Patient-derived small intestinal myofibroblasts direct perfused, physiologically responsive capillary development in a microfluidic Gut-on-a-Chip Model

Abstract

The development and physiologic role of small intestine (SI) vasculature is poorly studied. This is partly due to a lack of targetable, organ-specific markers for in vivo studies of two critical tissue components: endothelium and stroma. This challenge is exacerbated by limitations of traditional cell culture techniques, which fail to recapitulate mechanobiologic stimuli known to affect vessel development. Here, we construct and characterize a 3D in vitro microfluidic model that supports the growth of patient-derived intestinal subepithelial myofibroblasts (ISEMFs) and endothelial cells (ECs) into perfused capillary networks. We report how ISEMF and EC-derived vasculature responds to physiologic parameters such as oxygen tension, cell density, growth factors, and pharmacotherapy with an antineoplastic agent (Erlotinib). Finally, we demonstrate effects of ISEMF and EC co-culture on patientderived human intestinal epithelial cells (HIECs), and incorporate perfused vasculature into a gut-ona-chip (GOC) model that includes HIECs. Overall, we demonstrate that ISEMFs possess angiogenic properties as evidenced by their ability to reliably, reproducibly, and quantifiably facilitate development of perfused vasculature in a microfluidic system. We furthermore demonstrate the feasibility of including perfused vasculature, including ISEMFs, as critical components of a novel, patient-derived, GOC system with translational relevance as a platform for precision and personalized medicine research. Blood vessels supply oxygen and nutrients needed to sustain life. As such, their structure and response to physiological stimuli are crucial to nearly all aspects of human biology. This includes normal developmental and pathological conditions affecting the SI such as short gut syndrome, inflammatory bowel disease, and cancer 1-6 .

EGFR in enterocytes & endothelium and HIF1α in enterocytes are dispensable for massive small bowel resection induced angiogenesis

“…We have shown that patient-derived ISEMFs have angiogenic properties and form a vascular network when co-cultured with ECs in microfluidic devices. Additionally, we have shown that inhibition with Erlotinib, a receptor tyrosine kinase inhibitor of EGFR, prevents vasculogenesis in these co-cultures [44]. Given these prior findings, and the fact that we have shown that EGFR was strongly expressed in ISEMFs, we used this ex vivo microfluidic model of human small intestine to study the effects of EGFR signaling from ISEMFs on ECs.…”

“…Media was changed daily. It has been previously confirmed that ECs alone do not form perfused capillary networks in the microfluidic device and that ISEMFs are part of the capillary network scaffolding [45]. All cultures were performed in a 5% oxygen, 5% CO2 incubator.…”

“…Co-culture experiments in transwell plates and microfluidic devices were performed as previously described [44]. After expansion and confirmation of EC and myofibroblast identities via RT-PCR, cells were co-cultured in a transwell or microfluidic device.…”

“…Microfluidic devices were fashioned as previously described [44]. In brief, devices were fabricated using a SYLGARD 184 Silicone Elastomer Kit (NC9285739, Dow Corning; Midland, MI) at a 10:1 PDMS curing agent ratio, plasma bonded to glass slides, and sterilized by autoclave and exposure to UV light overnight.…”

“…This led us to explore one alternative source of EGFR signaling during angiogenesis-SI stromal cells. We chose to explore this because we have previously demonstrated that ISEMFs are necessary for EC capillary development in an ex vivo microfluidic culture system [44]. We interrogated the role of ISEMF EGFR by performing ISEMF and EC co-culture experiments (Fig 7A).…”