CO2 delivery system

In our setup, we chose to use the sublimation of dry ice to supply the CO₂ because dry ice is readily available in the United States. This approach is highly flexible and can accommodate the different incubator volumes needed. Our measurements indicated that 50 mL of CO₂ is produced per minute from the sublimation of dry ice inside a thermos, as measured by a bubble flow meter (Optiflow 520 Digital Volumetric Flowmeter). The ability of the PrintrLab incubator to grow and possibly transport a large volume of flasks/plates with temperature control also makes it a viable option for the transport of cell therapies products over other commercially portable incubators [3]. Although storing dry ice in a capped thermos is not recommended for safety reasons, we have a simple and low-cost method to provide a lid and venting mechanism to prevent excessive pressure buildup. We capped the insulated stainless-steel thermos containers (Thermos Brand, 24 or 40 oz.) with a flexible seal made of a latex balloon that is cut at the bulb end to cover the thermos. The flexible latex material from the balloon allows the CO₂ to expand slightly as the dry ice sublimates so that pressure does not build up in the thermos (Fig 2). Moreover, it acts as a safety pressure release since the balloon will detach from the thermos if the pressure becomes too high. During normal operation, the CO₂ generated from sublimation leaks slightly through the balloon seal. Therefore, we have not seen the balloon inflated significantly. Using a monometer (Risepro HT-1890), we measured the pressure build up and it did not rise above 0.22 pound per square inch (PSI) (S1 Fig). A fully inflated balloon gave a PSI value less than 0.3 PSI (S2 Fig). As shown in Fig 1, we also made a passive venting system using a three-way polyurethane air hose pipe. We covered the third opening with flexible latex plastic as a relief valve. This further reduced the pressure inside the thermos to less than 0.01 PSI (S3 Fig). Overall, these features allow positive pressure to push the CO₂ into the incubator when the solenoid valve is opened without allowing excessive pressure build up in the thermos when the solenoid valve is closed.

The thermos thus can store dry ice safely while providing CO₂ to the incubator. The capacity to store more dry ice in a larger thermos allows uninterrupted use for over four days.

To regulate the delivery of CO₂ to the chamber and achieve stable percentage readings, a solenoid valve (¼-inch DC 12V 2-Way Normally Closed Electric Solenoid Air Valve, Amazon) was placed between the thermos and the chamber, which is controlled by the system to deliver bursts of CO₂ to the chamber based on the reading from the nondispersive infrared sensor (NDIR) CO₂ sensor (ExplorIR^®-W 20% CO₂ Sensor, CO2Meter.com). Polyurethane tubing (4 mm OD) and quick-release connectors (Pneumatic 4 mm or 5/32-inch OD, 2.5 mm ID Polyurethane Air Hose Pipe Tubes) were used to connect the CO₂ reservoir to the solenoid valve and the incubation chamber. A small hole (1/32-inch [0.7938 mm]) was drilled in the lid to provide passive venting to avoid pressure and moisture buildup. Although 0.2 to 0.4 μm air filters for incubators are commercially available and can be connected to our system to avoid bacterial or viral contaminations, we did not use them in our experiment because we mainly focused on showing growth.