A new technique for use in the study of the microbiome: An evaluation of a three-dimensional cell culture technique in maintaining the gastrointestinal microbiome of four Balb/c female mice and implications for future studies

Castaneda, E. U.; Brady, J.; Speshock, J.

doi:10.1101/734145

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bioRxiv · 10.1101/734145

A new technique for use in the study of the microbiome: An evaluation of a three-dimensional cell culture technique in maintaining the gastrointestinal microbiome of four Balb/c female mice and implications for future studies

Castaneda, E. U.·Brady, J.·Speshock, J.

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Abstract

Fluctuations in oxygen, pH, nutrients, or other factors such as food or pharmaceuticals, may perturb the microbiota of the gastrointestinal (GI) tract. This environmental variation is a cause for concern given dysbiosis of the microbiome is correlated with disease states; thereby, model organisms are utilized to study microbial communities during, after, or before shifts in microbes since intact ex vivo microbiomes have historically been challenging to utilize. The objective of this study is to culture an explant microbiome of 4 Balb/c, laboratory bred mice to develop an ex vivo tool for future microbiome studies. We cultured homogenates of the distal colon of 4 mice in three dimensional, 24 well plate culture dishes. These dishes were incubated for 24 hours in two different oxygen concentration levels, 0% and 20%. The pH of the plate was tested before and after incubation. To analyze the integrity of the microbiome, we utilized 16S sequencing. Further, we utilized 16S metagenomics to characterize fecal samples and colon samples to speculate whether future studies may utilize feces in constructing an explant microbiome to spare animal lives. We found that pH and familial relationship had a profound impact on community structure while oxygen did not have a significant influence. The feces and the colon were similar in community profiles, which lends credence to utilizing feces in future studies. In addition, our efforts successfully cultured archaea, which included difficult to culture strains such as Miscellaneous Crenarchaeota group (MCG) and Methanobacteria. Ultimately, further attempts to culture and preserve an animals microbiome needs to control for and maintain stable pH.

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Castaneda, E. U., Brady, J., Speshock, J.. 2019-08-13. A new technique for use in the study of the microbiome: An evaluation of a three-dimensional cell culture technique in maintaining the gastrointestinal microbiome of four Balb/c female mice and implications for future studies. https://doi.org/10.1101/734145

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