1、 Preparation before operation
1. Cleaning and disinfection
Disassembly and cleaning: After disassembling the various components of the reactor (tank body, pipelines, electrodes, etc.), soak and clean them separately with detergent and 1% sodium hydroxide solution, and rinse them with purified water for more than 10 times.
Disinfection treatment: Soak the cleaned parts in disinfectant (diluted proportionally) for 30 minutes, and avoid direct contact with the surface after drying.
Sensor maintenance: Soak the pH electrode in a saturated KCl solution, replace the electrolyte and eliminate bubbles for the dissolved oxygen (DO) electrode, and polarize for more than 6 hours.
2. Tank assembly
Sealed installation: Align the tank cover with the tank base, use an Allen tool to tighten the screws diagonally and evenly to ensure sealing.
Pipeline connection: Connect replenishment bottles, exhaust bottles, sampling bottles, etc. through silicone hoses or quick connectors, and manually tighten the electrodes after inserting them into the corresponding interfaces.
2、 Loading operation
1. Cell inoculation
Mix the pre cultured cell suspension evenly, count it, transfer it to the inoculation bottle, and sterile connect it to the reactor inlet tube.
Use compressed air to pressurize the cell suspension into the reactor to avoid cell sedimentation and clumping.
2. Parameter initialization
Environmental settings: Set the temperature to 37 ℃ (mammalian cells), dissolved oxygen to 40%, pH to around 7.0, and initiate automatic adjustment through the control panel.
Feeding and ventilation: The peristaltic pump controls the rate of fluid replenishment, and the intake system adjusts the oxygen pressure to 0.1 MPa to ensure stable dissolved oxygen.
3、 Cultivation process control
1. Dynamic monitoring
Sensor linkage: Real time monitoring of temperature, pH, DO and other parameters, controller automatically adjusts mixing speed (100-300 rpm) and feeding rate.
Online sampling: Samples are taken every 24 hours to detect cell density and metabolic status. After sampling, the end of the pipeline is immersed in 75% alcohol to prevent contamination.
2. Pollution prevention and control
Maintain a sterile environment throughout the operation, use flame to disinfect the connection port, and prevent external impurities from entering.
4、 Operation and maintenance of the lower tank
1. Termination of cultivation
Turn off the automatic control module, discharge the culture medium into the waste liquid tank through compressed air, and disconnect the electrode connection.
2. Cleaning and Storage
Disassemble the tank components, rinse with purified water, and soak overnight in 0.1M NaOH solution to thoroughly remove any biological residues.
After drying, store in a well ventilated area, and store the electrodes separately in a dedicated packaging box.
5、 Key precautions
1. Sealing inspection: After assembly, a pressure test is required to avoid leakage during the cultivation process.
2. Parameter calibration: pH and DO electrodes need to be calibrated before each use to ensure data accuracy.
3. Scale up: Following a constant ventilation volume to liquid volume ratio (VVM) to ensure consistent dissolved oxygen transfer efficiency.
Through the above steps, the stainless steel cell bioreactor can achieve stable cell culture from laboratory to pilot scale, suitable for high-precision biological processes such as antibody production and vaccine development.