Overall Solution

The bioreactor protocol consists of 5 main parts: (1) sanitizing preparation, (2) cell culture preparation (3) bioreactor setup (4) experiment (5) and clean up.

1. Sanitizing preparation

This step is performed at least 1 day prior to the experiment. On all equipment, two main sanitary steps occur (1) autoclave (2) 70% ethanol washes. All equipment and parts for the bioreactor undergoes autoclaving and normal temperature and pressure specifications. Once the equipment is autoclaved, it is stored in a sanitary fashion either sealed or in closed containers until the day of the experiment. On the day of the experiment, we perform the second sanitation step which is the ethanol washers.We first place the pumps, tubing, all adapters, and fittings into the biosafety cabinet. Next, we then set up three dishes with the first being deionized water and the last two being 70% EtOH. The approach is to place all the adapters and fittings into the first dish containing dH₂O and remove any residual media and debris. Once this is clean, we move the pieces into the next dish containing 70% EtOH. This will remove any contaminants or bacteria that are stuck in the crevices of the adapters and fittings. We then place all the pieces into the last dish containing 70% EtOH, which will essentially increase the sanitation efficiency. Once this is finished, the sanitation preparation is complete. This should be performed the day of the experiment prior to setting up the bioreactor.

2. Cell culture preparation

The day prior to the experiment, we must culture the cells and allow them to properly adhere to the cell microfluidic chips. Refer to the “adherence cells setup” protocol of paragraph C design solution section regarding this step.

3. Bioreactor Setup

Once everything is sanitized, we begin our setup of the bioreactor within a standard biosafety cabinet. This will reduce the possibility of contamination. For this step, we will assemble the tubings, pumps, and all associated parts prior to placing the microfluidic chips. Once this is complete we will close the bioreactor system from the environment by sealing the microfluidic chips, and sealing all loose ends of any tubings. We will then move this to setup into the incubator to start the experiment.

4. Experiment

The experiment will be performed within a standard incubator with the peristaltic pumps connected to a standard 120 VAC outlet. Depending on the experiment, the length will vary. For this particular course, we want to examine the growth rate of C2C12 cells with different serine and glycine conditions. We will measure the output of different metabolites of these three different conditions at different timepoints. For a control, we will compare this data to a batch reactor and examine the concentration of cells for each system. Once complete we will immediately begin the clean up stage.

5. Clean up

The clean stage is quite simple. Immediately after finishing the experiment. We will dispose of the media, cells, and all other consumables appropriately. We will then perform the ethanol wash steps used for sterilization. Refer to sanitation preparation step 2 for more details.