1、 Preparation before canning
1. Preparation of cell suspension: Collect pre cultured cells from shake flasks or culture dishes, mix well, and take 1mL for cell counting to ensure appropriate inoculation density.
2. Equipment sterilization: Check whether the reactor tank, pipelines, and electrodes have been sterilized (such as 121 ℃ high-pressure steam treatment) to avoid microbial contamination.
3. Culture medium and buffer: Prepare sufficient culture medium and pre adjust the pH to the target range (such as 7.2-7.4), sterilize and cool for later use.
2、 Vaccination and Initial Cultivation
1. Aseptic operation: In the biosafety cabinet, transfer the cell suspension into the reactor through sterile tubing, while introducing compressed air to assist in mixing.
2. Parameter settings:
Temperature: Mammalian cells are typically set at 37 ℃.
Dissolved oxygen (DO): initially set at 40%, maintained stable through gas mixing (O ₂/N ₂/CO ₂).
PH control: Use a peristaltic pump to add acid/alkali or introduce CO ₂ to adjust and maintain the pH between 6.5-7.0 (depending on the cell type).
3、 Monitoring of the cultivation process
1. Regular sampling: online sampling every 24 hours to detect cell density, pH, and metabolite (such as glucose, lactate) concentration.
2. Flow feeding: Based on nutrient consumption, concentrated culture medium or specific components (such as amino acids) are added to prolong the logarithmic growth phase of cells.
3. Shear force control: Low speed stirring (such as 50-100 rpm) or wave reactor is used to avoid mechanical damage.
4、 Harvesting and Cleaning
1. Termination of cultivation: When the cells enter the senescence phase or the accumulation of products reaches its peak, the automatic control system is turned off.
2. Drainage and collection: The culture medium is discharged into the waste liquid tank through compressed air to separate cells from the supernatant.
3. Cleaning and sterilization: Rinse the tank and components with purified water, soak in 0.1M sodium hydroxide overnight, and ensure no residue.
5、 Precautions
1. Aseptic operation: Strictly avoid contamination throughout the process, disinfect gloves and tools before operation.
2. Parameter calibration: Regularly verify pH and dissolved oxygen electrodes to ensure accurate data.
3. Scaling up production: Batch operation can be directly scaled up to industrial scale (such as 12000L), but requires optimization of stirring and mass transfer efficiency.
Through the above steps, the operation of cell culture bioreactors can be efficiently completed, which is suitable for the production of biological products such as monoclonal antibodies and vaccines.