Cell Culture Protocols PDF

Protocol 1 - Aseptic Technique and Good Cell Culture Practice Aim To ensure all cell culture procedures are performed to a standard that will prevent contamination from bacteria, fungi and mycoplasma and cross contamination with other cell lines. Materials 70% (v/v) alcohol in sterile water...

Protocol 1 - Aseptic Technique and Good Cell Culture Practice Aim To ensure all cell culture procedures are performed to a standard that will prevent contamination from bacteria, fungi and mycoplasma and cross contamination with other cell lines. Materials 70% (v/v) alcohol in sterile water Sodium Hypochlorite Equipment Personal protective equipment (sterile gloves, laboratory coat, safety visor, overshoes, head cap) Microbiological safety cabinet at appropriate containment level Procedure 1. Sanitise the cabinet using 70% alcohol before commencing work. 2. Sanitise gloves by spraying them with 70% alcohol and allowing to air dry for 30 seconds before commencing work. 3. Put all materials and equipment into the cabinet prior to starting work. Equipment in the cabinet or that which will be taken into the cabinet during cell culture procedures (media bottles, pipette tip boxes, pipette aids) should be wiped with tissue soaked with 70% alcohol prior to use. 4. Whilst working do not contaminate gloves by touching anything outside the cabinet (especially face and hair). If gloves become contaminated re-spray with 70% alcohol as above before proceeding. 5. Discard gloves after handling contaminated cultures and at the end of all cell culture procedures. 6. Movement within and immediately outside the cabinet must not be rapid. Slow movement will allow the air within the cabinet to circulate properly. 7. Speech, sneezing and coughing must be directed away from the cabinet so as not to disrupt the airflows. 8. After completing work disinfect all equipment and material before removing from the cabinet. Spray the work surfaces inside the cabinet with 70% alcohol and wipe dry with tissue. 9. Liquid cell culture waste should be discarded in sodium hypochlorite (10,000 ppm) and must be kept in the cabinet for a minimum of two hours (preferably overnight) prior to discarding to the drain with copious amounts of water. 10. Periodically clean the cabinet surfaces with a disinfectant or fumigate the cabinet according to the manufacturers instructions. However, you must ensure that it is safe to fumigate your own laboratory environment due to the generation of gaseous formaldehyde, consult your on-site Health and Safety Advisor. Protocol 2 - Subculture of Adherent Cell Lines Aim Adherent cell lines will grow in vitro until they have covered the surface area available or the medium is depleted of nutrients. At this point the cell lines should be subcultured in order to prevent the culture dying. To subculture the cells they need to be brought into suspension. The degree of adhesion varies from cell line to cell line but in the majority of cases proteases, e.g. trypsin, are used to release the cells from the flask. However, this may not be appropriate for some lines where exposure to proteases is harmful or where the enzymes used remove membrane markers/receptors of interest. In these cases cells should be brought into suspension into a small volume of medium mechanically with the aid of cell scrapers. Materials Media – pre- warmed to 37°C (refer to the ECACC cell line data sheet for the correct medium) 70% (v/v) isopropanol in sterile water PBS without Ca2+/Mg2+ 0.05% trypsin/EDTA in HBSS, without Ca2+/Mg2+ Soyabean Trypsin Inhibitor Trypan blue (vital stain) Equipment Personal protective equipment (sterile gloves, laboratory coat, safety visor) Waterbath set to appropriate temperature Microbiological safety cabinet at appropriate containment level Incubator Pre-labelled flasks Inverted phase contrast microscope Centrifuge Haemocytometer or automated cell counter like Muse® Cell Analyzer or Scepter™ Cell Counter Marker Pen Pipettes Ampoule rack Tissue Procedure 1. View cultures using an inverted microscope to assess the degree of confluency and confirm the absence of bacterial and fungal contaminants. 2. Remove spent medium. 3. Wash the cell monolayer with PBS without Ca2+/Mg2+. Repeat this wash step if the cells are known to adhere strongly. 4. Pipette trypsin/EDTA onto the washed cell monolayer using approximately 1ml per 25cm2 of surface area. Rotate flask to cover the monolayer with trypsin. Decant the excess trypsin. 5. Return flask to the incubator and leave for 2-10 minutes. 6. Examine the cells using an inverted microscope to ensure that all the cells are detached and floating. The side of the flasks may be gently tapped to release any remaining attached cells. 7. Resuspend the cells in a small volume of fresh serum-containing medium to inactivate the trypsin. Remove 100-200μl and perform a cell count (see Protocol 3 – Cell Quantification). In the case of cells cultured in serum-free media, use a trypsin inhibitor e.g. soyabean trypsin inhibitor to inactivate the trypsin. 8. Transfer the required number of cells to a new labelled flask containing pre-warmed medium (refer to the appropriate ECACC Cell Line Data Sheet for the required seeding density). 9. Incubate as appropriate for the cell line. 10. Repeat this process as demanded by the growth characteristics of the cell line.