Bioreactor Assembly for Continuous Culture of Complex Fecal Communities

We study host bacteria interactions and how intrinsic and extrinsic factors influence health, focusing on bacterial effects on cancer development, progression, and treatment response. We studied anti-PD-1 immune checkpoint inhibitors in bacterial communities, finding that bacterial metabolites enhance anti-PD-1 therapy efficacy.

Isolating host factors' impact on the microbiome in vivo is challenging due to multiple influences. The microbioreactor array allows controlled examination of specific processes, clarifying their direct effects on microbial communities. This protocol offers a cost-effective method to cultivate fecal communities under controlled conditions while enabling researchers to run experiments in parallel relatively quickly.

[Instructor] To begin, obtain a 3D-printed six-well reactor array. Cut twelve 25-millimeter strips from 1/8 inch PTFE tubing and use a razor to shave ends. Insert one end of each strip into the screw thread opening of a male Luer with a 25 to 28-thread fitting. Securely screw the fittings into the designated waste and sample openings of the array. Now, insert a microstir bar into each reactor well. Screw a male 1/4-28 thread fitting into the source holes of each reactor well. Screw female Luer hose bar adapter fittings into the thread fittings for the waste and source connections of each reactor well. Then screw female Luer X to 3-32/2-inch hose barb adapter fittings into the 1/4-28 thread fittings for the sample ports. Place a rubber septum over the hose barb adapters and fold the top over. Next, cut two sets of E-LFL 2.06 millimeter tubing for the waste and source lines. Use hot water to soften the tubing for easier attachment. Attach the tubing to the female Luer X to 1/8 inch hose barb adapter fittings for the source and waste connections. Now fix a male Luer with a lock ring X to 1/8 inch hose barb adapter fitting to the other end of the tubing. Screw a female Luer X to 1/16th inch hose barb adapter fitting into it. Connect the appropriate size two-stop tubing to the corresponding source and waste connections. Then fix another female Luer X to 1/16th inch hose barb adapter fitting to the other end of the tubing. After repeating this for all source and waste fittings, insert a male Luer with lock ring X to 1/8 inch hose barb adapter fittings to a piece of E-LFL 2 tube. Attach one end to the unoccupied female Luer X to 1/16th inch hose barb adapter at the end of each two-stop tubing section for all pieces. After repeating this for all source and waste fittings, connect the E-LFL 2.06 millimeter tubing for the source lines of reactor wells one and two, using opposite ends of a female Luer T. Then fix a five centimeter piece of tubing with male Luer with lock ring X to 1/8 inch adapter fittings on each end to the middle outlet of the T, leading to a second T junction. Now connect the E-LFL 2.06 millimeter source tubing for the third reactor to the opposite side of the second T. Attach another five centimeter tubing segment having a male Luer with lock ring X to 1/8 inch adapter on one end and a female Luer with lock ring X to 1/8 inch adapter on the other. Next, attach a male Luer with lock ring X to 1/8 inch hose barb adapter fitting to one end of a 25 centimeter piece of E-LFL 2.06 millimeter tubing. Fix a female Luer X to 1/8 inch hose barb adapter fitting to the other end and cap it with aluminum foil. To assemble the waste collection bottle, loosely secure a two-hole cap onto a two-liter bottle. Insert male 1/4-28 thread fittings into both holes of the cap. Then attach a female Luer with 1/8 inch hose barb adapter to the male thread fitting that does not connect to the pre-attached PTFE tubing in the cap. Connect a five centimeter piece of E-LFL 2.06 millimeter tubing to the hose barb adapter on the bottle. Attach a male Luer with lock ring X to 1/8 inch hose barb adapter to the other end of the tubing before covering it with foil. Finally, cover the entire bottle cap with aluminum foil before autoclaving at 121 degrees Celsius and 15 psi for 45 minutes. Once Autoclaving is complete, tighten the fittings and foil seals and place the reactors in an anaerobic chamber for 48 hours before use. Align the reactor array inside the anaerobic chamber over a magnetic stir plate set to 1,600 RPM. Then fit the two-stop tubing for both the source and waste lines into the clamps on the corresponding peristaltic pumps. Lock the clamps in place if this is done less than 25 hours before use. Otherwise, leave it loose to reduce the wear on the tubing. Now remove the foil cap from the source bottle. Screw the fittings into the male Luer to 1/4-28 thread fittings, ensuring that these openings are connected to the internal PTFE tubing. Remove the foil caps, and attach a 0.22 micrometer syringe filter to the five centimeter vent line of both the source bottle and the waste bottle. Begin the flow at a moderate rate to begin filling the reactor. Ensure that the waistline tubing inside each reactor well is positioned lower than the source tubing to prevent overfilling or the complete emptying of each well. As the reactors begin to fill, check that all wells reach the same volume and that media flows into the waistline before leaving the reactors to degas overnight. Start by transferring the experimental materials to the anaerobic chamber for 24 hours. After calculating the amount of fecal inoculum needed, transfer the preserved fecal samples in cryogenic vials into the anaerobic chamber and allow them to thaw for 15 minutes. With a wide bore pipette tip, transfer the fecal material from cryogenic vials to a 50-milliliter conical tube. Centrifuge the sealed tube at 200 g for five minutes to settle particulates. Transfer the tubes back into the anaerobic chamber. Then remove the supernatant. Use an autoclave laboratory spatula to transfer the 5.7 grams of the fecal solids into a new 50-milliliter conical tube. Add 17.1 milliliters of anaerobic sterile saline to the tube and seal tightly. Now vortex the tube for five minutes before centrifuging as demonstrated earlier. Use a 10% bleach laboratory wipe to sterilize the septa on top of each reactor for at least two minutes. After returning the samples to the anaerobic chamber, pipet out the supernatant to new 50-milliliter conical tubes. Now transfer 3.8 milliliters of fecal slurry supernatant into six separate five-milliliter syringes fitted with 16-gauge 1.5-inch needles. Remove the bleach wipe from the reactor septa. Then insert the needle through the septum and slowly inject 3.8 milliliters of fecal slurry into each reactor before leaving the reactors to sit overnight. After a 16 to 18-hour incubation, start the flow to the reactor. Set the magnetic stir plate to maximum speed immediately after injecting the fecal slurry. The microbial community composition in both replicate reactors changed over time with different species showing variable relative abundance across days. Fastidious anaerobic bacteria, such as Clostridium genera, were consistently detected in each reactor at all time points.