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Gantt Chart
Gantt Chart
This was our preliminary plan for the entirety of the project. However, various bottlenecks arose and due to COVID-19, we had to suspend some experiments and work that was to be performed in Dr. Christian Metallo's lab.
Bottlenecks
Bottlenecks
Finding a cell culture lab
Running experiments took multiple consecutive days, and we needed to be flexible in working around the schedule of classes using this space. We also took into account university holidays and weekends where the lab will be inaccessible.
Complete thorough calculations when modeling and predicting the operation of the bioreactor
These calculations must be accurate to ensure we order the correctly sized parts and obtain the desired parameters of operation.
Ordering parts
We were subject to shipping time and possible delays that can affect the rest of our workflow. Parts should be able to be ordered again in case we exhaust our resources and need more. However, many parts were shipped quickly and samples were sent for us to use.
Optimizing the bioreactor and microfluidic chip before performing in vitro experiments
Unfortunately, we did not complete the in vitro experiments as soon as we expected due to various flow and system problems we encountered. By the time the problems were resolved, the stay-at-home orders began and we were not able to complete the experiments.
Parts List
Parts List
*Many of the waived components were single parts that we were able to receive samples of from the manufacturer, or were gifts from UCSD labs.
Full Breakdown
Full Breakdown
Preparation
Preparation
Preparation of Cell Line
Firstly, our team must familiarize ourselves with the C2C12 cell line. These are immortalized murine fibroblasts. Our team must thaw and passage these cells successfully in order to validate our cell culture protocol. In addition, our team must determine the optimal seeding density, growth rate, and optimal metabolite (serine and glycine) conditions.
Experiment 1
To determine the ideal serine and glycine concentrations for C2C12 cells, we must run a preliminary experiment. In a 12-well plate, different serine and glycine media concentrations were tested. Testing concentrations: 0mM Ser Gly, 0.1mM Ser, 0.4 mM Ser, 1 mM Ser. Each row was removed for a cell count at the respective time with row A = day 0 (baseline), B = day 1, C = day 4.
Bioreactor Metrics Consideration
Bioreactor Metrics Consideration
In order to determine the success and efficiency of the bioreactor, we must have a control to which we can compare. A perfusion bioreactor is an example of continuous culture where new media is continuously fed into the system whilst old media is being removed at a constant rate such that the volume of media remains constant. Meanwhile, the standard batch culture method is where a limited amount of stagnant media is provided for cell growth.
Suspension & Adherent Cell Consideration
Suspension & Adherent Cell Consideration
Suspension cells consideration
Due to the flow rate of a perfusion bioreactor, cells that are not properly adhered, such as suspension cells, will most likely get flushed out of the microfluidic chips into the waste reservoir. Furthermore, when dealing with suspension cells specifically, if the dilution rate (D) is greater than the cell specific growth rate(g), the cells will get flushed out of the bioreactor by the derived equation.
Cell washout occurs when:
As a result, when dealing with suspension cells within a perfusion bioreactor, we must have the flow rate be less than the cell specific growth rate scaled by the bioreactor volume in order to keep the suspension cells within the bioreactor: F < V*ug.
Adherent cells
In order to minimize cell washout, we will use adherent cells for our bioreactor. Before inputting the cells into this perfusion system, the ideal plan is to culture cells overnight in the microfluidic chips within a standard incubator. Once they are adhered, we will then setup the bioreactor within a biosafety cabinet and lastly install the microfluidic chips.
Bioreactor Protocol
Bioreactor Protocol
Bioreactor Protocol
The bioreactor protocol consists of 5 main parts: (1) sanitizing preparation, (2) cell culture preparation (3) bioreactor setup (4) experiment (5) and clean up.
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.
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.
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.
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.
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.
Time Stamps Consideration
Time Stamps Consideration
Time Stamps for metabolic rates
As mentioned before in the experimental breakdown, there is a need to be able to measure the output of different metabolites of these three different conditions at different timepoints. The flow rate indicator near each cell culture chamber will also include a stopcock that will be able to allow the user to remove media when necessary. For example, and will likely be the case, if the client decided to run a 3 day trial and wanted to measure the amino acid concentration at least once a day to get a trendline, the user can simply turn the stopcock, remove some media, and take it for testing.
Concentration Measurement Considerations
Concentration Measurement Considerations
Amino acid concentration measurement/Cell count
For the determination of cell number, we use standard cell counting procedure using a hemocytometer and trypan blue dye. We can count the cells at the end of the experimentation from each slide in order to determine survival rate and growth. We use the media from each slide at different time points throughout the experiment to determine amino acid concentration (specifically serine and glycine). We may use the mass spectrometer and YSI in Dr. Metallo’s lab. Dr. Metallo’s graduate student Jivani is willing to run these analyses with us. From the stopcock’s shown in the design, we may take used media samples to either be immediately analyzed or frozen down for future analysis.
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