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| Coculture type | Description | Pros | Cons | Reference |
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| Monolayer | An intestinal cell line (or ex vivo enteroids) is grown in monolayers on standard cell culture plate or transwell. Bacteria are added to the media and cocultured for hours or days. | (i) Assay effects of single bacterial strain or pathogen on IECs
(ii) Quick growth
(iii) Good reproducibility
(iv) Bacteria exposure remains apical
(v) Easily multiplexed
(vi) Coculture IECs with other intestinal niche cell types available
(vii) Easy genetic manipulated in culture via transfections or infection
(viii) Certain assays are more easily applied to monolayers | (i) Cell lines are somewhat homogenous and poorly reflect niche cell behavior
(ii) Poorly reflect the regional specificity of the intestine
(iii) Bacteria can quickly outgrow epithelial cells
(iv) Monolayers poorly reflect IE conditions or mucus layer physiology
(v) Certain niche cells lack representative cell lines | [129–134, 148, 155] |
|
| 3D-scaffold | Intestinal cell lines (or ex vivo enteroids) are seeded onto a fabricated 3D-scaffold. Bacteria are added to the media and cocultured for hours, days, or months. | (i) Assay effects of single bacterial strain or pathogen on IECs
(ii) Coculture IECs with other intestinal niche cell types
(iii) Quick growth
(iv) Depending on model, can better replicate movement, morphology, rigidity, oxygen, and nutrient gradients relative to monolayer models | (i) Difficult setup and/or specialized materials or parts
(ii) May be difficult to multiplex | [135–138, 156–159] |
|
| Mucosal explant | Intestinal tissue biopsies or slices are taken, and mucosa/submucosa can be isolated and plated on cell culture plates or transwell inserts. Selected bacterial strains are added to the media and cocultured for hours or days. | (i) Can easily assay effects of single bacterial strain or community on primary tissue
(ii) Better replicates in vivo environment than monolayer, cell line cultures
(iii) Good viability in presence of commensal microbes
(iv) Produce the wide range of metabolites and cytokines found in vivo | (i) Cannot be passaged or replicated
(ii) May require specialized media and expensive growth factors
(iii) Difficult to identify cell-type-specific effects/responses to microbiota | [149, 150, 160, 161] |
|
| Enteroids/organoids | IESCs or crypts are isolated fresh or derived from induced pluripotent or embryonic stem cells and suspended in a collagen-rich matrix (Matrigel). Growth factors are added to the media to support their growth. Bacteria should be injected into the lumen or added to the media, as enteroids/organoids form with the villi on the inside and crypts projecting outward. | (i) Assay effects of single bacterial strain or community on primary tissue
(ii) Better replicates in vivo environment than monolayer, cell line cultures
(iii) Produce the wide range of metabolites and cytokines found in vivo
(iv) Can be passaged indefinitely and cryopreserved
(v) Can easily be genetically manipulated in culture via transfections, gymnosis, or infection
(vi) Can generate from patient-derived tissue or any available genetic model | (i) Injection of bacteria requires specialized equipment and expert technical skill
(ii) May require specialized media and expensive growth factors | [38, 61, 139, 152, 162–165] |
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| Introduction model | Animals are derived or maintained in a GF (gnotobiotic) facility. Selected bacterial strains or mixed microbiota (such as reconstituted fecal matter) are introduced to the animals. | (i) Can assay effects of mono- or polycolonization
(ii) Can colonize with patient-derived microbiota
(iii) Variables can be tightly controlled
(iv) GF animals can be maintained under GF conditions indefinitely
(v) Highly reproducible | (i) GF mice have altered development and physiology
(ii) Limited number of genetic models readily available at most gnotobiotic facilities
(iii) Expensive to generate and house | [85, 166–171] |
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| Depletion model | CR or specific-pathogen free animals are given broad spectrum antibiotics, typically in drinking water, to remove measurable traces of microbiota. Microbiota may be reintroduced to the animals passively, or through forced colonization. | (i) Assay effects of single bacterial strain or community on primary tissue
(ii) No need for gnotobiotic facility or equipment
(iii) Very affordable to conduct
(iv) More realistic in terms of human disease and physiology
(v) Any available genetic model can be used | (i) Antibiotic treatment alters host gene expression independent of microbiota [172]
(ii) Does not fully eliminate all microbes | [42, 91, 172–174] |
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