Table 1.

Possible methods for gut microbiota sampling for in vitro culture

	Methods	Pros	Cons	Main application
FECES	Patient collect their stool, usually in bulk and at home. Commercial containers (e.g., Fisherbrand™ Commode Specimen Collection System, EasySampler®, Fe‐Col® or BioCollector™) limit their discomfort, prevent sample contamination from urine, toilet water and sanitizers, and provide closed containers for transportation	‐ Simplicity, repeatability on daily basis, affordability and noninvasiveness ‐ Guidelines and protocols for donor selection, stool collection, transportation and DNA extraction (Wu et al., 2019)	‐ Difficult collection for diarrhea or baby stool (Videnska et al., 2019) ‐ Use of protectant media (Bellali et al., 2019, 2020; Lagier et al., 2015; Martínez et al., 2019; Million et al., 2020) to maintain anaerobic bacteria ‐ Daily interindividual variations lead to greater differences in microbiota analysis than stool handling (e.g., homogenization or freezing; Cheng & Ning, 2019; Hsieh et al., 2016; Lozupone et al., 2012; Rinninella et al., 2019; Scepanovic et al., 2019; Vandeputte et al., 2016; Zarrinpar et al., 2014) ‐ Possible incomplete removal of fecal bacteria from intestinal flora (Tang et al., 2020) ‐ Samples treated with stabilization buffers (e.g., RNALater®, OMNIgene®·GUT, and FTA cards) to prevent DNA/RNA degradation are unsuitable for culturing and continuous colonic fermentation models (Chan et al., 2016; Morjaria et al., 2019)	‐ Study of the more transient luminal bacteria of the large intestine. Unsuitable to recapitulate the spatial organization of gut microbiota communities (Jones et al., 2018)
ENDOSCOPIC PROCEDURES	A) Mucosal biopsy: A flexible endoscopy reaches the sampling site from the mouth or the anus. With respect to standard forceps, the Brisbane Aseptic Biopsy Device reduces sample contamination and diversity (Shanahan et al., 2016). In this device, a membrane covers the tip of the endoscope. The biopsy forceps penetrate the membrane, that retracts when the forceps is advanced	‐ Suitable for the diagnosis of disease type in IBDs (Salvatori et al., 2012)	‐ Unfriendly, possible bleeding ‐ Bacteria in not‐sampling sites are dragged to sampling sites ‐ Small sampling area, leading to sample deviation ‐ Not enough DNA, RNA or proteins for multi‐omics technologies ‐ Large amounts of contaminated host DNA	‐ Assess the composition of mucosal microbiota in different GI (Tang et al., 2020; Zhang et al., 2017)
	B) Luminal brushing: Initially developed to sample lung microbiota (Wimberley et al., 1979) it couples mucosal biopsy to protected specimen brushing. A plug and sheath protect the sample in the colonoscope working channel (Lavelle et al., 2013)	‐ Reduced bleeding and infection, more representative mucosal samples with respect to biopsy ‐ Large ratio of bacterial to host DNA	‐ The general ones of endoscopic procedures	‐ Study of luminal‐associated microbiota (e.g., spatial variations between luminal and mucosal microbiota)
	C) Laser capture microdissection: An infrared laser beam allows for the adhesion of the tissue section on the surface of a biopsy to a thin, transparent film. Then, the film is removed and the sample (e.g., DNA, RNA, proteins) is treated Emmert‐Buck et al.,1996)	‐ Isolation of pure sections ‐ Punctual analysis (a small focal region is transferred to the film) ‐ Easy, specific and accurate sampling (resolution: 5 µm, (Nava et al., 2011)	‐ Biopsy samples are required ‐ Biopsy preparation before laser treatment (Baarlen et al., 2008) ‐ Nuclei acid degradation and low sample amount ‐ Not for large‐scale analyses, but suitable for precision medicine	‐ Study of mucosal‐ and crypt‐associated microbial communities (Nava et al., 2011; Pédron et al., 2012; Richard et al., 2018; Wang et al.,) ‐ Study of mucus layers and mucus‐associated bacteria (Chassaing & Gewirtz, 2019; Lavelle et al., 2015)
ASPIRATION OF INTESTINAL FLUID	A) Capsules: For example, stainless steel capsules connected to a negative‐pressure source by a tube: (Shiner, 1963)	‐ Prevention of contamination (after collection, the sample is isolated from the external environment)	‐ Technically challenging	‐ Culture of small bowel microflora for the diagnosis of SIBO (Choung et al., 2011) ‐ In vitro evaluation of orally administered drug products (Litou et al., 2020)
	B) Tubes: For example, tubes for enteral feeding and ingestible tubes (Lavelle et al., 2010; Minekus et al., 1999)	‐ Prevention of contamination	‐ Difficult and time‐consuming ‐ Possible occlusions due to the viscosity of intestinal fluid
	C) Endoscopic aspiration: (Rao & Bhagatwala, 2019)	‐ Alternative to endoscopic biopsy	‐ Patient discomfort ‐ Contamination of the endoscopic channel by oropharyngeal and GI content ‐ Time‐consuming ‐ Possible unsuccessful suction due to the sparseness of fluid aspirates	‐ Most popular method for intestinal fluid collection
CAPSULE ENDOSCOPY	Swallowable pills move actively or passively through the GI tract by peristalsis. They offer recognition, anchoring and bio‐sensing capabilities (Amoako‐Tuffour et al., 2014; Hale et al., 2014). Some of them can also collect tissue samples (at least 400 µm in size or aspirate intestinal fluid (> 200 µl) (Cui et al., 2008; Park et al., 2008)	‐ Low invasiveness ‐ Accurate location of sampling points ‐ Reduction of patient comfort ‐ High technological content (e.g., locomotion mechanisms; wireless connection; temperature, pH, pressure, oxygenation, oxidation/reduction, conductivity sensors; multi‐axial accelerometers and gyroscopes for inertial navigation and positioning)	‐ Risk of capsule aspiration and retention ‐ Possible sample contamination by intestinal fluid from noncollected sites (Cui et al., 2008b) ‐ High costs of the medical procedures ‐ High costs for fabrication, but a reduction is possible with 3D printing (Rezaei Nejad et al., 2019) ‐ Commercial products, but they do not perform biopsy (Pan et al., 2019)	‐ Study of small bowel diseases (e.g., celiac disease, CD, cancers), assess mucosal activity ‐ Intestinal microbiome sampling and preservation (Koziolek et al., 2015) ‐ Imaging the distal duodenum, jejunum and ileum (Moglia et al., 2009) ‐ Drug release (Cui et al., 2008b)

Abbreviations: CD, Crohn's disease; DBE, double‐balloon enteroscopy; IBD, inflammatory bowel diseases; GI, gastrointestinal; MEMS, microelectromechanical; SBE, single‐balloon enteroscopy; SIBO, small intestinal bacterial overgrowth.