Table 1. Advantages and disadvantages of current culturing techniques with respect to host–microbiome interaction.

Type of assay	Advantages	Disadvantages
2D ethos	Inexpensive, easy-to-use, data generated quickly [9,10]. Retain many genetic, epigenetic, and gene expression features of human cancer [9,10].	Established from metastatic tumours, restricting their use in cancer progression studies and early drug interventions [149]. Not an accurate representation of the in vivo 3D microenvironment [3,11]. Drug doses that are effective in 2D culture often result in poor efficacy when scaled in to humans [10-13]. 2D culture exerts selection pressures on cells that alter morphology and gene expression [10].
Transwell	Easily implemented, low-cost assay [145]. High throughput [145]. Can be used to compare metastatic potential of cells [145]. Provides thorough analysis of cells ability to migrate and sense a chemo-attractant [145]. Can be used to investigate cell invasion [146].	Low physiological relevance [79]. Migration and invasion assays can result in conflicting data [82]. Unstirred water layer may decrease the permeability rate of lipid soluble molecules [82]. Cells are grown in a static media, and there is no shear stress forced on the cells [147].
Organoids	Near-physiological model for epithelial tissue containing heterogeneous cell populations — maintaining integrity of the in vivo structure [49]. Enable analysis of cell–cell and cell–matrix interactions, while maintaining mechanical properties, gene expression, and metabolic profiles [148]. Ex vivo culture bridges gap between cell culture and animal modelling [149]. Can microinject microbes into the lumen of organoids to study host–pathogen interactions [63]. Organoid/enteroids can be grown in 2D monolayers on Transwell inserts such that microbes can be exposed to the apical cell surface simultaneously with immune cells at the basolateral surface, more representative of the in vivo human microenvironment, and permits human host–pathogen studies [42,75–77].	Reductionist approach — epithelial cells grow without its native microenvironment [49]. Wrong side of epithelium exposed to compounds/microbiome when added in the media [43].
SynVivo® (SynTumour)	Realistic vasculature morphology and physiology with diffusion transport [81]. SynVivo's inflammatory model has been successfully validated against in vivo studies [85,86]. Excellent correlation with rolling velocities, adhesion patterns, and migratory processes [85,86]. A SynVivo produced chip can partly mimic the in vivo blood–tumour barrier [82].	Still cannot be used without conjunction of animal testing [150]. Hard-to-remove bubbles can form — rendering the chip unusable [81]. Not yet applicable to studying microbiome interaction.
Tissue slices	Has the ability to assess the role of the tumour microenvironment on tumour growth and survival [92]. Freshly resected tissue slices are viable for at least 24 h [92]. Contains a higher level of biological organisation, and has cell–cell interaction, which may better reflect the response of the target organ [151]. Can be used to represent all regions within a tissue [91,92]. Effect of microbiome and pathogenic bacteria on epithelial integrity, permeability, and contractility can be examined when tissue slices are used in conjunction with Ussing chambers [95,98,101].	Tissues start to deteriorate after 3 weeks in culture (tissue-dependent) [152]. Donor-to-donor variability [152]. Difficulty in monitoring cells beyond the depth of confocal microscopy (unless cells are isolated for analysis) [152]. The system does not reflect the effects of in vivo systemic factors [152]. Further work is required to maintain anaerobic microbes in intestine [94].
Organ/tissue baths	Versatile, simple, and reproducible assay that is suitable for all organ sizes [103,104]. Ability to measure concentration-dependent changes to isometric contractions [103]. Experiment is in real time — can rapidly make conclusions and troubleshoot [103]. 3R's — multiple tissues can be prepared from one animal [103]. Scope to study probiotic and pathogenic microbes ex vivo [107].	Organs/tissues may experience different degrees of damage during surgical removal [103]. Organs/tissues may also have different viability lengths [103]. Responsiveness of tissues may alter — time controls may be necessary [103]. New tissue required for each experiment [105,106] hindering studies that follow genetic backgrounds.
Organ-on-a-chip	Enables research into physiological and pathophysiological responses of tissues, with interaction from microbes [153,154]. Can be exploited to complement animal studies and even bypass animal modelling in the future [153]. Accurately predict pharmacological effects in patients [153,154]. Raw materials used are relatively cheap [153]. Cheap, versatile, precise, and reliable system [114].	Microengineering complexities [155], cleanrooms, and pumps can be costly [153]. Surface area and roughness of chip and formation of bubbles can affect capillary forces and flow rate of the microfluid, which can damage cells [154]. Matrix degrades when culturing time increases, affecting cell functionality [153]. Cannot reflect chronic diseases, adaptive immune responses, or mirror complicated behaviours of the endocrine, skeletal, or nervous systems [156]. Human testing is still necessary [155,156].
Quasi-Vivo® (Kirkstall)	Provides a flexible and easy system that enables long-term co-culture of multiple cell types and improves cell viability [157]. Has the ability to apply high flow rates and create high nutrient turnover to cells without imposing high shear stress or turbulent flow [158]. Enables microwell protocols to be transferred directly to the bioreactor modules without the need to redesign cell culture [130]. Control of oxygen tension to visually represent conditions at the surface and within tumours [130].	Currently has a limited ability to model all tissue types [130]. The allometric aspects of dosage are unknown [130]. Not applicable to microbiome studies.
HuMiX module	Can simultaneously establish aerobic and anaerobic conditions and measure oxygen levels in real time [133]. Effects of individual and combinations of bacteria on host physiology are possible [134]. Can co-culture individual cell lines that are in close proximity to other cell types, yet can be exposed to independent conditions — great for understanding microecology of gut [134]. Ecology of communities can be manipulated, meaning transition states can be studied — potential for identification of early biomarkers in disease linked with dysbiosis [134].	Cell types cannot fully represent the cellular makeup of the entire intestinal tract [133,134].