introduction-types-methods-applications/  Primary Cell Culture Outgrowth of cells from a piece of tissue or from tissue that is disaggregated by enzymatic and/or mechanical methods. Primary cells are morphologically similar to the parent tissue. Limited number of cell divisions Cells will enter a non-proliferative state called senescence and eventually die out. Extension of proliferative capacity of cells  introduction of viral transforming genes (e.g., the SV40 transforming-antigen genes). The phenotype of these cultures is intermediate between finite cultures and continuous cultures. The cells will proliferate for an extended time, but usually the culture will eventually cease dividing, similar to senescent primary cells. * finite / continuous cell cultures https://www.qiagen.com/br/resources/molecular-biology-methods/animal-cell-culture/  Two main types of cells Depending on their origin, animal cells grow either: Adherent cells – Are anchorage-dependent and propagate as a monolayer attached to the cell culture vessel. – Most cells derived from tissues are anchorage- dependent. Suspension cells can survive and proliferate without being attached to a substratum.  Handling cell cultures Adherence to good laboratory practice when working with cell cultures is essential: To reduce the risk of exposure of the worker to any potentially infectious agent(s) in the cell culture To prevent contamination of the cell culture with microbial or other animal cells http://www.bionique.com/mycoplasma-resources/technical- articles/better-aseptic-technique.html  Handling cell cultures Aseptic technique and the proper use of laboratory equipment are essential when working with cell cultures. Always use sterile equipment and reagents, and wash hands, reagent bottles, and work surfaces with a biocide or 70% ethanol before beginning work.  Cell culture conditions Culture media and serum Different cell types have highly specific growth requirements, and the most suitable medium for each cell type must be determined experimentally. http://mousecells.easycgi.com/productslist2.aspx?sousuo=ENDOTHELIAL% 20CELLS  Cell culture conditions Common basal media include Eagle minimal essential medium (MEM), Dulbecco’s modified Eagle medium (DMEM), RPMI 1640, and Ham F10. These contain a mixture of amino acids, glucose, salts, vitamins, and other nutrients, and are available either as a powder or as a liquid from various commercial suppliers.  Basic components of media Bicarbonate buffering system – CO2 balances with the HCO3- content of the culture medium. – Cultures using bicarbonate buffering system needs to be maintained in an atmosphere of 5-10% CO2 in air usually supplied by CO2 incubator. – Bicarbonate buffering system is generally low cost and non-toxic  Basic components of media HEPES buffering system – Chemical buffer HEPES has a superior buffering capacity in the pH range 7.2-7.6 – HEPES is relatively expensive and toxic at higher concentration for some cell types. – HEPES has also been shown to greatly increase the sensitivity of media to phototoxic effects induced by exposure to fluorescent light  Basic components of media Phenol Red – Most of the commercially available culture media include phenol red as a pH indicator which allows constant monitoring of pH. – At low pH, phenol red turns the medium yellow, while at higher pH levels it turns the medium purple. – However, there are certain disadvantages of using phenol red: why ???  Serum Provides several growth factors and hormones involved in growth promotion and specialized cell function. Contains several binding proteins like albumin, transferrin, which can carry other molecules into the cell. For example: albumin carries lipids, vitamins, hormones, etc into cells. Supplies proteins, like fibronectin, which promote attachment of cells to the substrate. It also provides spreading factors that help the cells to spread out before they begin to divide. Provides protease inhibitors which protect cells from proteolysis. Contains minerals, like Na+, K+, Zn2+, Fe2+, etc. Increases the viscosity of medium and thus, protects cells from mechanical damages during agitation of suspension cultures.  Serum Inhibitors: – Serum may contain substances that inhibit cell proliferation – Heat inactivation removes complement proteins from the serum and reduces the cytotoxic actions of immunoglobulins without damaging other growth factors – Heat inactivation is carry out by incubating serum for 30 minutes at 56°C  Serum Advantages of serum Disadvantages of serum Contains various growth factors and Batch variation hormones which stimulates cell Testing needs to be done to maintain growth and functions. the quality of each batch before Helps in attachment of the cells using Acts as a spreading factor May contain some of the growth Acts as a buffering agent which helps inhibiting factors in maintaining the pH of the culture Increase the risk of contamination media Presence of serum in media may Functions as a binding protein interfere with the purification and Minimizes mechanical damages or isolation of cell culture products damages caused by viscosity  Serum free media Serum-free media (SFM) circumvents issues with using animal sera by replacing the serum with appropriate nutritional and hormonal formulations. Serum-free media formulations exist for many primary cultures and cell lines, including recombinant protein producing lines of Chinese Hamster Ovary (CHO), various hybridoma cell lines, the insect lines Sf9 and Sf21, and for cell lines that act as hosts for viral production and others.  Requirements of a tissue culture lab Three categories: Essential: you cannot perform a job without them Beneficial: the work would be done better, more efficiently with less labour Useful: they would make life easier, improve working conditions, enable more sophisticated analysis to be made  Requirements of a tissue culture lab Basic equipment Cell culture hood (laminar-flow hood or biosafety cabinet) Incubator (humid CO2 incubator recommended) Water bath Centrifuge Refrigerator and freezer (–20°C) Cell counter (hemocytometer) Inverted microscope Liquid nitrogen (N2) freezer or cryostorage container Sterilizer (autoclave)  Requirements of a tissue culture lab Beneficial equipment Aspiration pump (peristaltic or vacuum) pH meter Tube racks Pipettes Plate reader  Requirements of a tissue culture lab Useful additional equipment Glassware washing machine Confocal microscope Flow cytometer Culture vessels Sterile containers Syringes and needles  Cell culture hoods Air flow characteristics Cell culture hoods protect the working environment from dust and other airborne contaminants by maintaining a constant, unidirectional flow of HEPA-filtered air over the work area. The flow can be horizontal, blowing parallel to the work surface, or it can be vertical, blowing from the top of the cabinet onto the work surface. Adapted from Cell culture basics, Invitrogen **HEPA filter: a fine mesh that traps harmful particles such as pollen, pet dander, dust mites, and tobacco smoke http://517477.homepagemodules.de/t107f15-Sicherheitsbelehrung-Gentechnik.html  Cell culture hoods Air flow characteristics Depending on its design, a horizontal flow hood provides protection to the culture (if the air flowing towards the user) or to the user (if the air is drawn in through the front of the cabinet by negative air pressure inside). Vertical flow hoods, on the other hand, provide significant protection to the user and the cell culture.  Cell culture conditions Incubation conditions Cell cultures should be incubated in an incubator with a tightly regulated temperature (e.g., a water-jacketed incubator) and CO2 concentration. The purpose of the incubator is to provide the appropriate environment for cell growth. Frequent cleaning of the incubator is essential to avoid contamination of cell cultures. There are two basic types of incubators, dry incubators and humid CO2 incubators. http://ihcp.jrc.ec.europa.eu/our_labs/nanob_lab/nanobiotech- lab-photo-gallery/DSC_5605.jpg/view  Cell culture conditions Cell culture vessels Sterile, disposable dishes and flasks that have been treated to allow attachment of animal cells to the growing surface are available commercially. http://www.bdbiosciences.com/ca/cellculture/dishes/index.jsp http://us.gbo.com/bioscience/products/5/  Cell culture conditions Substrates and matrices Cells are able to attach to acid-washed glass, polystyrene treated by electric ion discharge, plastics with a net positive charge Question: How do cells attach? Cell secretes matrix products that adhere to the substrate and provide ligands for the interaction of matrix receptors such as integrins Treat the substrate with a matrix product, such as collagen type IV, fibronectin, or laminin, to promote the adhesion of cells  After studying this lecture, you should understand able to: 1. Discuss the principles of animal cell culture 2. Demonstrate animal cell culture techniques 3. Relate animal cell culture to specialized applications 4. Understand the importance of aseptic technique (Sterile work area, Personal hygiene, Sterile reagents and media) 5. Disuses the principles of cryopreservation and cell banking