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Huang et al. Microbiome Res Rep 2024;3:30 https://dx.doi.org/10.20517/mrr.2024.14 Page 3 of 13

F. prausnitzii actively produces butyrate and exerts anti-inflammatory effects by modulating the expression
[15]
of inflammation-related genes in the NF-κB pathway . However, it is unclear whether the GuMI system is
feasible for other donors and what metabolites beyond butyrate are modulated by F. prausnitzii. Here, we
tested the coculturing of F. prausnitzii and colonic epithelium from multiple donors. We further applied
targeted metabolomics to identify metabolites produced by F. prausnitzii in the GuMI system and compared
the metabolite profiles with that in GuMI without bacteria and under conventional Static culture.

METHODS
Bacterial culture
[19]
F. prausnitzii A2-165 (DSM 17677 or JCM 31915, now renamed F. duncaniae A2-165 ) was obtained from
the Harvard Digestive Disease Center. Bacterial culturing and identity confirmation were performed
according to a previous study . Bacteria from glycerol stock were plated in yeast casitone fatty acid (YCFA)
[5]
agar (Anaerobe Systems, AS-675). After 24-48 h being cultured at 37 °C in the incubator inside the anerobic
chamber (Coy Laboratory), a colony was picked and cultured in liquid YCFA medium (Anaerobe Systems,
AS-680). O in the anerobic chamber was constantly removed by the Palladium Catalyst (Coy Laboratory,
2
#6501050), which was renewed biweekly by incubating in the 90 °C oven for two days.

Organoids, monolayers, and GuMI experiments
The organoid was derived from patients following the protocol approved by the Institutional Review Board
of Boston Children’s Hospital (protocol number IRB-P00000529), the Koch Institute Institutional Review
Board Committee, and the Massachusetts Institute of Technology Committee, and informed consent was
obtained from the patients. The monolayers were prepared according to the protocols described
previously [16,20] with more details in the Supplementary Material.

TEER measurement
EndOhm-12 chamber with an EVOM2 meter (World Precision Instruments) was used to measure the
TEER values.

Sample preparation and targeted metabolomic analysis
Media collected from the apical compartment of GuMI was centrifuged to pellet the bacterial cells. Five
microliters of the supernatant were mixed with 195 μL extraction mix (50:30:20 methanol:acetonitrile:water
containing 0.2 μg/mL internal standards (Metabolomics Amino Acid Mix MSK-A2-1.2, Cambridge Isotope
Laboratories, Inc.) obtained from the core facility and stored at -20 °C), followed by media extraction
protocol for polar metabolites (50:30:20 method). Briefly, the mixture was vortexed for 5 min in the cold
room and then centrifuged for 10 min at maximal speed and 4 °C. After that, supernatants were transferred
to a new 1.5-mL tube and stored at -80 °C until analysis.

For liquid chromatography-mass spectrometry (LC-MS) analysis, 2 μL of each sample was injected onto a
ZIC-pHILIC 2.1 mm × 150 mm (5 μm particle size) column (Millipore Sigma), connected to a Q-Exactive
orbitrap mass spectrometer. The mobile phases were 20 mM ammonium carbonate, 0.1% ammonium
hydroxide (buffer A), and pure acetonitrile (buffer B). The chromatographic gradient was run at a flow rate
of 0.150 mL/min as follows: 0-20 min: linear gradient from 80% to 20% B, 20-20.5 min: linear gradient from
20% to 80% B; 20.5-28 min: hold at 80% B. The mass spectra were recorded in full-scan and polarity-
switching mode with the following parameters: spray voltage at 3.0 kV, the heated capillary at 275 °C, and
the HESI probe at 350 °C. The sheath gas flow was set to 40 units, the auxiliary gas flow was set to 15 units,
and the sweep gas flow was set to 1 unit. The MS data acquisition was performed in a range of 70-1,000 m/z,
with the resolution set at 70,000, the automatic gain control target at 106, and the maximum injection time
at 80 msec. Relative quantitation of polar metabolites was performed with Tracefinder 4.1 (Thermo Fisher