Evaluating live microbiota biobanking using an ex vivo microbiome assay and metaproteomics

Abstract

Biobanking of live microbiota is becoming indispensable for mechanistic and clinical investigations of drug–microbiome interactions and fecal microbiota transplantation. However, there is a lack of methods to rapidly and systematically evaluate whether the biobanked microbiota maintains their cultivability and functional activity. In this study, we use a rapid ex vivo microbiome assay and metaproteomics to evaluate the cultivability and the functional responses of biobanked microbiota to treatment with a prebiotic (fructo-oligosaccharide, FOS). Our results indicate that the microbiota cultivability and their functional responses to FOS treatment were well maintained by freezing in a deoxygenated glycerol buffer at −80°C for 12 months. We also demonstrate that the fecal microbiota is functionally stable for 48 hours on ice in a deoxygenated glycerol buffer, allowing off-site fecal sample collection and shipping to laboratory for live microbiota biobanking. This study provides a method for rapid evaluation of the cultivability of biobanked live microbiota. Our results show minimal detrimental influences of long-term freezing in deoxygenated glycerol buffer on the cultivability of fecal microbiota.

An economic and robust TMT labeling approach for high throughput proteomic and metaproteomic analysis

“…Human stool samples were collected from healthy volunteers with a protocol approved by the Ottawa Health Science Network Research Ethics Board at the Ottawa Hospital (Ottawa, Canada). Protocols for stool collection, pre-processing, live microbiota bio-banking, and ex vivo microbiome culturing were described previously [4, 15].…”

“…Human stool samples were collected from healthy volunteers with a protocol approved by the Ottawa Health Science Network Research Ethics Board at the Ottawa Hospital (Ottawa, Canada). Protocols for stool collection, pre-processing, live microbiota bio-banking, and ex vivo microbiome culturing were described previously [4,15]. Protein extractions and trypsin digestion from microbiota and Caco-2 cells were performed using different protocols to demonstrate the wide applicability of the established dry TMT workflow.…”

RapidAIM 2.0: a high-throughput assay to study functional response of human gut microbiome to xenobiotics

“…In the previous workflows, we described a 96-well based workflow to study the functional responses of individual gut microbiomes to xenobiotics in vitro. The development of the RapidAIM 2.0 protocol consists of three major parts, 1) optimized the culture medium and establishing and validating the 96-well based scalable culturing model [11,24]; 2) established a 96-well based metaproteomic sample processing and data analysis workflow [10]; and most recently, we 3) developed and validated a live microbiota biobanking workflow that is helpful to increase the reproducibility of experiments [23]. A limitation of the previous protocol was the considerably large sample size and LC-MS/MS time consumption for metaproteomics analysis.…”

“…Either fresh human fecal samples or -80 o C stored biobank samples can be used for the culturing step. We previously showed that our culturing protocol maintains the functionality of individual microbiomes with both sample types [10,23]. Supplementary Methods 1 and 2 provide details for collecting and processing fresh stool samples, and collecting, processing and biobanking of the samples, respectively.…”

“…The RapidAIM 2.0 approach is an updated version of the previous RapidAIM series approaches developed and used in research studies by our laboratory [10, 23]. In the previous workflows, we described a 96-well based workflow to study the functional responses of individual gut microbiomes to xenobiotics in vitro .…”