**9. Overall challenges: cell culture**

Culturing techniques for CEF cells or BHK-21 or others is always challenging. The followings are the most important points by maintaining these points researchers or laboratory personnel easily can overcome any possible challenges. The points that need to pay attention such as a). Lab requirement is in the first, set up all the required materials, chemicals, instruments before the work or research b). Novice personnel should be avoided c). Skilled manpower and staff should be appointed for the work and as a trainer for new one d). Interpretation of work daily e). Establish a strong QC department f). Store all the true data and metadata because it is important to identify problems and also help to design a research g). Calibration of pH meter regular basis is mandatory and pH level follow up because cell health depends on it h). Need to take necessary precautions during culture media preparation i). Maintain and check the required CO2 percentage based on cell j). Incubation time and temperature vary from cell to cell and it may be vary based on research work k). Transportation process, medium, time, precautions should be describing in transportation SOP l). Pay attention during thawing of the cell because it causes stress on the cell resulting cell might be death m). More careful and be cautious during preservation of cells, changing of media, passaging, trypsinization, harvesting of cells, mycoplasma contamination, and antibiotics treatment (for bacterial contamination) because these are the fundamental steps in cell culture technology. A non-technical or unskilled or novice personnel can easily ruin all the effective work at a time during the above points. That's why need to be more cautious about this n). Certificate of Analysis (COA) is essential for all the work like media preparation, pH level check, cell morphology, cell count, splitting, transportation, cell purchase or sell, and so on. It reduces the risk O). Based on international reference lab or manual SOP should be developed, SOP of how to prepare a good SOP and working guideline can minimize problems and it will take part to handle challenges. Current Good Manufacturing Practice (cGMP), Documentation of lab work, Training facility both for researchers and staff, Validation of work, calibration of instruments regularly can eradicate the problems and smoothly handle all the challenges.
