Abstract
The in vitro study of the pathogenesis of inflammatory bowel disease (IBD) requires a cell model which closely reflects the characteristics of the in vivo intestinal epithelium. This study aimed to investigate the application of L-pNIPAM hydrogel as a scaffold to develop a long-term 3D co-culture model of Caco-2 and HT29-MTX cells under conditions analogous to inflammation, to determine its potential use in studying IBD. Monocultures and co-cultures were layered on L-pNIPAM hydrogel scaffolds and maintained under dynamic culture conditions for up to 12 weeks. Treatments with IL-1β, TNFα, and hypoxia for 1 week were used to create an inflammatory environment. Following prolonged culture, the metabolic activity of Caco-2 monoculture and 90% Caco-2/10% HT29-MTX co-cultures on L-pNIPAM hydrogels were increased, and finger-like structures, similar in appearance to villi were observed. Following treatment with IL-1β, TNFα and hypoxia, ALP and ZO-1 were decreased, MUC2 increased, and MUC5AC remained unchanged. ADAMTS1 was increased in response to hypoxia. Caspase 3 expression was increased in response to TNFα and hypoxic conditions. In conclusion, L-pNIPAM hydrogel supported long-term co-culture within a 3D model. Furthermore, stimulation with factors seen during inflammation recapitulated features seen during IBD.
Highlights
Inflammatory bowel disease (IBD) such as Crohn’s disease is characterized by increased intestinal permeability due to intestinal mucosal barrier dysfunction, which may be a critical factor in the pathogenesis of IBD1,2
When Caco-2 and HT29-MTX cells were co-cultured on a 3D Poly-lactic-co-glycolic acid (PLGA) scaffold which was designed to replicate the architecture of the intestinal villi; the co-cultured cells migrated to the tips of the villi and underwent differentiation when stimulated by epidermal growth factor (EGF)[35]
When Caco-2 monocultures were layered on the surface of L-pNIPAM hydrogel scaffolds under dynamic culture, there was a significant increase in metabolic cell activity by week 2 to week 7 (P < 0.0001) (Fig. 1A)
Summary
Inflammatory bowel disease (IBD) such as Crohn’s disease is characterized by increased intestinal permeability due to intestinal mucosal barrier dysfunction, which may be a critical factor in the pathogenesis of IBD1,2. In vitro monocultures and co-cultures of Caco-2 and HT29-MTX cells have been successfully developed to study the intestinal permeability[27,30,31] When these models are used in monolayer the cells fail to develop the crypt-villus architecture seen in the small intestine. Our research group has previously established 3D in vitro culture models of the intestinal epithelium, consisting of monocultures of Caco-2 and HT29-MTX cells layered on L-pNIPAM hydrogel scaffolds studied under dynamic culture conditions[36]. This model supported the 3D culture of these cells generating villus-like structures and promoted differentiation, mimicking the native intestinal epithelium[36]. These studies did not include mucus-producing cells
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
