细胞培养 cell culture procedures.docVIP

4.2 Cell culture procedures The following important conditions must be satisfied to achieve successful cell culture: ??????????Incubation temperature should be 36°C. ??????????The pH for growth should be between 7.2 and 7.4. The levels of glucose and L-glutamine can influence cell growth, and correct levels for each cell line should be checked before attempting to put it into culture (typical levels for glucose and L-glutamine are 1-4 mM and 2 mM, respectively). A range of inorganic ions, amino acids and vitamins are essential for cell survival and will usually be included in basal growth media from proprietary sources. Both oxygen and carbon dioxide are essential and are provided either as a mixture of CO2?and air supplied to the culture vessel or by sealing the vessel tightly to retain the CO2?produced by cell metabolism. 4.2.1 ?? Aseptic technique Skill in aseptic technique is important to maintain sterility during media preparation and cell cultivation procedures. Furthermore, it is a vital component in ensuring operator protection from infectious agents that may be present in culture materials. Some important elements in aseptic technique are: ??????????Sterilize all glassware for handling cell cultures and media?(see below). ??????????Avoid splashes, spills and aerosols. ??????????Avoid liquid transfer by pouring. ??????????When adding (or replacing) medium, never touch the neck of the culture flasks with the bottle containing the medium or use the same pipette to transfer medium to more than one bottle. Ideally, aliquot the total amount of medium required for each batch of culture bottles being handled and store the remainder at 4–8°C. Dedicate separate medium for each cell line. ??????????Separate clean and contaminated materials in the BSC II. ??????????Minimize exposure of sterile media and cell cultures to open air (even within the BSC?II). ??????????Perform any final preparation of sterile media (i.e. addition of serum or other additives) before dealing