Human colon function ex vivo: Dependence on oxygen and sensitivity to antibiotic

Abstract

Background Human intestines contain a heterogeneous collection of cells that include immune, neural and epithelial elements interacting in a highly complex physiology that is challenging to maintain ex vivo. There is an extreme oxygen gradient across the intestinal wall due in part to microbiota in the lumen and close to the gut wall, which complicates the design of tissue culture systems. The current study established the use of an organotypic slice model of human intestinal tissue derived from colonoscopy biopsies to study host-microbial interactions after antibiotic treatment, and the influence of oxygen concentration on gut wall function. Methods Organotypic slices from human colon biopsies collected during routine colonoscopy provided three-dimensional environments that maintained cellular morphology ex vivo. Biopsy slices were used to study impacts of oxygen concentrations and antibiotic treatments on epithelial proliferation rates, and metabolites from tissue culture supernatants. Results Immune function was validated via demonstration of a T lymphocyte response to Salmonella enterica serovar Typhimurium. Following 24 h of Salmonella exposure there was a significant increase in CD3 + T-lymphocytes in biopsy slices. Metabolite profiling of tissue culture supernatants validated the influence of antibiotic treatment under varied oxygen culture conditions on both host and microbiome-mediated metabolism. Epithelial health was influenced by oxygen and antibiotic. Increased epithelial proliferation was measured in lowered oxygen conditions (1% = 5.9 mmHg) compared to atmospheric conditions standard at 5000 feet above sea level in Colorado (~17% = 100 mmHg). Antibiotic treatment reduced epithelial proliferation only in 5.9 mmHg oxygen cultured slices. Conclusions A human colon organotypic slice model was established for applications ranging from gut epithelial proliferation to enteric pathogen influence on mucosal immune functions ex vivo. The results further support the need to account for oxygen concentration in primary tissue cultures, and that antibiotic use impacts gut-microbe-immune interactions.

The Past, Present and Future of Intestinal In Vitro Cell Systems for Drug Absorption Studies

“…111 Furthermore, in proof-of-concept studies, murine and human intestinal slice cultures were used to study the effect of oxygen and microbiota on segmental contractility, epithelial proliferation and mucosal immune functions ex vivo. 112,113 Integration of ex vivo slices into microfluidic perfusion chips opens new possibilities to increase the functional life span of organotypic slice cultures. Richardson and colleagues presented a microfluidic organotypic device (MOD) that supports the culture of mammalian explants, including muscular, neural, immune, and epithelial components for 72 h 114 up to 8 days.…”

Modeling Immunity In Vitro: Slices, Chips, and Engineered Tissues

“…Samples of human gut tissue can be collected during colonoscopies and can thus provide a valuable platform for translational study. For example, Pender et al and Schwerdtfeger et al (122,123) used human gut cultures to investigate the effects of antibiotics and immunoprotection on exposure to Salmonella enterica bacteria. Thus, ex vivo gut culture has the potential to facilitate deep investigations of the complex interactions between the gut and the immune system.…”

Oxygen and Metabolism: Digesting Determinants of Antibiotic Susceptibility in the Gut

“…These microhabitats are shaped by gradients that create ecological niches and determine both microbial structure and function ( Figure 1 A). For example, oxygen ( Zheng et al, 2015 ; Thermann et al, 1985 ; Kelly and Colgan 2016 ; Schwerdtfeger et al, 2019 ) and antimicrobial concentrations ( Kim et al, 2018 ) decrease from the small intestine to the colon while bacterial load ( Gu et al, 2013 ; Dieterich et al, 2018 ; Sender et al, 2016 ; Donaldson et al. 2016 ), pH ( Nugent et al, 2001 ; Ilhan et al, 2017 ), and transit time ( Donaldson et al, 2016 ) increase, typically selecting for facultative or obligate anaerobes ( Roediger 1980 ; Thursby and Juge 2017 ).…”

“…These microhabitats are shaped by gradients that create ecological niches and determine both microbial structure and function (Figure 1A). For example, oxygen (Zheng et al, 2015;Thermann et al, 1985;Kelly and Colgan 2016;Schwerdtfeger et al, 2019) and antimicrobial concentrations decrease from the small intestine to the colon while bacterial load (Gu et al, 2013;Dieterich et al, 2018;Sender et al, 2016;Donaldson et al 2016), pH (Nugent et al, 2001;Ilhan et al, 2017), and transit time (Donaldson et al, 2016) increase, typically selecting for facultative or obligate anaerobes (Roediger 1980;Thursby and Juge 2017). Facultative anaerobes that are bile acid tolerant, such as Lactobacillaceae and Enterobacteriaceae, constitute a majority of the microflora in the small intestine (Zoetendal et al, 2012; Antibiotic-induced dysbiosis specifically decreases diversity of the gut microbiome and creates a nutrient niche vacuum that allows opportunistic pathogens to colonize and cause local infections (Dollive et al, 2013;Blaser and Falkow 2009;Chang et al, 2008).…”