(M-491) Microfluidic colon organoids: patient-specific ex vivo gut serotonin characterization

Introduction:: Generalized Anxiety Disorder (GAD) and Major Depressive Disorder (MDD) are two of the most common mental illnesses in the United States, affecting 24.1 million adults annually1,2. Selective serotonin reuptake inhibitors (SSRIs) are the most common form of antipsychotic treatment, however patient treatment response is highly variable in terms of both efficacy and side-effect prevalence. Recent insight into the gut-brain axis, the interactions between emotional and cognitive functions in the brain and the gut system, has suggested the importance of the gut microbiome as a key influencer of mood, cognition, and mental health, which could play a role in treatment response to SSRIs3,4,5. To investigate this interaction, this study proposes a gut organoid microfluidic culture device for high-throughput screening of SSRI modulation of the gut microbiome, as well as gut serotonin production on a patient-specific basis.


Materials and Methods:: Adult stem cell-derived colon organoids (colonoids) were cultured in well plates as representative models of the patient gut. These samples were anonymized and provided by the Calvin Kuo Lab, as well as WENS culture media and EN maturation media6. Once matured, the colonoids exhibited an array of intestinal epithelial cells types, including serotonin-secreting enterochromaffin cells. Organoids were cultured and grown in the Stanford Uytengsu Teaching Lab using a Thermo Scientific CO₂ Incubator Steri-Cycle, at 37°C and 5% CO₂.

Colonoid serotonin expression was validated using an EagleBio Serotonin ELiSA Assay to measure serotonin concentration within colonoid culture media. Wells of colonoids grown from one donor cell line were then treated with several SSRIs (Prozac, Lexapro, Zoloft) in solution at respective c_max concentrations. The ELiSA Assay was then used to measure media serotonin concentration over a six hour period and compared to untreated control colonoid media.

Following this, the colonoid culture was replicated in a preliminary model of the microfluidic chip, designed with guidance from the Stanford Microfluidics Foundry. Mature colonoids were suspended in Cultrex Matrigel and injected into the chip’s culture chamber with general purpose laboratory syringes, followed by perfusion of colonoid growth media. Over a three day incubation period, culture media was refreshed regularly by syringe perfusion and colonoid growth was monitored via microscopy.


Results, Conclusions, and Discussions:: Results:

A preliminary version of the microfluidic chip facilitated successful injection of adult stem cells into a culture chamber and perfusion of colonoid media, allowing successfully maturation of organoids within the chip, as evidenced in the microscopy images resembling organoid growth in standard well plate environments. Results showed that at all time points across all experimental samples treated with SSRIs, serotonin levels were significantly lower at all time points against the mean of the control sample.



Conclusion:

Colonoid serotonin secretion is impacted differently by treatment with different SSRIs, as evidenced in the provided graphs. While it was expected that the serotonin levels to be greater in SSRI treatment cases compared to the control, a noticeable difference is promising for future studies involving multiple cell lines and a greater number of wells. Successful growth and maturation of the organoids within the chip greenlight further testing within integrated microfluidic chips and elaboration upon existing designs.



Discussion:

While this study compared SSRI effects on a single donor cell line and used a single replicate for each drug, future research will involve increased quantities of replicates, and compare response profiles across multiple donor lines. Continued experimentation would include perfusing SSRI solution through the chip and measuring colonoid serotonin production and, in the long-term, integrating human gut microbiota for co-culture and monitoring of microbe-host interactions during SSRI treatment.




Acknowledgements (Optional): : Dr. Roel Polak and Dr. Qinghui Mu from Calvin Kuo Lab at Stanford University

Dr. Jennifer Ortiz Cardenas from Stanford Microfluidics Foundry and Polly Fordyce Lab

Dr. Ross Venook and Dr. Emma Lundberg


References (Optional): : 1. Generalized anxiety disorder (GAD): Anxiety and depression association of america, ADAA. Generalized Anxiety Disorder (GAD) | Anxiety and Depression Association of America, ADAA. (n.d.).https://adaa.org/understanding-anxiety/generalized-anxiety-disorder-gad#:~:text=GAD%20affects%206.8%20million%20adults,between%20childhood%20and%20middle%20age
2. Depression statistics. Depression and Bipolar Support Alliance. (2019, July 12). Retrieved March 15, 2023, from https://www.dbsalliance.org/education/depression/statistics/
3. Appleton J. The Gut-Brain Axis: Influence of Microbiota on Mood and Mental Health. Integr Med (Encinitas). 2018 Aug;17(4):28-32. PMID: 31043907; PMCID: PMC6469458.
4. Dinan TG, Cryan JF. Melancholic microbes: A link between gut microbiota and depression? Neurogastroenterol Motil. 2013;25(9):713-719.
5. Evrensel A, Ceylan ME. Gut-brain axis: The role of gut microbiota in the psychiatric disorders. Curr Approach Psychiatry. 2015;7:461-472.
6. Calvin Kuo Laboratory at Stanford University