AVIAN ENTEROIDS

Abstract

There is provided an in vitro three dimensional cell construct for use as a model of the avian intestine derived from avian intestinal tissue comprising avian cells organised into intestinal villi and crypts. Suitably the construct comprises an exterior surface that mimics the apical surface of a chicken intestine. Also provided are methods of making the cell construct and use of the construct as an in vitro intestinal model system to examine an agent including, but not limited to a microbe, a vaccine, a pharmaceutical, a feed additive, a toxin, a pre-biotic, post-biotic, pre pro post biotic, therapeutic, a cell, gene construct, protein, immune-modulator, an intestinal effector agent, a candidate intestinal effector agent, cell signalling inhibitor, or cell signalling activator.

Claims

1. An in vitro three dimensional cell construct for use as a model of the avian intestine derived from avian intestinal tissue comprising avian cells organised into intestinal villi and crypts.

2. The in vitro cell construct of claim 1 wherein the construct comprises a surface that mimics the apical surface of a chicken intestine.

3. The in vitro cell as claimed in claim 1 wherein the construct comprises an exterior surface that mimics the apical surface of a chicken intestine.

4. The in vitro cell construct as claimed in claim 1 wherein the cell construct is an enteroid.

5. The in vitro cell construct of claim 1 comprising (a) a core (b) an exterior comprising an apical epithelial cell surface.

6. A method of culturing an in vitro three dimensional cell construct comprising avian cells organised into intestinal villi and crypts, the method comprising the steps a) providing isolated cells from intestinal tissue from an avian to culture media to provide culture media with seeded cells, b) expanding the seeded cells floating in the culture media to form at least one enteroid.

7. The method of claim 6 wherein the method does not require providing an extracellular matrix.

8. The method of claim 6 wherein the isolated cells are isolated from avian intestinal villi or intestinal crypt.

9. The method of claim 6 wherein the isolated cells are seeded by floating in culture media, optionally wherein the media is a basic media composed of DMEM/F12 and B27 supplement.

10. A method of screening an agent for activity the method comprising the steps of a. providing at least one cell construct of claim 1, b. contacting said agent to the cell construct in vitro, c. determining the activity or effect of the agent on cells of the cell construct.

11. A device for use in the method of claim 6 wherein the device comprises: a. a microfluidic device comprising a chamber and at least a first channel in fluid communication with the chamber, b. a cell construct as claimed in claim 1, optionally c. growth media in the chamber.

12. A method of growing a cell construct of claim 1, the method comprising a. providing a device of claim 11, b. providing growth media in the chamber at first time point to promote growth of the cells.

13. Use of an in vitro three dimensional cell construct comprising avian cells organised into intestinal villi and crypts as claimed in claim 1 in at least one selected from a group comprising of: examining microbe interactions, culturing of microbes, vaccine and pharmaceutical development, feed additive screening, toxicology studies and developmental studies, screening of pre-biotics, screening of post-biotics, screening of pre pro post biotics, screening of an intestinal effector agent, screening of an candidate intestinal effector agent and regenerative medicine.

14. A method of determining the efficacy of one or more therapies for one or more medical conditions, diseases or disorders comprising the step of exposing one or more therapies to the cell construct of claim 1.

15. Use of an in vitro three dimensional cell construct comprising avian cells organised into intestinal villi and crypts as provided by claim 6 in at least one selected from a group comprising; examining microbe interactions, culturing of microbes, vaccine and pharmaceutical development, feed additive screening, toxicology studies and developmental studies, screening of pre-biotics, screening of post-biotics, screening of pre pro post biotics, screening of an intestinal effector agent, screening of an candidate intestinal effector agent and regenerative medicine.

16. A method of determining the efficacy of one or more therapies for one or more medical conditions, diseases or disorders comprising the step of exposing one or more therapies to the cell construct as provided by any of the methods of claim 6.

17. A method of claim 14, wherein the efficacy of the one or more therapies are monitored by assaying at least one of the cell barrier integrity, assaying the gene expression of one or more genes, assaying the protein levels and/or identity of one or more proteins and/or assaying the histology, assaying the immune response of the enteroid culture.

18. A method of claim 14 wherein the method further comprises the step of providing one or more microbes to a cell construct.

19. A method of providing an in vitro intestinal model system, the method comprising: exposing an enteroid of claim 1 or as provided by a method of claim 6 with an agent monitoring the response of the enteroid to the agent, wherein the response of the enteroid to the agent is a model of the avian intestine.

20. The method of claim 19 wherein an agent is selected from a group comprising; a microbe, a vaccine, a pharmaceutical, a feed additive, a toxin, a pre-biotic, post-biotic, pre pro post biotic, therapeutic, a cell, gene construct, protein, immune-modulator, an intestinal effector agent, a candidate intestinal effector agent, cell signalling inhibitor, or cell signalling activator.

Description

[0095] Embodiments of the present invention will now be described with reference to the accompanying figures by way of example only, in which

[0096] FIG. 1 illustrates the establishment of floating chicken enteroids (a) Matrigel-embedded embryonic chicken spheroids increase in size but lack budding at day 1 and (b) day 7 of culture. (c) Floating large multi-lobulated chicken enteroid structures develop over 3 days and are maintained at day 9 of culture (d). Scale bar: 50 μm. (e) Time-lapse images showing the formation of budding crypt-like structures (marked by *) in floating embryonic enteroids. Images are representative of data from at least 20 independent cultures each containing 2-3 embryos. Scale bar: 100 μm.

[0097] FIG. 2 illustrates the reverse polarisation of avian floating enteroids. Confocal images of whole-mount enteroids stained to detect F-actin-expressing brush borders (arrows) and DAPI to visualize cell nuclei. a Floating embryonic chicken enteroids at 2 days showing epithelial cells polarised with the apical brush border (closed arrow) facing the media and basal lamina (open arrow) sits on a central core of cells. b Embryonic chicken enteroids at 7 days of culture showing ‘inside-out’ polarisation. c Tissue isolated from embryonic chicken intestine to from enteroids are villi. d Embryonic enteroids cultured in Matrigel for 2 days with epithelial cells polarised so the apical brush border is facing a central lumen. e Embryonic enteroids cultured in Matrigel for 7 days. f Isolated crypts from 9 week old chicken intestine. g Enteroids from 9 week old chickens mimic embryonic chicken enteroid polarisation at 2 days and 7 days (h) in floating culture. i Matrigel-embedded enteroids from 9 week old chickens at 2 days and 7 days (j). k Enteroids derived from 1 week old quail also show ‘inside-out’ polarisation at 2 days and 7 days (l) of culture. m Isolated crypts from adult mouse intestine. n Matrigel-embedded mouse enteroid with internal lumen at 2 days in culture. o Floating mouse enteroid at 2 days in culture showing dissociated crypt cells. Scale bar: 20 μm. Images a-e, and f-o are representative of 3 cultures composed of 3 chicken embryos and 1 mouse per culture respectively.

[0098] FIG. 3 illustrates the multicellular composition of chicken enteroids. Confocal images of a-d embryonic jejunum, e-h 6 week old chicken jejunum, and i-l, o embryonic chicken enteroids at 2 days of culture. All cells are counterstained with DAPI. The cells are stained for Lysozyme C (a, e, i, Paneth cells), Muc5AC (b, f, j, goblet cells), SOX9 (c, g, k, proliferating cells) and Chromogranin A (d, h, l, enteroendocrine cells) and indicated by arrows. i-l Chicken enteroids stained to detect F-actin-expressing brush border. m Transmission electron microscopy of chicken enteroids (4 h in culture) demonstrates an enterocyte (closed arrow) and Paneth cell (open arrow). n TEM of chicken enteroids (7 days in culture) demonstrates a goblet cell. o Confocal image of chicken enteroid stained for villin (arrow, epithelial microvilli). p TEM of chicken enteroid 7 days in culture, enterocyte basal lamina (closed arrow) and microvilli (open arrow). Scale bar: a-l, o 20 μm. m, n, p 2 μm. Images a-p are representative of data from at least 3 independent cultures each containing 2-3 embryos. q Expression of intestinal epithelial cell lineage-specific genes in freshly isolated villi (0 h) and enteroids at 3 and 7 days of culture compared by RNA sequencing analysis. Heat maps show the relative expression levels (log 2 counts per million reads) of a range of mammalian epithelial cell lineage-related genes. TA: transit amplifying cells, ECepr: early enterocyte precursor cells, EClpr: late enterocyte precursor cells. RNA sequencing data is representative of 3 independent experiments, each comprising of 2 technical replicates, each containing 3 embryos.

[0099] FIG. 4 illustrates site-specific chicken enteroids demonstrate multicellular composition. a-l Confocal images of embryonic chicken enteroids at 2 days of culture grown from the duodenum, jejunum and caeca. Stained for Lysozyme C (a, e, i, Paneth cells), Muc5AC (b, f, j, goblet cells), Sox9 (c, g, k, proliferating cells), and Chromogranin A (d, h, l, enteroendocrine cells) as indicated by arrows. All counterstained with DAPI (blue). b, f, j stained to detect F-actin-expressing brush border. Scale bar: 20 μm. Images are representative of data from at least 3 independent cultures, each containing 2-3 embryos.

[0100] FIG. 5 illustrates chicken enteroids display epithelial barrier integrity, express cell-junction related genes and minimally alter stress-related genes. a Transmission electron microscopy of a chicken enteroid (7 days of culture) demonstrates tight junctions (closed arrow) and desmosomes (open arrows). b-c Confocal images of chicken enteroids (2 days of culture) stained for E-cadherin (b adherens junctions) and ZO-1 (c tight junctions) as indicated by arrows and counterstained with DAPI. d Confocal images of chicken enteroids (2 days of culture) immersed in FITC-dextran 4 kDa showing epithelial barrier integrity in untreated (d) and loss of barrier integrity after EDTA-treatment (e). Scale bar: a 2 μm, b-e 20 μm. Images are representative of data from at least 3 independent cultures each containing 2-3 embryos. f-g Expression of epithelial cell junction-related genes (f) and cell stress-related genes (g) in freshly isolated villi (0 h) and chicken enteroids at 3 and 7 days of culture was compared by RNA sequencing analysis. f Heat maps show the expression levels (log 2 counts per million reads) of a range of epithelial cell junction-related genes. DM: desmosomes HD: hemi-desmosomes. g Heat map shows minimal change in expression levels of a range of mammalian cell stress-related genes over 7 days of culture. RNA sequencing data is representative of 3 independent experiments comprising 2 technical replicates each containing 3 embryos.

[0101] FIG. 6 illustrates the immune cell component of chicken enteroids. Confocal images of chicken enteroids stained for leukocyte markers (arrows) at 2 or 7 days of culture (a-i). All enteroids are counterstained with DAPI and Phalloidin. a-c Enteroids stained for CD45 showing leukocytes in the lamina propria and epithelium (b). Enteroids at 2 days of culture stained for CD3 (d), CD4 (e), CD88 (f), chB6 (g), TCR2 (h; chicken αβ.sub.1 TCR), and TCR3 (i, chicken αβ.sub.2 TCR). Enteroids cultured from CSF1R-eGFP transgenic embryos at day 2 (j) and day 7 (k1 magnification of k) of culture. Scale bar: 20 μm. Images are representative of data from at least 3 independent cultures each containing 2-3 embryos. | Expression of immune cell-related genes in freshly isolated villi (0 h), 3 day and 7 day chicken enteroids was compared by RNA sequencing analysis. Heat maps show the expression levels (log 2 counts per million reads) of a range of immune cell-related genes. RNA sequencing data is representative of 3 independent experiments each comprising of 2 technical replicates each containing 3 embryos.

[0102] FIG. 7 illustrates chicken enteroid propagation. a Brightfield images of representative enteroid cultures supplemented with EGF, R-spondin and Noggin at day 1 and (b) day 9 compared to FOM-only at (c) day 1 and (d) day 9 of culture. Chicken enteroids at (e) day 4 culture, (f) immediately post-passage and (g) day 3 post-passage in plain and (h) growth factor supplemented media. Cryopreserved crypts at (i) point of thaw and (j) after 4 days of culture compared to (k) freshly isolated crypts then (l) cultured for 4 days. Images a-i are representative of at least 3 independent cultures each containing 2-3 embryos. Scale bar: 200 μm.

[0103] FIG. 8 illustrates chicken enteroids as a model for host-bacterial interactions a-f Representative z-axis projections of chicken enteroids 2 days in culture whole-mount stained to detect cell nuclei (DAPI) and F-actin-expressing brush border. Enteroids incubated with a-c wild type S. Typhimurium-GFP (arrows) and d-f mutant non-invasive S. Typimurium-GFP (arrows) at 4 hpi. Magnified images of al actin remodelling, 131 intracellular bacteria and dl, f1 lack of actin remodelling and intracellular bacteria. Images are representative of data from at least 3 independent cultures each containing 2-3 embryos. Scale bar: 20 μm. g Bacterial net replication assay confirmed Salmonella counts were significantly increased for enteroids infected with wild-type versus mutant Salmonella strains. ***p<0.0002, W=55, 95.5% CI for n1-n2 is (−419850, −189760) using a Mann-Whitney U test (two-sided). The assay also showed wild-type Salmonella replicated in the enteroids over 0-8 h. ***p<0.0001, R.sup.2=0.73, df=29 using a linear regression test. Bars represent bacterial count from −800 infected enteroids post high-dose gentamicin treatment. Data represent mean±SD derived from 5 independent experiments with 2-3 embryos per culture.

[0104] FIG. 9 illustrates chicken enteroids as a model for host-viral interactions a-b Representative z-axis projections of chicken enteroid 3 days in culture whole-mount stained to detect cell nuclei (DAPI), F-actin-expressing brush border and virus nucleoprotein (arrows) after incubation with influenza A virus (PR8) for 24 h. al Magnified image of a. b2 Magnified image of b. c Isotype control. Images are representative of data from at least 3 independent cultures each containing 2-3 embryos. Scale bar: 20 μm. d Bars represent viral titers, determined by plaque assay in supernatant from −800 infected enteroids at 0 and 48 hpi. Data represent mean±SD derived from 4 independent experiments each containing 2-3 embryos and ˜800 seeded enteroids/well. **p<0.001, T=−14.98, 95% CI for mean difference (−514720, 334344), df=3 using a paired t-test (two-sided).

[0105] FIG. 10 illustrates chicken enteroids as a model for host-protozoal interactions a Brightfield image of sporozoites (arrows) entering caecal enteroid at 1 dpi. b-f Representative z-axis projections of chicken caecal enteroids whole-mount stained to detect cell nuclei (DAPI), F-actin-expressing brush border after incubation with PKH-67 labelled E. tenella (arrows). b Sporozoite at 2 dpi within enteroid epithelial cell and (c) migrating through basement membrane into lamina propria. d E. tenella trophozoite-like structures. e-f Schizogeny within enteroid epithelial cell at 9 dpi. Scale bar: a-d, f 20 μm, e 10 μm. Images are representative of data from 2 independent experiments each with 2-3 technical replicates containing>3 embryos.

[0106] FIG. 11 illustrates development of sexual stages of Eimeria tenella using PCR of gamete marker EtGAM56 in infected caecal enteroid. Positive (+) control is chicken caecal tissue from 6 dpi and 13 dpi after in vivo infection with Eimeria tenella. Caecal enteroids were infected with Eimeria tenella for 2 days, 5 days, 7 days and 9 days. Band at 178 bp where EtGAM56 expected in positive control as well as 5, 7 and 9 dpi. No band evident at 2 dpi or in water control.

[0107] FIG. 12 illustrates the induction of proinflammatory cytokine IL-6 mRNA after inoculation of 3D enteroids with wild type (w/t) Salmonella Typhimurium and a non-invasive mutant strain defective in the Salmonella pathogenicity island 1 (SPI1)-encoded T3SS.

[0108] FIG. 13 illustrates that incubation of 3D enteroids with the TLR4 ligand LPS (lipopolysaccharides derived from Salmonella enterica) induces modest upregulation of proinflammatory cytokines IL-6 but not IL-8 mRNA at 6 hours post stimulation.

DETAILED DESCRIPTION OF THE INVENTION

Examples

Example 1—Animals

[0109] Experiments were performed using ED18 to 9 week old Hy-Line Brown chickens (Gallus gal/us), ED17 CSF1R-eGFP transgenic chickens and 2 day old quail (Coturnix coturnix) obtained from the National Avian Research Facility, Edinburgh, UK. Five month old C57BL/6 mice were provided by the Biological Research Facility, University of Edinburgh, UK. All animals were housed in premises licensed under a UK Home Office Establishment License in full compliance with the requirements of the Animals (Scientific Procedures) Act 1986 and with approval from The Roslin Institute Animal Welfare Ethics Review Board.

Example 2—Isolation of Avian Intestinal Stem Cells Containing Tissue

[0110] The small intestine was removed post-mortem, cut open longitudinally then into 5 mm sections and collected into Ca.sup.2+- and Mg.sup.2+-free Phosphate-buffered saline (PBS) and washed. The tissue was digested in Dulbecco's Modified Eagle's Medium (DMEM) (Thermo Fisher Scientific) with 0.2 mg/mL Collagenase from Clostridium histolyticum Type IA (Merck) at 37° C. The tube was shaken vigorously, tissue allowed to settle then supernatant collected. These steps were repeated to generate 4 fractions. Fractions were centrifuged at 100×g for 4 min and tissue integrity assessed. The crypts/villi were counted and resuspended in FOM at ˜200/mL; Advanced DMEM/F12 (Thermo Fisher Scientific) supplemented with 10 mM HEPES (Thermo Fisher Scientific), 2 mM L-Glutamine (Thermo Fisher Scientific), 50 U/mL Penicillin/Streptomycin (Merck) and 2% B27 supplement (50×; Thermo Fisher Scientific). Where indicated the enteroid cultures were supplemented with 25 ng/mL EGF (Prepotech), 25 ng/mL Noggin (Enzo Life Sciences), 250 ng/mL R-spondin (R&D Systems), 100 mM Y-27632 (Cambridge Bioscience), 100 mM SB202190 (Enzo Life Sciences) and 5 mM LY2157299 (Cambridge Bioscience). Differentiation of avian enteroids occurred at 37° C., 5% CO.sub.2 with media changed every 2 days. For duodenal, jejunal, caecal and quail enteroids the isolation and culture protocols were kept the same.

[0111] To seed chicken intestinal crypts/villi in Matrigel (Corning), the isolated material was resuspended in equal volumes of FOM and ice-cold Matrigel to allow for 50 per 50 μL and cultured as described for mouse intestinal crypts, using FOM instead of Intesticult medium (Stemcell Technologies).

Example 3—Infection of Organoids

[0112] Infection of Enteroids with Eimeria tenella Sporozoites

[0113] Frozen purified Eimeria tenella sporozoites were washed in warm DMEM and labelled with PKH67 Green Fluorescent Cell Linker kit (Sigma-Aldrich) according to manufacturer's protocol. ˜5×10.sup.4 sporozoites were added to each well containing fifty 2 day old chicken enteroids. These were incubated at 37° C., 5% CO.sub.2. Fresh caecal enteroids, cultured for 2 days, were added to the cultures at 4 and 6 dpi to provide fresh epithelial cells for the merozoite stages. Enteroids were collected for analysis at 1, 2, 4, 7 and 9 dpi.

Infection of Enteroids with Salmonella Typhimurium

[0114] Salmonella enterica subspecies enterica serovar Typhimurium strain 4/74 carrying a chromosomal pFVP25.1::gfp fusion linked to the naladixic acid resistance gene was utilised for infections of the chicken enteroids and compared to a defined mutant, ST4/74 nal.sup.R ΔprgH::kan. This prgH mutant is confirmed to have reduced Type 3 secretion by analysing secretion of SipC, a Salmonella type III secretion system effector protein, and was also transformed with the plasmid pFVP25.1 which constitutively expresses GFP. Strains were cultured overnight in Luria-Bertani (LB) broth with 50 μg/mL kanamycin (not used for wild-type ST4/74 nal.sup.R), 50 μg/mL ampicillin and 20 μg/mL naladixic acid at 37° C. Wells containing 50 enteroids were inoculated with 5×10.sup.4 bacteria in antibiotic-free FOM and incubated statically at 37° C., 5% CO.sub.2 before samples were collected at 0.5 and 4 hpi for analysis. Bacterial replication was measured by incubating enteroids with the Salmonella strains at 37° C., 5% CO.sub.2 for 1 h, then high-dose gentamicin (50 μg/mL) was added to the wells for 30 min. Enteroids were washed and incubated with low-dose gentamicin (10 μg/mL) added to FOM (without Penicillin/Streptomycin). Enteroids were collected at 0, 3 and 8 h post high-dose gentamicin treatment and disrupted using steel beads in a Tissue-Lyser. Serial dilutions were plated on naladixic acid containing LB agar in duplicate and incubated at 37° C. overnight.

Infection of Enterioids with Influenza Virus A

[0115] Fifty enteroids were incubated with 2×10.sup.7 PFU H1N1 virus (A/Puerto Rica/8/34 (PR8) in DMEM supplemented with 50 U/mL Penicillin/Streptomycin and 50× v/v B27 at 37° C., 5% CO.sub.2 for 1 h. Control cultures either had PBS or allantoic fluid from uninfected chicken eggs added to the media. The enteroids were then washed and reseeded in DMEM media supplemented with 2 μg/mL TPCK-trypsin and collected at 48 hpi for analysis. Supernatants were harvested at 0 and 48 h post incubation and titrated by plaque assay on MDCK cells.

Example 4

[0116] To determine the structure and composition of the cell constructs provided, a number of methods were utilised.

Transmission Electron Microscopy (TEM)

[0117] Enteroids were fixed in 3.0% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.3, for 2 hours and processed as described as would be known in the art. Ultra-thin sections (60 nm) were stained in uranyl acetate and lead citrate and imaged using a JEOL JEM-1400 Plus TEM. Images were analysed using ImageJ (Fiji).

Whole Mount and Immunohistochemical (IHC) Staining

[0118] Details of the sources, clone numbers and concentrations of the primary and secondary antibodies used for IHC are provided in Table 1. Enteroids were fixed with 4% paraformaldehyde then blocked with 5% v/v goat serum in permeabilisation solution (0.5% v/v bovine serum albumin and 0.1% v/v Saponin in PBS) and stained with primary and secondary antibodies at 4° C. DNA was stained with 4′, 6-diamidino-2-phenylindole (DAPI; Thermo Fisher Scientific) and, where indicated, F-actin was visualised with Alexa Fluor conjugated Phalloidin (Thermo Fisher Scientific). Enteroids were then mounted in ProLong® Diamond Antifade Mountant (Thermo Fisher Scientific). Isotype and negative controls were prepared for each staining.

[0119] Intestinal tissue was snap frozen in liquid nitrogen, 10 μm sections were prepared on a Leica Cryostat CM1900 and mounted on Superfrost Plus slides (Thermo Fisher Scientific). Tissues were fixed in 50% methanol then blocked and stained as described for staining of whole mount enteroids.

TABLE-US-00001 TABLE 1 Primary antibodies used for immunohistochemistry. Antibody Catalog Target details Clone number Dilution Mucin 5AC Mouse anti- 45M1 Ab212636 20 μg/ml mucin 5AC .sup.a Lysozyme C Rabbit anti- polyclonal Ab391 20 μg/ml lysozyme .sup.a Chromogranin Rabbit anti- polyclonal 20085 1.3 μg/ml A[SP1] chromogranin A .sup.b Sox9 [phospho Rabbit anti- polyclonal Ab59252 2 μg/ml S181] SOX9 .sup.a Villin Mouse anti- 1D2C3 Sc-58897 4 μg/ml villin .sup.c E-Cadherin Mouse anti-E- 36/E- 610181 5 μg/ml Cadherin .sup.d Cadherin ZO-1 tight Rabbit anti- polyclonal Ab216880 10 μg/ml junction ZO1 .sup.a protein Virus Rabbit anti- polyclonal n/a 1:1000 nucleoprotein NP .sup.e CD45 Mouse anti- AV53 n/a 1:100  CD45.sup.f ChB6 Mouse anti- AV20 839502 2 μg/ml Bu-1.sup.g CD3 Mouse anti- CT-3 820009 2 μg/ml CD3.sup.h

RNA Isolation and Sequencing

[0120] Enteroids from three cultures were collected at 0, 3 and 7 days of culture, lysed in RLT buffer (Qiagen) containing 10 μg/mL 2-mercaptoethanol (Sigma-Aldrich) and homogenised using Qiashredder columns (Qiagen). Each culture (biological replicate) arose from 3 pooled embryos. Two duplicate plates were cultured for each biological replicate, and samples were taken from each plate as technical replicates. RNA was extracted using the RNeasy mini kit (Qiagen) according to the manufacturer's protocol including DNase I treatment. The RNA quality and concentration was assessed using D1000 Screentape Agilent System (Agilent Technologies) then stored at −80° C. Libraries were prepared and sequenced on Illumina Novaseq 6000 by using 150-bp paired-end sequencing. Obtained reads were trimmed for quality and to remove adaptor sequences using Cutadapt. Reads after trimming were required to have a minimum length of 50 bases. Paired-end reads from Illumina sequencing were aligned to the Gallus gallus genome (Gallus_gallus-5.0) using STAR. The annotation used for counting was the standard GTF-format annotation for that reference (annotation version 91). Raw counts for each annotated gene were obtained using the featureCounts software (version 1.5.2). Differential gene expression analysis was performed within the Bioconductor edgeR package (version 3.16.5). Comparison of the embryonic enteroid transcriptome at 0, 3 and 7 days post cultivation revealed that there were no differentially expressed genes between the technical replicates (FDR<0.05), demonstrating the consistency and reproducibility of the enteroid system. The sumTechReps function in EdgeR was used to merge technical replicates. All subsequent steps were performed on the merged samples. The raw counts table was filtered to remove genes consisting predominantly of near-zero counts, filtering on counts per million (CPM) to avoid artefacts due to library depth. Statistical assessment of differential expression was carried out with the likelihood-ratio test. Differentially expressed genes were defined as those with FDR<0.05 and log FC>2. Heatmaps were constructed in R using the pheatmap package (v. 1.0.10).

[0121] Development of chicken enteroids after villi or crypt isolation using a Matrigel-based culture system resulted in poor representations of the in vivo chicken intestine (FIG. 1). Tissue fragments seeded in Matrigel quickly form spheroid structures that did increase in size over 7 days of culture; however the architecture remained basic with no obviously defined crypt-villus development.

Floating chicken enteroids demonstrate a unique ‘inside-out’ phenotype

[0122] Self-organising chicken and mammalian enteroids embedded in Matrigel demonstrate a single sheet of epithelial cells that are polarized so the microvilli surface is facing into a central lumen. In contrast, fluorescent staining of the embryonic floating chicken enteroids of the invention at 2 and 7 days after cultivation showed their epithelial cells had an atypical reversed polarity (FIG. 2a, b). The F-actin positive apical brush border could be visualised on the external epithelial surface facing the media (FIG. 2a closed arrow). The basal surface of the epithelial cells, in contrast, abutted a dense central cellular core (FIG. 2a open arrow). The inventors identified that the isolation procedure from embryonic chicken intestine resulted in collection of villus structures with an external brush border and cell-dense internal structure rather than crypts (FIG. 2c). In contrast, staining of digested and fractionated adult avian and mouse small intestine for F-actin showed the expected isolated crypts with an internal dense brush border (FIG. 2f, m).

[0123] To confirm the ‘inside-out’ phenomenon was unique to floating enteroids, the isolated embryonic chicken villi were seeded into Matrigel domes and imaged at 2 and 7 days of culture (FIG. 2d, e). At both time-points orientation of the Matrigel-embedded chicken enteroids reflected their mammalian Matrigel-embedded enteroid counterparts, with the basal cell surface polarised towards the outside, touching the Matrigel, and enterocyte brush borders forming the central luminal surface. The direction of the Matrigel-embedded enteroids' epithelial apical-basolateral polarity was in stark contrast to that of the floating enteroids (FIG. 2a, b).

[0124] Similar staining was performed on 2 and 7 day chicken enteroids derived from 9 week old birds to investigate if the reverse polarity was related to the age and type of progenitor enteroid tissue (FIG. 2f-j). Enteroids developed from the crypts of 6-9 week old chickens displayed the same phenotypes when grown in Matrigel (internal brush border) or floating (external brush border) as enteroids developed from the villi of embryos. In mature birds, the enteroids developed fewer buds and long-term viability of cultures was lower compared to those prepared from embryonic tissue.

[0125] In order to explore whether the development of ‘inside-out’ enteroids from floating crypts/villi was a species-specific phenomenon, F-actin staining of floating enteroids from 2 day old quail was performed at 2 and 7 days of culture (FIG. 2k, l). The external brush border of quail enteroids mirrored data obtained from chickens, demonstrating that formation of ‘inside-out’ enteroids from isolated villi in floating culture extends across at least two avian species.

[0126] The inventors then expanded the range of species analysed to explore the phenotypic plasticity of mammalian intestinal crypts in a liquid environment. Mouse crypts (FIG. 2m) seeded in Matrigel predictably developed into enteroids with an internally polarised lumen, forming spheroid structures by 2 days of culture (FIG. 2n) and multiple budding structures by 7 days. In contrast, isolated murine crypts cultured in Mouse IntestiCult medium alone failed to maintain structural integrity and at 2 days of culture no enteroid-like structures were visible (FIG. 2o).

Floating Chicken Enteroids Reproduce the Cellular Diversity of the Intestinal Epithelium In Vivo

[0127] To investigate whether the multilobulated floating chicken enteroids displayed the array and of cell types that would be expected in vivo, immunofluorescent staining and TEM were performed at various time-points from isolation to 7 days of enteroid culture and compared with embryonic and immunologically mature chicken jejunal tissue sections.

[0128] In human and mouse small intestinal epithelium, lysozyme C is synthesized and secreted by crypt dwelling Paneth cells, in the embryonic chicken small intestine and floating chicken enteroids, lysozyme C-expressing epithelial cells were observed scattered throughout the epithelium (FIG. 3a, i; TEM FIG. 3m). Assuming chicken Paneth-like cells play a similar stem-cell supportive role as their murine counterparts, this staining pattern could also reflect the multiple sites of proliferation expected along the villi in late stage embryos and newly hatched chicks. Lysozyme C was not detected by immunohistochemistry in the 6 week old gut sections (FIG. 3e). This could reflect a reduction in expression with age, or, as a more recent publication has reported Lysozyme C mRNA expression up to 8 weeks post-hatch, reduction of protein expression through complex gene regulatory mechanisms. SOX9, which is expressed by stem cells, transit-amplifying cells and terminally differentiated Paneth cells, was concentrated in cells lining the embryonic villi and mature chicken crypts (FIG. 3c, g). Localisation of SOX9 in embryonic enteroid buds (FIG. 3k) indicates these villus-like structures are sites of proliferation and differentiation of intestinal stem and progenitor cells. Goblet cells (Muc5AC+ cells, FIG. 3b, f, j; TEM FIG. 3n) and enteroendocrine cells (chromogranin A+ cells, FIG. 3d, h, l) were scattered throughout the enteroids, and in embryonic and mature chicken gut sections. Uniformly polarized enterocytes with clear apical villin-expressing brush borders (FIG. 3o), external microvilli (TEM, FIG. 3p open arrow) and internal basal lamina (FIG. 3p closed arrow) lined the enteroid epithelial surface. Globally the distribution patterns of the cell types in the enteroids appeared to be similar to that observed in the embryonic intestine in vivo.

[0129] The transcriptional profile of embryonic enteroids at 0, 3 and 7 days post cultivation suggested expression of gene sets characteristically associated with mammalian Paneth cells, enterocytes, goblet cells and enteroendocrine cells (FIG. 3q). In addition, expression of classical markers for cell subpopulations that could not be detected by microscopy, including stem cells, transit amplifying cells and tuft cells, were identified. The expression of these genes for most cell-types was relatively stable during the cultivation period, suggesting that the enteroids accurately recapitulate the cellular diversity of the in vivo epithelium for at least 7 days.

[0130] In order to provide site-specific models for in vitro infection studies, differentiated chicken duodenal, jejunal and caecal enteroids were individually prepared (FIG. 4). Characterisation of these enteroids showed they contained a similar abundance of cell types, as well as an ‘inside-out’ conformation. The caecal enteroids utilised the same growth requirements as small intestinal enteroids, but developed fewer shorter buds which is reflective of the in vivo villi characteristics.

Example 5

Chicken Intestinal Organoids are Susceptible to Infection by Bacteria, Eukaryotic Parasites and Viruses

[0131] Once an in vitro chicken enteroid culture system and its reversed polarisation had been established, the inventors tested if they could be infected by different classes of pathogens. The ‘inside-out’ phenotype facilitated uncomplicated infection studies by simply adding microorganisms to the media, using a range of important avian and zoonotic pathogens. Enteroids were incubated for 4 h with either a wild-type S. Typhimurium strain or a non-invasive mutant strain, defective in the Salmonella pathogenicity island 1 (SPI1)-encoded T3SS. S. Typhimurium uses effector proteins translocated by the SPI1 T3SS to induce host-cell actin remodelling on the apical surface of polarized epithelial cells. These membrane ‘ruffles’ are a well-characterised feature of Salmonella virulence, promoting internalization of the pathogen by non-phagocytic cells. After 30 min of wild-type S. Typhimurium incubation with enteroids, the bacteria were visualised in contact with the apical epithelial surface. At 4 hours post infection (hpi), dense actin rings surrounded individual bacteria (FIG. 8a1) and large numbers of bacteria were disseminated intracellularly throughout the enteroids (FIG. 8b1). In contrast, few non-invasive mutant S. Typhimurium were found in contact with the microvilli, no dense cytoskeletal modifications were visualised (FIG. 8d1,f1) and only occasional intracellular bacteria were identified by 4 hpi. Bacterial net replication assays confirmed significantly increased numbers of wild-type Salmonella in enteroids at 1 hpi, versus those incubated with the mutant strain, and only the wild-type strain demonstrated significant net replication (FIG. 8g).

[0132] Influenza A Viruses that affect poultry are primarily respiratory pathogens, but will readily infect the intestines of many avian species. Invasion of PR8 (a mouse adapted H1N1 labstrain) into epithelial cells is the basis for virus replication and this process was confirmed in the chicken enteroids by confocal microscopy. Expression of viral nucleoprotein (NP) was detected within the epithelium of the enteroids at 24 hpi (FIG. 9). Viral replication in the enteroids was verified by measuring infectious virus titers in supernatant of infected enteroids, by plaque titration on MDCK cells. Titers increased from 0 to 48 hpi compared to mock infected controls (FIG. 9d).

[0133] The use of avian enteroids as models for viral infection is particularly advantageous as it provides for a model to study influenza infection. Some Influenza A virus strains survive and replicate in the intestine of waterfowls, spreading through fecal matter to cause an epidemic potential. Enteroids as discussed herein, for example chicken enteroids are considered to provide a representative experimental model for studying the gastrointestinal interactions of avian influenza virus in waterfowl. The inventors have detected H1N1 virus non-structural protein, NP, by immunofluorescence at 48 hpi throughout the organoid structures and confirmed replication through plaque assays, demonstrating PR8 successfully infected the enteroids and confirming these are capable models to recreate viral infection of the avian intestinal mucosa.

Successful Invasion of the E. tenella Sporozoites in Chicken Enteroids

[0134] The apicomplexan protozoa of the genus Eimeria are one of the major parasitic diseases of poultry. Following oral infection in vivo, the E. tenella sporozoites enter the caecal epithelial cells, migrate through the lamina propria to undergo multiple rounds of asexual multiplication at the base of the crypts, before eventually undergoing sexual multiplication. Since each developmental stage of Eimeria harbours a distinct number of parasitic divisions, the inventors used a combination of brightfield and fluorescence microscopy to determine whether the enteroid cultures could support parasite replication. In order to visualise the parasites, E. tenella sporozoites were stained with a fluorescent cell-membrane tracking dye, PKH67.

[0135] As shown in FIG. 10, at 1 day post-infection (dpi) sporozoites were identified in contact with the enteroid apical epithelial surface (FIG. 10a) and by 2 dpi they were observed inside enteroid cells (FIG. 10b, c). E. tenella subsequently divided within the enteroid cells (FIG. 10d-f), as determined by the size and increased number of PKH67+ parasites within a singular cell. The fluorescent membrane divisions in the enteroid cells correlated with what would be expected for distinct parasite life-cycle stages (FIG. 10g). To confirm the sexual replication stage was reached, expression of EtGAM56, which encodes a macrogamete specific protein incorporated into the oocyst wall, was demonstrated in E. tenella infected enteroids at 5, 7, and 9 dpi, but not at 2 dpi (FIG. 11).

Example 6

Epithelial Barrier Integrity and Cell Stress

[0136] The innermost layer of the intestinal luminal surface consists of a single cell thick epithelial lining which acts as a barrier, preventing the entry of harmful molecules and microbes while still allowing the selective passage of dietary nutrients, ions, and water. Tight junction proteins together with adherens junctions and desmosomes are essential gut epithelia barrier components which maintain physiological homeostasis. By immunostaining for two major cell-adhesion molecules and using TEM, the inventors demonstrated the presence of these junctions in chicken enteroids. Desmosomes and tight junctions (FIG. 5a) were visualised with TEM, adherens junctions were identified by intercellular E-cadherin expression (FIG. 5b) and the tight junction-associated protein ZO-1 was expressed in the epithelial layer (FIG. 5c). In order to determine whether the cell-cell junctions were functional, the enteroids were immersed in 4 kDa FITC-dextran. Enteroids treated with EDTA, which disrupts tight junctions, were used as a positive control. This analysis showed that untreated enteroids excluded the FITC-dextran, demonstrating mechanical integrity through intact intercellular junctions (FIG. 5d). The EDTA-treated enteroids, in contrast, allowed permeation of FITC-dextran through the intercellular spaces, indicating breakdown of the epithelial barrier (FIG. 5e).

[0137] Transcriptional analysis demonstrated that the enteroids expressed a large range of genes encoding components of mammalian focal adhesions, tight junctions, gap junctions, adherens junctions and desmosomes (FIG. 5f). Expression of these genes was generally stable throughout the culture period. In addition, although a range of cell-stress associated genes (derived from murine studies) were expressed in the enteroids, there was no significant evidence of modulation of their expression across the time points (FIG. 5g). Steady expression of both gene sets throughout the time-points is indicative of stable enteroid cultures over 7 days.

[0138] Floating chicken enteroids were found to develop and survive for a period of time. Without wishing to be bound by theory, it is considered that, for example with use of B27 media without additional exogenous growth and/or inhibitory factors, the cells in the isolated tissue and/or accompanying fibroblasts appear to initially supply the required factors for stem cell proliferation and propagation of intestinal epithelium. A unique feature of avian enteroids grown floating in culture is their ‘inside-out’ conformation, with the apical brush border facing the media. Intestinal stem cells contained within avian embryonic villi or mature crypts successfully self-organise to form enteroids with 3D multilobulated structures that mimic the in vivo architecture and differentiated cell-types of the in vitro avian intestinal epithelium.

[0139] Floating avian crypts rapidly orientate themselves so their basal epithelial surface rests on a dense central core of cells, thereby re-establishing integrin signalling. This positional change was not visualised in murine cultures and so the inventors consider this is an avian-specific phenomenon.

[0140] The inventors have demonstrated that the chicken enteroid in vitro model is closely akin to the in vivo intestine and will therefore provide more valuable data than single cell cultures as well as providing cost and ethical benefits to the poultry industry by avoiding the need for in vivo studies. The classical matrix-embedded enteroid, as determined for mammalian enteroids, possesses an internal lumen which proves limiting for host-pathogen studies where fragmenting the enteroids cannot guarantee the route of pathogen entry, and microinjections and monolayers add increasing layers of complexity and cost to the infection process. The novel externally accessible epithelial surface of the chicken enteroids allows for uncomplicated replication of the natural infection process.

[0141] The method to isolate crypts and derive differentiated enteroids with an accessible epithelial layer from the chicken small and large intestine as discussed herein allows for inexpensive and uncomplicated techniques to study host-pathogen interactions, pharmaceutical, nutritional, food additive and developmental studies.

[0142] As these enteroids reflect the 3D architecture and cellular composition of their in vivo counterparts they provide an effective in vitro model of the chicken intestinal epithelium.

Example 7—Immune Cell Component of Enteroid

[0143] Since embryonic enteroids develop from intestinal villi the inventors determined whether they also contained immune cells derived from the intestinal lamina propria. Using immunohistochemistry the inventors identified CD45+ leukocytes scattered throughout the central cell-dense core of the enteroids (FIG. 6a-c) and occasionally within the epithelium (FIG. 6b) at 2 days (FIGS. 6a, b) and 7 days (FIG. 6c) of culture. Subsequent immunostaining reflected the presence of cytoplasmic CD3+ cells, typical of NK cells which have been detected in embryos from ED14, CD4+ cells and CD8p+ cells (FIG. 6d-f). ChB6+ cells were identified which are indicative of both B cells and NK cells (FIG. 6g). Occasional chicken αβ1 TCR+(TCR2) and chicken αβ2 TCR+(TCR3) cells were found scattered through the enteroid lamina propria core (FIG. 6 h-i), appearing in the embryonic intestine a couple of days earlier than previous studies have reported. It is considered the could reflect breed variation or general changes to laying stock in the intervening 30 years of genetic selection. Embryonic chicken intestines cultured from CSF1R-reporter transgenic chicken embryos, which express eGFP in cells of the myeloid lineage, were used to visualise tissue mononuclear phagocyte. Imaging of CSF1R-eGFP transgenic chicken enteroids showed the presence of multiple CSF1R transgene+ cells, representing macrophages and dendritic cells, within the enteroid core at 2 days (FIGS. 6j) and 7 days (FIG. 6k, kl) of culture.

[0144] Further transcriptional analysis of mRNA from floating enteroids showed the expression of gene sets encoding various leukocytes of the mammalian enteric immune system (FIG. 6l), with relatively stable expression across the 7 days of culture. The inventors analyses confirmed strong expression of macrophage-related genes CSF1R, CTSB, LRP1, CKB, UQCRC1, PHB2 and HADHB. Gene-sets associated with NK cells, T cells, dendritic cells, and B cells were also represented, but their expression profiles suggest they are present in lower numbers than macrophages.

Uses of 3D Enteroids to Study Immune Responses after Interaction with Micro-Organisms and Pharmaceutical/Vaccine Components

[0145] The inventors have determined methods to successfully differentiate self-organising, extensively budding avian enteroids that mimic the in vivo architecture and eplithelial characteristics of avian intestine without the use of a gel scaffold. Strikingly, the avian enteroids grown floating in culture adopt an “inside-out” confirmation, witht the apical brush border facing the media. Additionally these enteroids comprise leukocytes that makes them a useful, natural epithelial-leukocyte co-culture model.

[0146] An example of the tests conducted on the enteroids provided by the invention which show that these are useful as a model system is discussed below.

[0147] Incubation of 3D enteroids with live Salmonella Typhimurium, wild type invasive bacteria and mutant non invasive resulted in upregulation of proinflammotory cytokine IL-6 mRNA (FIG. 12). As expected, purified LPS (lipopolysaccharides) derived from Salmonella enterica induced a modest increase in IL-6 but not IL-8 mRNA at 6 hours post stimulation. These data comparing induction of immune responses using live bacteria and purified LPS suggest that the 3D enteroids distinguish LPS from live bacteria (FIG. 13), micking the in vivo gut. Additional experiments in which enteroids are stimulated with other TLR agonists (viral and bacterial) or live pathogens have indicated responses micking the in vivo gut.

[0148] Although the invention has been particularly shown and described with reference to particular examples, it will be understood by those skilled in the art that various changes in the form and details may be made therein without departing from the scope of the present invention.