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Microscale Bioreactors for in situ characterization of GI epithelial cell physiology.

ABSTRACT: The development of in vitro artificial small intestines that realistically mimic in vivo systems will enable vast improvement of our understanding of the human gut and its impact on human health. Synthetic in vitro models can control specific parameters, including (but not limited to) cell types, fluid flow, nutrient profiles and gaseous exchange. They are also "open" systems, enabling access to chemical and physiological information. In this work, we demonstrate the importance of gut surface topography and fluid flow dynamics which are shown to impact epithelial cell growth, proliferation and intestinal cell function. We have constructed a small intestinal bioreactor using 3-D printing and polymeric scaffolds that mimic the 3-D topography of the intestine and its fluid flow. Our results indicate that TEER measurements, which are typically high in static 2-D Transwell apparatuses, is lower in the presence of liquid sheer and 3-D topography compared to a flat scaffold and static conditions. There was also increased cell proliferation and discovered localized regions of elevated apoptosis, specifically at the tips of the villi, where there is highest sheer. Similarly, glucose was actively transported (as opposed to passive) and at higher rates under flow.

SUBMITTER: Costello CM

PROVIDER: S-EPMC5624909 | biostudies-literature | 2017 Oct

REPOSITORIES: biostudies-literature

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Microscale Bioreactors for in situ characterization of GI epithelial cell physiology.

Costello Cait M CM Phillipsen Mikkel B MB Hartmanis Leonard M LM Kwasnica Marek A MA Chen Victor V Hackam David D Chang Matthew W MW Bentley William E WE March John C JC

Scientific reports 20171002 1

The development of in vitro artificial small intestines that realistically mimic in vivo systems will enable vast improvement of our understanding of the human gut and its impact on human health. Synthetic in vitro models can control specific parameters, including (but not limited to) cell types, fluid flow, nutrient profiles and gaseous exchange. They are also "open" systems, enabling access to chemical and physiological information. In this work, we demonstrate the importance of gut surface to ...[more]

PMID: 28970586

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Project description:Studies investigating the feasibility of new, or improved, biotechnologies, such as wastewater treatment digesters, inevitably start with laboratory-scale trials. However, it is rarely determined whether laboratory-scale results reflect full-scale performance or microbial ecology. The Expanded Granular Sludge Bed (EGSB) bioreactor, which is a high-rate anaerobic digester configuration, was used as a model to address that knowledge gap in this study. Two laboratory-scale idealizations of the EGSB-a one-dimensional and a three- dimensional scale-down of a full-scale design-were built and operated in triplicate under near-identical conditions to a full-scale EGSB. The laboratory-scale bioreactors were seeded using biomass obtained from the full-scale bioreactor, and, spent water from the distillation of whisky from maize was applied as substrate at both scales. Over 70 days, bioreactor performance, microbial ecology, and microbial community physiology were monitored at various depths in the sludge-beds using 16S rRNA gene sequencing (V4 region), specific methanogenic activity (SMA) assays, and a range of physical and chemical monitoring methods. SMA assays indicated dominance of the hydrogenotrophic pathway at full-scale whilst a more balanced activity profile developed during the laboratory-scale trials. At each scale, Methanobacterium was the dominant methanogenic genus present. Bioreactor performance overall was better at laboratory-scale than full-scale. We observed that bioreactor design at laboratory-scale significantly influenced spatial distribution of microbial community physiology and taxonomy in the bioreactor sludge-bed, with 1-D bioreactor types promoting stratification of each. In the 1-D laboratory bioreactors, increased abundance of Firmicutes was associated with both granule position in the sludge bed and increased activity against acetate and ethanol as substrates. We further observed that stratification in the sludge-bed in 1-D laboratory-scale bioreactors was associated with increased richness in the underlying microbial community at species (OTU) level and improved overall performance.

Project description:BACKGROUND: At present, alphaherpesviruses gI gene and its encoding protein have been extensively studied. It is likely that gI protein and its homolog play similar roles in virions direct cell-to-cell spread of alphaherpesviruses. But, little is known about the characteristics of DEV gI gene. In this study, we expressed and presented the basic properties of the DEV gI protein. RESULTS: The special 1221-bp fragment containing complete open reading frame(ORF) of duck enteritis virus(DEV) gI gene was extracted from plasmid pMD18-T-gI, and then cloned into prokaryotic expression vector pET-32a(+), resulting in pET-32a(+)-gI. After being confirmed by PCR, restriction endonuclease digestion and sequencing, pET-32a(+)-gI was transformed into E.coli BL21(DE3) competent cells for overexpression. DEV gI gene was successfully expressed by the addition of isopropyl-?-D-thiogalactopyranoside(IPTG). SDS-PAGE showed that the recombinant protein His6-tagged gI molecular weight was about 61 kDa. Subsequently, the expressed product was applied to generate specific antibody against gI protein. The specificity of the rabbit immuneserum was confirmed by its ability to react with the recombinant protein His6-tagged gI. In addition, real time-PCR was used to determine the the levels of the mRNA transcripts of gI gene, the results showed that the DEV gI gene was transcribed most abundantly during the late phase of infection. Furthermore, indirect immunofluorescence(IIF) was established to study the gI protein expression and localization in DEV-infected duck embryo fibroblasts (DEFs), the results confirmed that the protein was expressed and located in the cytoplasm of the infected cells, intensively. CONCLUSIONS: The recombinant prokaryotic expression vector of DEV gI gene was constructed successfully. The gI protein was successfully expressed by E.coli BL21(DE3) and maintained its antigenicity very well. The basic information of the transcription and intracellular localization of gI gene were presented, that would be helpful to assess the possible role of DEV gI gene. The research will provide useful clues for further functional analysis of DEV gI gene.

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Microscale Bioreactors for in situ characterization of GI epithelial cell physiology.

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Microscale Bioreactors for in situ characterization of GI epithelial cell physiology.

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