Article introduction
In August 2021, American researchers published a report in the journalnature protocols （if:14.8）published an article entitled“coculture of primary human colon monolayer with human gut bacteria”Research Articles.
This study describes a protocol that can be used to co-culture primary human colonic monolayers with aerobic tolerant bacteria. The characteristics of the co-culture system show that in engineering BIn the presence of thetaiotaomicron, the barrier function remains intact. Bacteria stay near the mucus layer and respond to the inducers of the genetic circuit in a microenvironment-specific manner. Thus, this protocol provides a useful mucosal barrier system to assess bacterial cell responses to the colonic microenvironment, and can also be used to mimic human intestinal barrier properties and microbiome-host interactions in other contexts.
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Background: background
Genetically engineered microorganisms and non-engineered microorganisms have great potential in chronic diseases by influencing host physiology. A comprehensive evaluation of the performance of these potentially active microorganisms is critical and requires the use of both in vitro and in vivo models. The Human Gut Microbiome Ecosystem Simulator provides reproducible experiments, however, lacks key components of host-microbial interactions. To study the communication between bacteria and host mucosal barrier cells, and to assess the attachment of bacteria to cells and/or mucus produced by cells, various in vitro intestinal models have been developed. These intestinal models use immortalized human cancer cell lines or primary cells, expanded into organoids in static culture or in continuous microfluidic systems, and have been shown to be useful for drug delivery and delivery.
This study describes how colon proliferating cells from organoids can be used to generate primary human colonic monolayers in a standard cell culture membrane embedding device (Transwell) (Figure 1) and provides detailed instructions for colonic organoid passage and expansion, as well as cell seeding and differentiation of monolayers.
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Research ideas. methods
Establish organoids
Maintain, passage, and expand organoids
Isolate organoids to generate single cells and seed into transwells to generate a functional colon monolayer
Assessment of the proliferative status of monolayer cells
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Design of Experiments. design
Figure 1 is a schematic diagram of the protocol.
Figure 1 |Generate differentiated colonic epithelial monolayers and interact with engineered BSchematic diagram of the workflow for thetaiotaomicron co-culture.
The workflow includes initial organoid seeding (steps 1-14), organoid passaging (steps 15-34), organoid media exchange (steps 35-39), organoid processing for monolayer seeding (steps 40-62), monolayer differentiation (steps 63-80), bacteria-monolayer co-culture (steps 81-94), inducer introduction (steps 95-97), and downstream analytical sampling (steps 98-102). Sampling refers to the collection of apical medium containing bacteria, as detailed in steps 98-100 and references. In our previous co-culture experiments, engineered B. was usedThetaiotaomicron MT798, MT799 and MT800. Each strain carries a genetic circuit that exhibits an established response to the inducers dehydrated tetracycline (ATC, an antibiotic analogue) and deoxycholic acid (DCA, a secondary bile acid in the human colon). As a control, use an engineered B. that is able to stably express the luminescent signal thetaiotaomicron（mt768）。TEER measurements are optional, but we recommend performing them at critical time points such as D7, D10, and D14, especially before D14 is co-cultured with bacteria and after medium is collected at D14. The procedure for TEER measurements is described in steps 67-79.
This study describes how colon organoids can be maintained and monolayers of cells are prepared from these cells. Due to differences in monolayer cell formation between donors, the investigators propose to test the ability of some donors to grow as monolayers in transwells and remain intact after induced differentiation. The researchers found that organoids from diseased donors were more difficult to expand into organoids in Matrigel and less likely to produce stable monolayers in Transwells. To reduce inter-donor variability, it is important to keep the organoids in an undifferentiated state (i.e., the organoids show cavities and thin layers of cells) to increase cell viability during single-cell dissociation (>85%) and monolayer seeding. Second, it is critical to monitor organoid growth in Matrigel, which can be assessed based on shape, size, and overall organoid morphology. If the organoids are growing faster (or slower) than initially expected, the passage plan should be adjusted accordingly. Third, it is recommended to prepare more than the minimum number of monolayers required for the study to allow partial monolayer failure.
Figure 6 |Representative images of organoids, monolayers, and proliferating cells within monolayers. A, Growth of organoids on day 1 (D1), day 4 (D4), and day 7 (D7) in Matrigel. B, Brightfield images of colonic monolayer at days 3 (d3), 5 (d5) and 7 (d7) post-inoculation. C, TEER of the monolayer (n=4) on days 3 (D5) and 7 (D7) post-inoculation. D, EdU staining of monolayers of cells on days 3 (D3), 5 (D5) and 7 (D7) post-seeding. E, the proportion of EdU-positive cells decreased after differentiation, indicating a decrease in proliferating cells (n = 3). The image is at the same scale as b. Data in c and e are presented as mean and standard deviation.
Combine the colonic monolayer with BThetaoMICRON MT768, MT798, MT799 and MT800 co-cultured. After 2 hours of co-culture, the bacteria were placed under four induction conditions: no inducer, ATC, DCA and ATC+DCA. After 6 h of induction, collect the content containing bApical medium of thetaotaomicron to determine the effect of the inducer on bacteria in the medium. Most bacterial cells are located near the colonic epithelium. At the same time, the integrity of the monolayer is monitored by TEER measurements. A representative brightfield image of the monolayer after 8 h of co-incubation is shown in Figure 7a. Compared to the bilayer co-culture of the bioprinted primary human intestinal fibroblast and colorectal cancer cell line (CACO-2) monolayer, the monolayer appears intact, with well-defined cell boundaries and relatively high Teer values (Figure 7B). Further evaluation of the monolayer using immunofluorescence staining revealed a well-defined polygonal cell boundary between epithelial cells (Figure 7C) and bacterial cells co-cultured with epithelial cells (Figure 7D).
Three strains (bThetaoMICRON MT798, MT799, and MT800) co-cultures with relative bacterial promoter units (RPULs) showed that each strain responds to the corresponding inducer (e.g., strain MT798 preferentially responds to DCA inducers, while MT800 preferentially responds to ATC). These quantification results are similar to the bacterial-induced responses previously observed in loop validation assays (Figure 7e).
Figure 7 |Evaluate the integrity of the monolayer structure and post-co-culture bThetaiotaomicron response to inducers. a, and bRepresentative brightfield image of the monolayer after 8 h of co-incubation of Thetaiotaomicron MT798 with the addition of the indicated inducer in the last 6 h of culture. Scale bar, 750 m. b., with bThe Teer values of the monolayer co-cultured with Thetaiotaomicron MT800 (round), MT798 (upward triangle) and MT799 (downward triangle) under four different conditions (n = 2), corresponding to the measurements in E. Two separate experiments are depicted in the diagram. The raw data can be found in Source Data Fig 7。c, Nuclei (blue) and -actin (green) staining of monolayer epithelial cells. Scale bar, 30 m. D, 3D rendering of a monolayer of DAPI (blue) and -actin (green) staining; The bacteria appear above the green actin layer of epithelial cells (see white arrows). Note that the scale bar in the 3D rendered image is an approximation of the exact scale. e, b.Luminescence measurements of ThetaiotaoMicron MT800 (Output 1), MT798 (Output 2), and MT799 (Output 3) under four different conditions (n = 2). The dots indicate the values of the two independent experiments. Figures b, d, and e are taken from Ref. 10. The raw data and RPIL calculations are shown in Supplementary Table 2.
Figure 2 |Success rate in generating usable monolayers and survival of single cells in isolated organoids for monolayer seeding. The success rate of the three donors in generating usable monolayers varies. n indicates the number of attempts (weeks) to generate a monolayer of cells in 2019. Two donors were excluded, one because it was only used once, and the other because the culture was contaminated. b, single-cell viability obtained after treatment of organoids. In most cases, cell viability reaches >80%.
Figure 3 |Primary human colon organoid morphology. a, representative images of organoids exhibiting different morphologies during differentiation; Thick folds at the edges (all), dark cavities (two in the middle), and non-cystic shapes (two in the right). b, Representative image of undifferentiated cystic colon organoids. (c) Side-by-side comparison of differentiated and undifferentiated organoids within the same Matrigel droplet. Note that relatively large and undifferentiated organoids (highlighted by yellow asterisks) show cavities and compare them to differentiated organoids, which show thick edges, budding, and dark cavities (red rectangles highlighted).
Figure 4 |Sample of organoid expansion after each passage. At the time of the first passage after thawing, the number of organoids is small. Some organoids show thick cell layers and dark lumens (highlighted with white triangles). In subsequent passages, the number of organoids with thinner cell layers and clear lumen increases (highlighted with a yellow asterisk). After the third generation, the number of undifferentiated organoids with thin cell layer and clear lumen increased (yellow asterisk), and the number of differentiated organoids with thick cell layer and dark lumen decreased (white arrow). The dotted line represents the approximate boundary of the edge of the Matrigel droplet.
Figure 5 |Representative image of a failed monolayer with too many holes or dead cells after exposure to bacterial medium. A, Example of a monolayer of cells showing holes at the edge of transwells after induced differentiation. Note that the pored monolayer has a relatively high teer value (depending on the donor, the teer > 500 cm2); Therefore, we recommend examining each layer under an inverted microscope before adding bacteria. B, monolayer exposed to bacterial medium (TYG) for 6 h. The arrows point to the area where the monolayer of mesoepithelial cells detached from the transwell membrane, indicating that the epithelial cells died after exposure to TYG medium alone. Scale bar, 300 m.
Regarding the 120 specific steps of the experimental operation in this study, the primary school club will explain it in detail in the next tweet!
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References. zhang j, hernandez-gordillo v, trapecar m, wright c, taketani m, schneider k, chen wlk, stas e, breault dt, carrier rl, voigt ca, griffith lg. coculture of primary human colon monolayer with human gut bacteria. nat protoc. 2021 aug;16(8):3874-3900. doi: 10.1038/s41596-021-00562-w. epub 2021 jun 28. pmid: 34183870; pmcid: pmc9109719.
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