Updated Protocol for the Assembly and Use of the Minibioreactor Array (MBRA)

So our research focuses on the gut microbiome, specifically how it can be engineered to prevent the colonization of harmful pathogens. At the same time, we're also interested in understanding the ecological rules and mechanisms behind this colonization resistance. Technology, such as next generation sequencing, advanced bioinformatics, germ-free mice, and in vitro gut models, are all transforming the way we study microbial communities and their role in health and disease.

One of the biggest challenges with in vitro gut models is operating them at high throughput. What we need are systems that allow for the functional interrogation of microbial communities at scale. The minibioreactor array is a continuous flow culture system that was designed to address the lack of throughput capacity in other systems.

It allows us to scale up experiments while still capturing complex and reproducible microbial community behavior. To begin, ensure that the minibioreactor array strips are 3D-printed and contain six independent bioreactor chambers. Arrange all the components required for the assembly.

Using a 1/4-inch 28 NF fraction tap with a T-handle tap wrench. Thread the three 1/4-inch ports in each chamber to insert fittings. After washing the chamber with water, place a 10 by 3 millimeter magnetic stir bar into each chamber and add one milliliter of distilled water.

Then position a rubber washer on top of each port of the bioreactor. For each chamber, screw in one media straw threaded male lure, one waste straw threaded male lure, and one empty threaded male lure into the ports. Now insert six rubber septa onto 3/32-inch female lure barbs and fold the upper sleeve of each septum down to cover the neck.

Attach these to the designated ports of each chamber. Cut C-flex tubing strips of the desired length and number. Attach a 1/8-inch female lure barb to one end and a male lure lock connector to the opposite end of each length of tubing.

Then insert a 1/16-inch female lure barb into each end of the red two-stop E-lab tubing with a 1.14-millimeter inner diameter, and the orange two-stop E-lab tubing with a 0.89-millimeter inner diameter. Connect the prepared E-lab tubing to the C-flex tubing and ensure each of the six C-flex tubing lengths is connected to one red and one orange E-lab line via female lures. Next, cut the C-flex tubing to different lengths as required.

Attach 1/8-inch female lure barb and a male lure lock connector to both ends of one 3-inch piece and the 12-inch piece of C-flex tubing. Attach male lure lock connectors to both ends of the remaining pieces. Assemble the waste line tree according to the 3D diagram.

Attach the exposed ends of the red two-stop E-lab tubing to the terminal male lure locks on the waste line tree in ascending order based on C-flex tubing length. Then connect the 3-inch C-flex tubing with the 1/8-inch female lure barb and male lure lock connector to the top of the waste line tree. Assemble the feed line tree according to the 3D diagram.

Bridge the exposed ends of the orange two-stop E-lab tubing to the terminal male lure locks on the feed line tree in ascending order based on C-flex tubing length, and attach the 12-inch C-flex tubing to the top of the feed line tree. Attach the variable length C-flex tubing at the end of the feed line tree to the bioreactor, arranging them in ascending order from the shortest line on the left to the longest on the right. Attach the variable length C-flex tubing at the end of the waste line tree to the bioreactor strip in descending order with the longest line on the left and the shortest on the right to to accommodate pump placement.

Bundle all C-flex feed lines together on the left side of the strip and secure them with a twist tie. Form a loop with the orange two-stop E-lab tubing between the C-flex lines and secure the loop using autoclave tape, and repeat the process for the red two-stop E-lab tubing on the waste side of the bioreactor strip. Cover the female lure at the end of the waste and feed line trees with foil to prevent contamination.

Loosen the male threaded lures with septa on each bioreactor chamber to allow steam to escape during autoclaving. After placing the assembly into an autoclave bin, stretch out the feed and waste line trees into separate bins adjacent to the one containing the MBRA strips. To attach the system to the pumps, remove the autoclave tape securing the E-lab tubing for both waste and feed lines, and untie the bundles of C-flex tubing.

Position the MBRA between the two pumps on top of the stir plate. Clamp it down using the 3D printed holders and align it with the marked stirring positions on the plate. Now attach the feed line E-lab tubing to the peristaltic pump cartridges and position the tubing stops into the cartridge slots.

Repeat the process for the waste line E-lab tubing on the pump located to the right of the stir plate. Then lock the peristaltic pump cartridges into the pump. Arrange the C-flex tubing neatly using the 3D printed tube holders.

Connect the end of the waste line tree to the tubing connected to the waste bottles. Next, attach the female lure on the feed line entry tubing to the male connector on the 12-inch tube from the media bottle cap. Turn on both pumps to start media flow and ensure both pumps are set to clockwise rotation when waste is positioned to the right of the pumps.

Observe the droplet size and cadence in each bioreactor chamber. If any variability or abnormality is noticed, replace the orange two-stop E-lab tubing connected to the affected chamber to reduce flow rate variation. Once the chambers are full, shut off both pumps and allow the bioreactors to sit for 24 to 48 hours to check for contamination before starting the experiment.

Switch the media input to a one liter container of 10%bleach in deionized water and increase the flow rate on both pumps to maximum to displace the contents of the bioreactor chambers with bleach. Once the chambers are clear of media, invert the MBRA to disinfect above the fill line for five minutes. After five minutes, right the system and wait an additional five minutes for sterilization.

After the chambers have been cleared of media and have been sterilized for 10 minutes, replace the bleach with one liter of deionized water and flush the system until the water has passed through. Then disconnect the bioreactor E-lab tubing from the pumps and remove the MBRAs. Remove the used septa from the bioreactor and drain each chamber until only one milliliter of water remains.

Replace the septa, the orange two-stop E-lab tubing, and autoclave the completely assembled strip as demonstrated previously. After three cycles of reuse, follow these steps. A human fecal sample was prepared and grown in the MBRA system.

After four days of continuous flow, the microbial community in all nine bioreactors was dominated by 18 bacterial genera, each comprising at least 2%of relative abundance in any replicate. 22 out of the 65 detected genera were present in all nine bioreactor replicates, demonstrating high reproducibility. Alpha diversity analysis showed minimal variation between replicates in both the observed operational taxonomic units and Shannon Diversity Index.