MEC-based cell culture instrument¶
Finally, to demonstrate that the MEC system can be used to create research-grade biological instruments, a MEC-based bioreactor was built. The bioreactor (Fig 5C) contains a square-shaped loop of MECs that contains 46 mL of cell culture media. This loop was constructed from a new family of MEC components that interconnect lengths of 0.5-inch diameter rigid plastic tubing. These interconnect MECs are fabricated to the MEC mechanical standards in both size and interface spacing. This allows them to easily mount to our standard baseboard as shown in Fig 5C. The media is recirculated (and cells are kept in suspension) using a stream of air bubbles, although pump MECs could also be used. The bioreactor uses the optical density MEC (OD1 in Fig 3B) to measure the optical density of the cell culture in the media loop.
After sterilizing the MEC bioreactor with a 70% ethanol solution, the bioreactor was filled with yeast extract peptone dextrose (YEPD) media, then seeded with a small amount of Saccharomyces cerevisiae yeast culture. The bioreactor was then placed in an incubator at 30°C for 24 hours. An inexpensive data acquisition card (NI USB-6008, National Instruments, Austin, TX) was used to record the voltage output of the optical density MEC, though an Arduino-based microcontroller MEC (under development) could also be used. Data from the optical density MEC is available in S1 File; this file includes the Python program used to analyze the data.
The output of the optical density MEC is plotted versus time in Fig 5D. The slight decrease in growth rate observed at 13 hours likely represents the diauxic shift, the point at which the glucose in the culture media is depleted and the yeast cells must switch their metabolism from glycolysis to the less-favored aerobic oxidation of ethanol. Additionally, the halt in growth observed after 15 hours indicates the depletion of ethanol in the media and the entry of the yeast cells into stationary phase (G0). These metabolic transitions are extremely important in yeast biology and even provide insights into human conditions like cancer and aging 10. The fact that our prototype MEC-based bioreactor is capable of observing these biologically and clinically meaningful metabolic transitions confirms that research-grade instruments can be built using the MEC system.
© A MEC-based bioreactor capable of culturing cells. The optical density macroMEC (OD1 in Fig 3B) measures the concentration (via optical density) of the cells as they grow and react to stimuli. (D) A growth curve obtained by using the bioreactor in C to culture Saccharomyces cerevisiae yeast cells. The bioreactor is sensitive enough to detect important metabolic checkpoints in the growing yeast, including the diauxic shift (when the yeast cells switch from glycolysis to the aerobic oxidation of ethanol) and the entry into G0 (when the yeast cells exhaust all nutrients and enter stationary phase).
The analytical bioreactor can be used for applications ranging from producing biofuels to curing diseases. One application envisioned is a personalized bioreactor to train a patients T-cells to attack cancer. Control of the cellular environment is needed for this type of application. Our Analytical bioreactor is created in a loop and developed with thermal and media flow control in that loop. The bioreactor has the ability to inject air or gases such as CO2 into the media. It also has a media and waste disposal system. The device houses a serial dispensing system with precise control over injection volumes of various biomaterials, small molecules, or nutrients as needed. Cell density can be maintained and controlled through an optical density sensor. An onboard minicomputer with a display and keypad takes care of basic operations. Another application would be in a class environment enabling high school or college students to grow cells and be motivated with the challenges of engineering real world solutions.