This study presents a protocol for generating bovine intestinal 2D monolayers from organoids, offering improved access for studying host-pathogen interactions. It includes methods for assessing membrane integrity and functionality, advancing in vitro models that mimic cattle’s gastrointestinal physiology. This approach promises significant biomedical and agricultural benefits, including enhanced treatment strategies.
Advancing knowledge of gastrointestinal physiology and its diseases critically depends on the development of precise, species-specific in vitro models that faithfully mimic in vivo intestinal tissues. This is particularly vital for investigating host-pathogen interactions in bovines, which are significant reservoirs for pathogens that pose serious public health risks. Traditional 3D organoids offer limited access to the intestinal epithelium’s apical surface, a hurdle overcome by the advent of 2D monolayer cultures. These cultures, derived from organoid cells, provide an exposed luminal surface for more accessible study. In this research, a detailed protocol is introduced for creating and sustaining 2D monolayer cultures from cells of bovine small and large intestinal organoids. This method includes protocols for assessing membrane integrity through transepithelial electrical resistance and paracellular permeability alongside immunocytochemistry staining techniques. These protocols lay the groundwork for establishing and characterizing a 2D bovine monolayer culture system, pushing the boundaries of these method applications in biomedical and translational research of public health importance. Employing this innovative approach enables the development of physiologically pertinent in vitro models for exploring both normal and diseased states of cattle intestinal physiology. The implications for biomedical and agricultural advancements are profound, paving the way for more effective treatments for intestinal ailments in cattle, thereby enhancing both animal welfare and food safety.
The culture of intestinal epithelial stem cells in three-dimensional (3D) cultures, known as intestinal organoids, marks a significant advancement in in vitro technology for investigating intestinal functions, nutrition, and interactions with pathogens1,2. These organoids mimic the complex structure of the in vivo intestinal epithelium by self-replicating and organizing into 3D formations that encompass various intestinal cell lineages3. This feature highlights their considerable potential to propel the understanding of intestinal biology forward.
The rising interest in applying intestinal organoid technology to farm animals necessitates the refinement of culture and maintenance techniques4,5. This technology's relevance is underscored by its potential impact on studying the gut health of farm animals, which plays a critical role in their productivity and, consequently, the economics of the food-animal industry by influencing animal welfare and operational costs6,7. Specifically, employing intestinal organoid cultures to explore the gut function of cattle is of paramount importance, given their role as reservoirs for zoonotic enteric pathogens, such as Salmonella spp. and Escherichia coli (E. coli) O157:H78. These pathogens are localized in particular segments of the gut, making it essential to differentiate intestinal organoid culture methods by gut segment to enhance precision in studies9.
A significant obstacle in the study of intestinal organoids is the restricted access to the epithelial cell's apical surface10. When cultured within an extracellular matrix (ECM), the cells naturally orient themselves so the basal surface faces outward, and the apical surface is directed inward10. To address this challenge, methods are presented that involve dissociating 3D organoids into single cells and seeding them into semi-permeable cell culture inserts. This setup establishes an interface between the apical surface and a basolateral compartment. This protocol demonstrates that cells derived from bovine intestinal organoids can form a coherent 2D monolayer, as evidenced by transepithelial electrical resistance (TEER) measurements and paracellular permeability assays. Additionally, the development of cellular polarity with brush borders and tight junctions in the organoid-derived 2D monolayer cells is confirmed through immunofluorescence and electron microscopy, reflecting the in vivo gut epithelium's properties.
In this study, the ileum represents the small intestinal tract, and the rectum signifies the large intestinal tract. These selections are based on relevant enteric pathogens such as Salmonella spp., which can translocate the ileum11, and E. coli O157:H7 known to primarily colonize the rectum9 in cattle. The selection of these specific intestinal segments highlights the necessity of tailoring intestinal organoid culture methods to the gut region for precision in research. These methods detail the procedure for effectively culturing an organoid-derived 2D monolayer interface from these intestinal segments, providing a robust model for exploring cattle gut health, pathogen infections, and interactions between the gut microbiome and host.
The health of the intestinal tract is paramount to both the productivity and overall well-being of cattle16. Leveraging organoid-derived 2D monolayer technology, scientists can now more accurately mimic the complex structure of the bovine gut epithelium within an in vitro setting5. This innovative approach not only reproduces the diverse cellular composition of the intestinal lining, including its multicellular lineages but also captures key functional characteristics, such as mucus secretion and the presence of microvilli, essential for understanding gut physiology and pathology3. The development of tailored culture protocols for segments of the ileum and rectum has given rise to an advanced platform that significantly enhances the capacity to study bovine gut health. This sophisticated approach enables detailed investigations into the interactions between zoonotic pathogens and the bovine intestinal environment. The ability to closely replicate and study the unique aspects of the bovine intestinal ecosystem in vitro is a significant stride towards developing targeted strategies for improving livestock health and mitigating the spread of zoonotic diseases.
Nevertheless, to ensure successful 2D monolayer development using bovine intestinal organoids, it is critical to maintain the health and vitality of both the organoids and their dissociated single cells. Careful handling and the minimization of stress are paramount in preserving cell integrity and functionality, which are essential for the effective growth of organoids and the subsequent creation of a functioning monolayer. Furthermore, achieving a uniform monolayer relies on the successful dissociation of organoids to single cells without forming large clumps. Such clumps can disrupt cell distribution and compromise the monolayer's structure. Therefore, employing precise techniques for smooth dissociation is crucial, resulting in a consistent single-cell suspension. Additionally, minimizing disturbances during cell adhesion and when washing away excess non-adherent cells becomes beneficial. This approach is particularly important for addressing potential issues with 3D morphogenesis, thus enhancing the overall quality of the monolayer.
A noted challenge with ECM-based hydrogels that are of biological origin is batch-to-batch variation in composition17. While this was not observed using the described protocols and materials, batch-to-batch variations in ECM composition could pose challenges to successful monolayer development. If monolayer formation is compromised when ECM products, brands, or lot numbers change, optimization steps may be necessary to determine the appropriate ECM concentration required for coating the cell culture inserts.
Moreover, adjusting the culture medium to room temperature before making any changes is a critical step that helps mitigate thermal shock, protect cell health, and maintain the quality of both the organoid and monolayer cultures. Gentle washing practices are also paramount in maintaining the monolayer's integrity during its formation and subsequent assays, and avoiding disruptions can prevent inaccuracies in results. Substituting PBS with Hank's Balanced Salt Solution (HBSS) appeared helpful in minimizing monolayer detachment when it became an issue during repeat washing or prolonged exposure to PBS, such as in the paracellular permeability assays. Finally, tailoring the culture medium to meet the specific needs of cells from different segments of the intestinal tract, such as the ileum and rectum, is essential for accurately replicating in vivo conditions. This specificity ensures optimal cell health and functionality, facilitating precise modeling of cattle gut physiology and interactions with pathogens, thereby highlighting these critical steps in organoid research.
Besides employing a gentle cell handling practice, building good technical competency associated with cell counting and TEER measurements is crucial for the successful development of a functioning 2D monolayer. Since both too-low and too-high seeding densities resulting from over- or under-counting of the cells, respectively, can lead to compromised monolayer growth. It is encouraged to carefully review cell counts and ensure appropriate seeding density in occasions where inaccurate seeding densities are suspected. Additionally, inadequate TEER measurement techniques can result in disruption of the monolayer by inadvertent scratches with the electrodes. Carefully introducing the electrodes to the apical chamber and paying particular attention to maintaining their vertical orientation relative to the membrane surface could help mitigate the risk of accidental damage to the monolayers.
The methods of paracellular permeability assay described here have been adapted from a previous protocol18. Modifications to the reported protocol, which include multiple samplings over 120 min and replacement of the sampled aliquot with equal amounts of PBS, are made to improve the accuracy and reliability of the results. Maintaining the total volume within the chamber is critical for several reasons: it preserves osmotic balance, ensures cell integrity, maintains the concentration gradient essential for accurate permeability assessment, and prevents alterations in hydrostatic pressure that could affect transport rates. This practice of replenishing the basolateral chamber with fresh PBS equivalent to the volume of the fluorescent tracer-containing PBS sampled is pivotal to preserving these conditions, enabling accurate and meaningful evaluations of monolayer permeability. The paracellular permeability assay serves as a complement to the TEER measurement by assessing the movement of tracer molecules through the monolayer directly. Furthermore, comparing TEER values across various laboratories may not yield relevant insights, as these values can be affected by numerous variables, such as temperature and the specific conditions under which cells are cultured, including cell types, passage numbers, and the composition of the culture medium19. The paracellular permeability assay provides a functional in vitro assessment of the effective expression of adherens and tight junctions within an epithelial barrier20.
While the development of 2D monolayers from 3D organoids represents a significant advance in culture technology, it is important to acknowledge the limitations associated with 2D monolayers. One major drawback is that this remains a static culture system, lacking the dynamic stimulation found in the in vivo environment. Additionally, modifying the oxygen content within the culture system presents challenges due to its open setup involving culture plates with lids, making it less suitable for long-term co-culture with anaerobic bacteria. These limitations could potentially be addressed by adopting more dynamic culture platforms, such as microfluidic systems21, which offers a more controlled and physiologically relevant environment. Furthermore, it is crucial to recognize that while current culture conditions are rich in nutrients beneficial for maintaining stem cell growth, they may not be optimal for inducing physiological differentiation of the epithelial cells. This discrepancy highlights a need for optimization in future research to closely mimic the in vivo conditions and support the differentiation process. By addressing these limitations and refining these approaches, the utility and applicability of organoid culture technologies are enhanced, moving closer to replicating the complex dynamics and interactions of the gastrointestinal tract in vitro.
The protocol for generating 2D monolayers from bovine ileal and rectal tissues offers researchers a valuable in vitro model of the luminal interface of both small and large intestinal epithelium. This model opens up vast possibilities for application in fundamental animal nutrition studies, particularly in examining how nutrients are absorbed under various conditions. A notable area of interest is the investigation of the leaky-gut syndrome, characterized by an abnormal increase in gastrointestinal permeability, often triggered by dietary shifts and extreme environmental temperatures22,23. Moreover, this model serves as an essential tool for exploring the complex interactions between the gut microbiome and its host. It allows for the study of how commensal microorganisms may affect the health of the host organism, addressing a crucial aspect of veterinary and medical science1,24. Additionally, human food-borne pathogens are frequently found as commensals in different segments of cattle gut8,9,25, this protocol enables detailed studies of the specific conditions that allow these zoonotic agents to thrive in their respective niches.
Throughout this study, it was observed that rectal and ileal organoid-derived monolayers require different conditions for successful development. Specifically, when rectal organoid-derived monolayers were initially seeded onto cell culture inserts prepared with 2% ECM-based hydrogel in basal media for 1 h, large holes and cell sloughing were noted. This issue was resolved by switching to a specialized rectal monolayer culture media and extending the incubation period to overnight before seeding, whereas ileal organoid-derived monolayers were successfully developed using a shorter preparation protocol. Furthermore, the addition of CHIR99021 to the culture medium consistently improved the establishment of rectal monolayers26 but was not necessary for ileal monolayers27. Additionally, ileal monolayers required a higher cell density for successful development compared to rectal organoids27. These optimized conditions (Table 1) have repeatedly developed monolayers that maintain resistant barrier integrity, underscoring the importance of tailoring culture conditions to the specific gut segment.
Access to a model that accurately reflects the multicellular lineage complexity of the in vivo gut is critical for these investigations. It allows researchers to closely mimic the natural conditions of the gut environment, providing a more reliable basis for experiments. With this protocol, investigators are equipped with a robust model that enhances their research capabilities, potentially leading to groundbreaking discoveries in their fields of study. This approach not only contributes to understanding gut health and disease but also aids in the development of strategies to improve livestock management and food safety.
The authors have nothing to disclose.
This study was supported in part by the Office of the Director National Institutes of Health (K01OD030515 and R21OD031903 to YMA) and WSU VCS Resident and Graduate Student Research Grant (to GDD). The authors would like to thank the participating slaughterhouse for supplying donor cattle.
Basal Medium | |||
Advanced DMEM/F12 (1X) | Gibco | 12634-010 | n/a |
GlutaMAX-I (100X) | Gibco | 35050-061 | 2 mM |
HEPES (1M) | Gibco | 15630-080 | 10 mM |
Pen Strep Glutamine (100X) | Gibco | 10378-016 | 1X |
Organoid Culture Medium (Supplements to Basal Medium) | |||
A-83-01 | Sigma-Aldrich | SML0788-5MG | 500 nM |
B27 Supplement (50X) | Gibco | 17504-001 | 1X |
[Leu15]-Gastrin I human | Sigma-Aldrich | G9145-.5MG | 10 nM |
Murine EGF | PeproTech | 315-09-500UG | 50 ng/mL |
Murine Wnt-3a | PeproTech | 315-20-10UG | 100 ng/mL |
N-Acetyl-L-cysteine | MP Biomedicals | 194603 | 1 mM |
N-2 MAX Media Supplement (100X) | R&D Systems | AR009 | 1X |
Nicotinamide | Sigma-Aldrich | N0636-100G | 10 mM |
Noggin Conditioned Medium | n/a | n/a | 10 vol/vol % |
Primocin | InvivoGen | ant-pm-2 | 100 µg/mL |
R-Spondin-1 Conditioned Medium | n/a | n/a | 20 vol/vol % |
SB202190 | Sigma-Aldrich | S7067-25MG | 10 µM |
Monolayer Culture Medium (Supplements to Organoid Culture Medium) | |||
CHIR99021 | Sigma-Aldrich | SML1046-5MG | 2.5 µM |
HI FBS | Gibco | 10438-034 | 20 vol/vol % |
LY2157299 | Sigma-Aldrich | SML2851-5MG | 500 nM |
Y-27632 | StemCellTechnologies | 72308 | 10 µM |
Reagents | |||
Alexa Fluor 488 Mouse anti-E-cadherin | BD Biosciences | 560061 | 1:200 dilution |
Alexa Fluor 647 Phalloidin | Invitrogen | A22287 | 1:400 dilution |
BSA | Cytiva | SH30574.02 | 2 w/vol % |
Cell Recovery Solution | Corning | 354253 | n/a |
DAPI Solution (1 mg/mL) | Thermo Scientific | 62248 | 1:1000 dilution |
DPBS (1X) | Gibco | 14190-144 | n/a |
Fluorescein Isothiocyanate–Dextran | Sigma-Aldrich | FD4-100MG | 0.5 mg/mL |
Matrigel Matrix | Corning | 354234 | n/a |
Paraformaldehyde Solution (4%) | Thermo Scientific | J19943K2 | n/a |
ProLong Gold antifade reagent | Invitrogen | P36930 | n/a |
SNA, EBL, Fluorescein | Vector Laboratories | FL-1301 | 1:100 dilution |
Triton X-100 | Thermo Scientific | A16046.AE | 0.3 vol/vol % |
TrypLE Express | Gibco | 12605-028 | n/a |
Trypan Blue Solution, 0.4% | VWR Life Science | K940-100ML | n/a |
Materials and Equipment | |||
0.4 µm Cell Culture Insert | Falcon | 353095 | |
24-well Cell Culture Plate | Corning | 3524 | |
48-well Cell Culture Plate | Thermo Scientific | 150687 | |
70 µm Sterile Cell Strainer | Fisher Scientific | 22-363-548 | |
96-well Cell Culture Plate | Greiner Bio-One | 655086 | |
Centrifuge | Eppendorf | 5910Ri | |
CO2 Incubator | Thermo Scientific | 370 | |
Epithelial Volt-Ohm Meter | Millipore | Millicell ERS-2 | |
Hemocytometer | LW Scientific | CTL-HEMM-GLDR | |
Inverted Confocal Microscope | Leica Microsystems | SP8-X | |
Inverted Phase-Contrast Microscope | Leica Microsystems | DMi1 | |
Microscope Cover Glass | Fisher Scientific | 12-540-B | |
Microplate Reader | Molecular Devices | SpecrtraMax i3x | |
Microscope Slides | Fisher Scientific | 22-034-486 | |
Pasteur Pipets | Fisher Scientific | 13-678-20C | |
Scalpel Blade | iMed Scientific | – | #11 carbon steel |
Vortex Mixer | Scientific Industries | SI-0236 | |
Software | |||
LAS X imaging software | Leica Microsystems | LAS X 3.7.6.25997 | |
Microplate Reader software | Molecular Devces | SoftMax Pro 7.1.2 |