A comparison of three Peyer’s patch “M-like” cell culture ...



A comparison of three Peyer’s patch “M-like” cell culture models: particle uptake, bacterial interaction, and epithelial histologyTauseef Ahmad a, Martina Gogarty a , Edwin Walsh a,,& David J. Brayden a*aUCD School of Veterinary Medicine and UCD Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland.*Corresponding author. Room 231, UCD Veterinary Sciences Centre, University College Dublin, Belfield, Dublin 4, Ireland. Tel: +3531 7166013; fax: +3531 7166204. E-mail address: david.brayden@ucd.ie AbstractIntestinal Peyer’s patch (PP) microfold (M) cells transport microbes and particulates across the follicle-associated epithelium (FAE) as part of the mucosal immune surveillance system. In vitro human M-like cell co-culture models are used as screens to investigate uptake of antigens-in-nanoparticles, but the models are labour-intensive and there is inter-laboratory variability. We compared the three most established filter-grown Caco-2/Raji B cell co-culture systems. These were Model A (Kerneis et al, 1997), Model B (Gullberg et al., 2000), and Model C (Des Rieux et al. 2007). The criteria used were transepithelial resistance (TEER), the apparent permeability coefficient (Papp) of [14C]-mannitol, M cell-like histology, as well as latex particle and Salmonella typhimurium translocation. Each co-culture model displayed substantial increases in particle translocation. Truncated microvilli compared to mono-cultures was their most consistent feature. The inverted model developed by des Rieux et al. (2007) displayed reductions in TEER and an increased (Papp), accompanied by the largest increase in particle translocation compared to the other two models. The normally-oriented model developed by Gullberg et al. (2000) was the only one to consistently display an increased translocation of salmonella typhimurium. By applying a double Matrigel? coating on filters, altering the medium feeding regime for Raji B cells, and restricting the passage number of B cells, improvements to the Gullberg model B were achieved, as reflected by increased particle translocation and improved histology. In conclusion, this is the first time all three designs have been compared in one study and each displays phenotypic features of M-like cells. While Model C was the most robust co-culture, the Model B protocol could be improved by optimizing several variables and is less complicated to establish than the two inverted models. Keywords: Peyer’s patch M cells, M-like cells, Caco-2, nanoparticle, follicle-associated epithelium, oral vaccine deliveryIntroduction Small intestinal Peyer’s patch (PP)-associated M cells are specialised epithelial cells within the FAE. They function as conduits to underlying B and T cells for pathogens, antigens, particulates and macromolecules, thereby playing a major role in immune-surveillance of the luminal environment with regard to initiation of either tolerance or mucosal immunity ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1111/imr.12400", "ISSN" : "1600065X", "PMID" : "27088918", "abstract" : "Secondary lymphoid tissues share the important function of bringing together antigens and rare antigen-specific lymphocytes to foster induction of adaptive immune responses. Peyer's patches (PPs) are unique compared to other secondary lymphoid tissues in their continual exposure to an enormous diversity of microbiome- and food-derived antigens and in the types of pathogens they encounter. Antigens are delivered to PPs by specialized microfold (M) epithelial cells and they may be captured and presented by resident dendritic cells (DCs). In accord with their state of chronic microbial antigen exposure, PPs exhibit continual germinal center (GC) activity. These GCs not only contribute to the generation of B cells and plasma cells producing somatically mutated gut antigen-specific IgA antibodies but have also been suggested to support non-specific antigen diversification of the B-cell repertoire. Here, we review current understanding of how PPs foster B-cell encounters with antigen, how they favor isotype switching to the secretory IgA isotype, and how their GC responses may uniquely contribute to mucosal immunity.", "author" : [ { "dropping-particle" : "", "family" : "Reboldi", "given" : "Andrea", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Cyster", "given" : "Jason G.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Immunological Reviews", "id" : "ITEM-1", "issue" : "1", "issued" : { "date-parts" : [ [ "2016" ] ] }, "page" : "230-245", "title" : "Peyer's patches: Organizing B-cell responses at the intestinal frontier", "type" : "article-journal", "volume" : "271" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "[1]", "plainTextFormattedCitation" : "[1]", "previouslyFormattedCitation" : "[1]" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }[1]. M cells have also been identified in the intestinal villous epithelium outside PP, where their role is less defined ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1073/pnas.0400969101", "ISBN" : "0027-8424 (Print)\\n0027-8424 (Linking)", "ISSN" : "0027-8424", "PMID" : "15071180", "abstract" : "M cells located in the follicle-associated epithelium of Peyer's patches (PP) are shown to be the principal sites for the sampling of gut luminal antigens. Thus, PP have long been considered the gatekeepers of the mucosal immune system. Here, we report a distinct gateway for the uptake of gut bacteria: clusters of non-follicle-associated epithelium-associated Ulex europaeus agglutinin (UEA)-1(+) cells, which we have designated intestinal villous M cells. Interestingly, villous M cells are developed in various PP [or gut-associated lymphoid tissue (GALT)]-null mice, such as in utero lymphotoxin beta receptor (LTbetaR)-Ig-treated, lymphotoxin alpha (LTalpha)(-/-), tumor necrosis factor/LTalpha(-/-), and inhibition of differentiation 2 (Id2)(-/-) mice. Intestinal villous M cells have been observed to take up GFP-expressing Salmonella, Yersinia, and Escherichia coli-expressing invasin, as well as gut bacterial antigen for subsequent induction of antigen-specific immune responses. Thus, the identified villous M cells could be an alternative and PP-independent gateway for the induction of antigen-specific immune responses by means of the mucosal compartment.", "author" : [ { "dropping-particle" : "", "family" : "Jang", "given" : "Myoung Ho", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Kweon", "given" : "Mi-Na", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Iwatani", "given" : "Koichi", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Yamamoto", "given" : "Masafumi", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Terahara", "given" : "Kazutaka", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Sasakawa", "given" : "Chihiro", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Suzuki", "given" : "Toshihiko", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Nochi", "given" : "Tomonori", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Yokota", "given" : "Yoshifumi", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Rennert", "given" : "Paul D", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Hiroi", "given" : "Takachika", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Tamagawa", "given" : "Hiroshi", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Iijima", "given" : "Hideki", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Kunisawa", "given" : "Jun", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Yuki", "given" : "Yoshikazu", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Kiyono", "given" : "Hiroshi", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Proceedings of the National Academy of Sciences of the United States of America", "id" : "ITEM-1", "issued" : { "date-parts" : [ [ "2004" ] ] }, "page" : "6110-6115", "title" : "Intestinal villous M cells: an antigen entry site in the mucosal epithelium.", "type" : "article-journal", "volume" : "101" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "[2]", "plainTextFormattedCitation" : "[2]", "previouslyFormattedCitation" : "[2]" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }[2]. Several intestinal pathogens exploit the sampling function of PP M cells to gain systemic entry as a route of infection and examples include salmonella, polio and reovirus [3]. The avidity of PP for particulate matter has generated interest in mimicking such pathogens by targeting human M cells for uptake of particles containing either vaccine antigens as part of oral delivery systems. For example, biodegradable microparticles containing an enterotoxogenic E. coli antigen induced an antibody response in some subjects in a Phase I trial [4]. Preclinical research using antigens in M cell-targeted microparticles using conjugated ligands on the surface of particles has shown some promise ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1021/acs.molpharmaceut.5b00265", "ISSN" : "15438392", "abstract" : "Orally ingested pathogens or antigens are taken up by microfold cells (M cells) in Peyer\u2019s patches of intestine to initiate protective immunity against infections. However, the uptake of orally delivered protein antigens through M cells is very low due to lack of specificity of proteins toward M cells and degradation of proteins in the harsh environment of gastrointestinal (GI) tract. To overcome these limitations, here we developed a pH-sensitive and mucoadhesive vehicle of thiolated eudragit (TE) microparticles to transport an M cell-targeting peptide-fused model protein antigen. Particularly, TE prolonged the particles transit time through the GI tract and predominantly released the proteins in ileum where M cells are abundant. Thus, oral delivery of TE microparticulate antigens exhibited high transcytosis of antigens through M cells resulting in strong protective sIgA as well as systemic IgG antibody responses. Importantly, the delivery system not only induced CD4+ T cell immune responses but also gen...", "author" : [ { "dropping-particle" : "", "family" : "Singh", "given" : "Bijay", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Maharjan", "given" : "Sushila", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Jiang", "given" : "Tao", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Kang", "given" : "Sang Kee", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Choi", "given" : "Yun Jaie", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Cho", "given" : "Chong Su", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Molecular Pharmaceutics", "id" : "ITEM-1", "issue" : "11", "issued" : { "date-parts" : [ [ "2015", "11", "2" ] ] }, "page" : "3816-3828", "publisher" : "American Chemical Society", "title" : "Combinatorial Approach of Antigen Delivery Using M Cell-Homing Peptide and Mucoadhesive Vehicle to Enhance the Efficacy of Oral Vaccine", "type" : "article-journal", "volume" : "12" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "[4]", "plainTextFormattedCitation" : "[4]", "previouslyFormattedCitation" : "[5]" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }[5]. In the light of this, there is a need for a practical in vitro screening bioassay for assessing targeted and untargeted particle uptake by human M cells. M cells are present in the human FAE at very low levels,representing just 1% of the surface area of the intestine [6]. Therefore, isolation and culture of sufficient human M cells for characterization and functional evaluation is especially challenging ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "ISSN" : "0309-0167", "PMID" : "8088713", "abstract" : "Ileocolonoscopy and biopsies of patients with spondylarthropathy reveal gut inflammation in 62% of cases. In order to better understand the pathogenetic mechanisms of spondylarthropathy-related gut inflammation, the follicle-associated epithelium was examined. Biopsies from nine controls and 18 patients with spondylarthropathy were studied by electronmicroscopy. Membranous (M) cells were investigated in normal and inflamed ileum. In normal mucosa, M-cells were scarce whereas in inflamed mucosa their number was increased (up to 24% of follicle-associated epithelial cells). They showed a thin rim of cytoplasm covering groups of lymphocytes. In chronic ileitis, necrotic M-cells, rupture of M-cells and lymphocytes entering the gut lumen were observed. The bursting of M-cells at the top of the lymphoid follicles leads to interruption of the gut epithelial lining and gives the luminal content access to the lymphoid tissue. This pathogenetic mechanism may cause aphthoid ulcers.", "author" : [ { "dropping-particle" : "", "family" : "Cuvelier", "given" : "C A", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Quatacker", "given" : "J", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Mielants", "given" : "H", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Vos", "given" : "M", "non-dropping-particle" : "De", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Veys", "given" : "E", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Roels", "given" : "H J", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Histopathology", "id" : "ITEM-1", "issue" : "5", "issued" : { "date-parts" : [ [ "1994", "5" ] ] }, "page" : "417-26", "title" : "M-cells are damaged and increased in number in inflamed human ileal mucosa.", "type" : "article-journal", "volume" : "24" }, "uris" : [ "" ] }, { "id" : "ITEM-2", "itemData" : { "ISSN" : "0019-9567", "PMID" : "9916113", "abstract" : "The biochemical features that distinguish human M cells from other intestinal epithelial cell types are important for understanding microbial pathogenesis and for targeting vaccines to the mucosal immune system. We applied a large panel of carbohydrate-specific monoclonal antibodies and lectins to Peyer's patch and cecum biopsy specimens from three normal individuals and a patient with inflammatory bowel disease. The results show that human M-cell glycosylation patterns are distinct from those of other species examined and that human M cells preferentially display the sialyl Lewis A antigen. This carbohydrate epitope is also present in a small subpopulation of enterocytes in the follicle-associated epithelium and in goblet cell mucins.", "author" : [ { "dropping-particle" : "", "family" : "Giannasca", "given" : "P J", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Giannasca", "given" : "K T", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Leichtner", "given" : "A M", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Neutra", "given" : "M R", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Infection and immunity", "id" : "ITEM-2", "issue" : "2", "issued" : { "date-parts" : [ [ "1999", "2" ] ] }, "page" : "946-53", "title" : "Human intestinal M cells display the sialyl Lewis A antigen.", "type" : "article-journal", "volume" : "67" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "[5,6]", "plainTextFormattedCitation" : "[5,6]", "previouslyFormattedCitation" : "[6,7]" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }[7]. Furthermore, interpretation of preclinical in vivo study data is confounded by the variability in proportion and phenotype of M cells between species [8], hence the difficulty in predicting human responses. Consequently, reproducible human M cell culture models would be useful both for screening M cell-targeted vaccine delivery constructs and to study of pathogen interactions. The first “M-like” cell culture model was established by Kerneis et al. in 1997 ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1126/science.277.5328.949", "ISSN" : "00368075", "author" : [ { "dropping-particle" : "", "family" : "Kern\u00e9is", "given" : "S", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Bogdanova", "given" : "A", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Kraehenbuhl", "given" : "JP", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Pringault", "given" : "E", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Science", "id" : "ITEM-1", "issue" : "5328", "issued" : { "date-parts" : [ [ "1997", "8", "15" ] ] }, "page" : "949-952", "title" : "Conversion by Peyer's Patch lymphocytes of Human Enterocytes into M Cells that Transport Bacteria", "type" : "article-journal", "volume" : "277" }, "uris" : [ "" ] }, { "id" : "ITEM-2", "itemData" : { "ISSN" : "1286-4579", "PMID" : "10967292", "abstract" : "Peyer's patch lymphocytes cocultured with Caco-2 cells trigger the phenotypic conversion of enterocytes into cells that express morphological and functional M-cell properties. We report a technical update for setting up this model, which will enable the study of M-cell biology, the identification by biochemical approaches of molecules involved in the interaction of microorganisms with M cells, and the development of vectors that would efficiently target the mucosal immune system.", "author" : [ { "dropping-particle" : "", "family" : "Kern\u00e9is", "given" : "S", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Caliot", "given" : "E", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Stubbe", "given" : "H", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Bogdanova", "given" : "A", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Kraehenbuhl", "given" : "J", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Pringault", "given" : "E", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Microbes and infection / Institut Pasteur", "id" : "ITEM-2", "issue" : "9", "issued" : { "date-parts" : [ [ "2000", "7" ] ] }, "page" : "1119-24", "title" : "Molecular studies of the intestinal mucosal barrier physiopathology using cocultures of epithelial and immune cells: a technical update.", "type" : "article-journal", "volume" : "2" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "[8,9]" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }[9], comprising a heterologous human Caco-2 epithelial monolayer with murine PP primary lymphocytes in co-culture. Caco-2 cells were grown on the underside of a filter, inverted, incubated in this format for a short period, and then co-cultured with either murine PP lymphocytes or (in a subsequent iteration) human Raji B cells [10] in the cup of the filter in normal orientation. This resulted in a Caco-2 monolayer with embedded lymphocytes migrating through the filter pores to intercalate within the epithelium on the opposite side of the filter. In comparison to Caco-2 monolayers, the converted “M-like” cells of the Kerneis model displayed truncated microvilli, a propensity to transport fluorescent latex microparticles and a capacity to internalise vibrio cholera. Subsequently, in 2000, Gullberg et al. ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1006/bbrc.2000.4038", "ISSN" : "0006-291X", "PMID" : "11162433", "abstract" : "The aim of this work was to establish a new, simplified in vitro model of the human M-cell. Cocultures of physically separated human intestinal epithelial Caco-2 cells and B-cell lymphoma Raji cells were established. The cocultures were characterized under the criteria of morphology, integrity, expression of M-cell markers and cell adhesion molecules (CAMs), and altered particle transport. Using this construct, the epithelial cells were transformed to cells with an M-cell-like morphology and had altered expression of potential human M-cell markers (alkaline phosphatase down-regulation and Sialyl Lewis A antigen up-regulation). The expression of intercellular adhesion molecule-1 and vascular cell adhesion molecule was altered and there was an increased binding of lectins wheat germ agglutinin and peanut agglutinin with a 40-fold increase in microparticle transport. The particle transport was size-dependent and could be inhibited at 4 degrees C or by replacing the Raji B-cells with Jurkat T-cells. This new coculture model will enable controlled studies of M-cell development and function in vitro.", "author" : [ { "dropping-particle" : "", "family" : "Gullberg", "given" : "E", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Leonard", "given" : "M", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Karlsson", "given" : "J", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Hopkins", "given" : "A M", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Brayden", "given" : "D", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Baird", "given" : "A W", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Artursson", "given" : "P", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Biochemical and biophysical research communications", "id" : "ITEM-1", "issue" : "3", "issued" : { "date-parts" : [ [ "2000", "12", "29" ] ] }, "page" : "808-13", "title" : "Expression of specific markers and particle transport in a new human intestinal M-cell model.", "type" : "article-journal", "volume" : "279" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "[10]", "manualFormatting" : "[11]", "plainTextFormattedCitation" : "[10]", "previouslyFormattedCitation" : "[11]" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }[11] focused on a variation of the model in which Caco-2 cells were initially differentiated on the filter in normal orientation, followed by incubation with Raji B cells in the basolateral medium of the well. Results from this model indicated that released soluble mediators from Raji B lymphocytes (but not Jurkat T cells) were sufficient for conversion of Caco-2 monolayers into the M-like cell phenotype without need for physical contact between the two cell types. Markers of M-like cells included loss of microvilli and reduced alkaline phosphatase expression, increased expression of the human M cell marker, Sialyl Lewis A antigen, and increased translocation of latex particles. Further studies with the Gullberg model indicated its suitability for discriminating the translocation of latex nanoparticles with different charges, as well as examining transcytotic mechanisms of particle uptake [12] Apart from the practicality and ease of culture in normally-oriented filter formats, the Gullberg model was also used for gene expression studies in the search for human M cell apical membrane receptor targets because the populations of Caco-2 and Raji B cells are in separate compartments ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1093/intimm/dxh011", "ISSN" : "1460-2377", "PMID" : "14688064", "abstract" : "Peyer's patch follicle-associated epithelium (FAE) regulates intestinal antigen access to the immune system in part through the action of microfold (M) cells which mediate transcytosis of antigens and microorganisms. Studies on M cells have been limited by the difficulties in isolating purified cells, so we applied TOGA mRNA expression profiling to identify genes associated with the in vitro induction of M cell-like features in Caco-2 cells and tested them against normal Peyer's patch tissue for their expression in FAE. Among the genes identified by this method, laminin beta3, a matrix metalloproteinase and a tetraspan family member, showed enriched expression in FAE of mouse Peyer's patches. Moreover, the C. perfringens enterotoxin receptor (CPE-R) appeared to be expressed more strongly by UEA-1(+) M cells relative to neighboring FAE. Expression of the tetraspan TM4SF3 gene and CPE-R was also confirmed in human Peyer's patch FAE. Our results suggest that while the Caco-2 differentiation model is associated with some functional features of M cells, the genes induced may instead reflect the acquisition of a more general FAE phenotype, sharing only select features with the M cell subset.", "author" : [ { "dropping-particle" : "", "family" : "Lo", "given" : "David", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Tynan", "given" : "Wendy", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Dickerson", "given" : "Janet", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Scharf", "given" : "Melinda", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Cooper", "given" : "Judith", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Byrne", "given" : "Daragh", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Brayden", "given" : "David", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Higgins", "given" : "Lisa", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Evans", "given" : "Claire", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "O'Mahony", "given" : "Daniel J", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "International Immunology", "id" : "ITEM-1", "issue" : "1", "issued" : { "date-parts" : [ [ "2004", "1", "1" ] ] }, "page" : "91-99", "title" : "Cell culture modeling of specialized tissue: identification of genes expressed specifically by follicle-associated epithelium of Peyer's patch by expression profiling of Caco-2/Raji co-cultures", "type" : "article-journal", "volume" : "16" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "[11]", "manualFormatting" : "[13]", "plainTextFormattedCitation" : "[11]", "previouslyFormattedCitation" : "[12]" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }[13]. The third iteration of the M-like cell model was developed by des Rieux et al. in 2007 ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1016/j.ejps.2006.12.006", "ISSN" : "0928-0987", "PMID" : "17291730", "abstract" : "An alternative in vitro model of human follicle-associated epithelium (FAE) to study nanoparticle transport mechanisms by M cells was developed and characterized. The previous in vitro model of human FAE has been improved by inverting inserts after Caco-2 cell seeding. Raji and M cells were identified only in inverted co-culture cell monolayers by immunohistochemistry, confocal microscopy, and electron microscopy. The M cell conversion rate evaluated by scanning electron microscopy ranged between 15 and 30% of cells. Transport of 200 nm carboxylated polystyrene nanoparticles was higher and more reproducible in the inverted model. Nanoparticle transport was temperature-dependent, not affected by the presence of EGTA or by potassium depletion, but inhibited by EIPA or nystatin, suggesting that it occurs most likely by macropinocytosis. The inverted model appears more physiologic, functional and reproducible than the normally oriented model.", "author" : [ { "dropping-particle" : "", "family" : "Rieux", "given" : "Anne", "non-dropping-particle" : "des", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Fievez", "given" : "Virginie", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Th\u00e9ate", "given" : "Ivan", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Mast", "given" : "Jan", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Pr\u00e9at", "given" : "V\u00e9ronique", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Schneider", "given" : "Yves-Jacques", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "European journal of pharmaceutical sciences", "id" : "ITEM-1", "issue" : "5", "issued" : { "date-parts" : [ [ "2007", "4" ] ] }, "page" : "380-91", "title" : "An improved in vitro model of human intestinal follicle-associated epithelium to study nanoparticle transport by M cells.", "type" : "article-journal", "volume" : "30" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "[12]" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }[14]. They calculated an improved conversion rate of 15-30% through promoting intimate contact between Caco-2 and concentrated B cells compared to the Gullberg model, where interaction relied largely on released Type 1 cytokine mediators. To achieve such contact Caco-2 cells were grown on the filter for an extended period in normal orientation, at which point the filters were inverted and placed in a petri dish with medium. A temporary basolateral compartment was created by attaching sterilised silicone tubing around the up-turned filters. Raji B cells were then added, thereby concentrating them on the opposing side of filter to the Caco-2 monolayers [14]. M-like cells were again identified in the des Rieux model by loss of microvilli and by increased latex particle translocation across the co-culture monolayers compared to Caco-2 monolayers. Table 1 shows the specifications of the models from the three original publications [9] [11] [14]. Importantly, the data in the des Rieux model [14] suggested that particle transport was especially increased in co-cultures created in the inverted format compared to those in the normally-oriented format (Gullberg) and that this model (des Rieux) might therefore be more reliable and reproducible. A comparison with the inverted Kerneis model has not been done before and, since both the des Rieux model and Kerneis models use Caco-2 cells on one side of the filter and B cells on the other, it is unclear if the former offers any advantage over it. In addition, it is unclear as to how each of the three models compare under set criteria under the same controlled conditions of Caco-2 and B cell origin and passage numbers in one laboratory. Our primary aim therefore was to (a) carry out a = head-to-head comparison of the three models by reducing variability in respect of cell culture and bioassay read-outs and to thereby achieve a rank order under set criteria and (b) to use the resulting data to optimize the Gullberg model over its 2000 version.Material and MethodsMaterialsThe human intestinal colonic epithelial cell line, Caco-2, and the Burkitt’s lymphoma cell line, Raji B cells, were obtained from the European Collection of Animal Cell Cultures (ECACC), Salisbury, UK. Latex particles, with mean diameters of 0.5 ?m (yellow-green; cat# F-8813) and 1.0 ?m (Nile Red; cat# F-8819) were obtained from Molecular Probes? (ThermoFisher Scientific). Transwell? polycarbonate filter membrane inserts (12 mm diameter; 3.0 ?m pore size, Cat# 3414) were purchased from Corning Costar (UK). MatrigelTM Basement Membrane Matrix was obtained from Becton Dickinson, Bedford, MA, USA. Glass petri dishes (diameter × height=200×50 mm) were obtained from VWR (Ireland); silicone tubing (internal diameter=14 mm) was from Labo-Moderne (Paris, France). All other tissue culture reagents were Gibco? products, obtained from Biosciences Ireland Ltd. [14C]-Mannitol was obtained from Perkin Elmer (UK). A Vi-CELL? Series Cell Viability Analyzer was obtained from Beckman Coulter, USA. The Titramax 1000 vibrating platform shaker for flux studies was from Heidolph Instruments GmbH & Co. KG, Germany.Caco-2 and Raji B cell cultureCaco-2 cells (passage 49-59) and Raji B cells (passages 5-18) were maintained in DMEM and RPMI, respectively, each supplemented with 10% foetal calf serum, 1% penicillin-streptomycin, 1% non-essential amino acids. Media was replenished every two days and cells were passaged at a 1:5 split ratio every week. 2.3 Generation of co-culture constructsTo obtain differentiated epithelial monolayers, Caco-2 cells were seeded onto either the upper or lower face of Transwell? filters (depending on the model) at a density of 300,000 cells per MatrigelTM (30 ?g/insert; 27 ?g/cm2)-coated insert and then grown for a total of ~21 days at 37°C and 5% CO2 in air [14]. Media was replaced every second day on both sides of inserts until co-cultures were initiated after 15-16 days by Raji B cell addition.Model A (Kerneis, [10])Caco-2 cells were seeded on the underside of Transwell? filters in 500 μl medium. To do this, filters were inverted in a sterile glass petri dish and a piece of sterile silicone tubing was attached to the upturned side to create a temporary upper chamber. This step standardised the methodology between Models A and C (des Rieux) in order to reduce variability by using silicone tubing to create separate compartments. Following attachment of cells (~12 h), filters with Caco-2 cells attached on the underside were returned to the normal orientation in the multi-well plate and grown for ~15-16 days following aseptic removal of silicone tubing. Instead of using PP lymphocytes [9], co-cultures were initiated by addition of Raji B cells to filter cups at a concentration of 500,000 cells/filter [10], suspended in co-culture media (RPMI: DMEM (1:2)) and co-cultured for ~5 days. Control Caco-2 monolayers were cultivated in an identical process, but without B cell addition (Fig. 1A). During co-culture, the apical medium bathing Caco-2 monolayers was changed daily, while the basolateral medium was changed every other day. In all models, feeding on the basolateral side where B cells were differentiating was achieved by carefully aspirating off most of the medium without disturbing the settled B cells.Model B (Gullberg ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1006/bbrc.2000.4038", "ISSN" : "0006-291X", "PMID" : "11162433", "abstract" : "The aim of this work was to establish a new, simplified in vitro model of the human M-cell. Cocultures of physically separated human intestinal epithelial Caco-2 cells and B-cell lymphoma Raji cells were established. The cocultures were characterized under the criteria of morphology, integrity, expression of M-cell markers and cell adhesion molecules (CAMs), and altered particle transport. Using this construct, the epithelial cells were transformed to cells with an M-cell-like morphology and had altered expression of potential human M-cell markers (alkaline phosphatase down-regulation and Sialyl Lewis A antigen up-regulation). The expression of intercellular adhesion molecule-1 and vascular cell adhesion molecule was altered and there was an increased binding of lectins wheat germ agglutinin and peanut agglutinin with a 40-fold increase in microparticle transport. The particle transport was size-dependent and could be inhibited at 4 degrees C or by replacing the Raji B-cells with Jurkat T-cells. This new coculture model will enable controlled studies of M-cell development and function in vitro.", "author" : [ { "dropping-particle" : "", "family" : "Gullberg", "given" : "E", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Leonard", "given" : "M", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Karlsson", "given" : "J", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Hopkins", "given" : "A M", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Brayden", "given" : "D", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Baird", "given" : "A W", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Artursson", "given" : "P", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Biochemical and biophysical research communications", "id" : "ITEM-1", "issue" : "3", "issued" : { "date-parts" : [ [ "2000", "12", "29" ] ] }, "page" : "808-13", "title" : "Expression of specific markers and particle transport in a new human intestinal M-cell model.", "type" : "article-journal", "volume" : "279" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "[10]", "plainTextFormattedCitation" : "[10]", "previouslyFormattedCitation" : "[11]" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }[11])Caco-2 cells were seeded on the normal ‘cup’ side of the Transwell? insert in normal orientation and cultured for ~15-16 days before being co-cultured with Raji B cells on the basolateral (well) side for 5 days (Fig. 1B). Medium inside the insert was changed every day while basolateral media was replenished every other day during co-culture. Generation of control Caco-2 monolayers involved an identical process, but without B cell addition.Model C (des Rieux ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1016/j.ejps.2006.12.006", "ISSN" : "0928-0987", "PMID" : "17291730", "abstract" : "An alternative in vitro model of human follicle-associated epithelium (FAE) to study nanoparticle transport mechanisms by M cells was developed and characterized. The previous in vitro model of human FAE has been improved by inverting inserts after Caco-2 cell seeding. Raji and M cells were identified only in inverted co-culture cell monolayers by immunohistochemistry, confocal microscopy, and electron microscopy. The M cell conversion rate evaluated by scanning electron microscopy ranged between 15 and 30% of cells. Transport of 200 nm carboxylated polystyrene nanoparticles was higher and more reproducible in the inverted model. Nanoparticle transport was temperature-dependent, not affected by the presence of EGTA or by potassium depletion, but inhibited by EIPA or nystatin, suggesting that it occurs most likely by macropinocytosis. The inverted model appears more physiologic, functional and reproducible than the normally oriented model.", "author" : [ { "dropping-particle" : "", "family" : "Rieux", "given" : "Anne", "non-dropping-particle" : "des", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Fievez", "given" : "Virginie", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Th\u00e9ate", "given" : "Ivan", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Mast", "given" : "Jan", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Pr\u00e9at", "given" : "V\u00e9ronique", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Schneider", "given" : "Yves-Jacques", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "European journal of pharmaceutical sciences", "id" : "ITEM-1", "issue" : "5", "issued" : { "date-parts" : [ [ "2007", "4" ] ] }, "page" : "380-91", "title" : "An improved in vitro model of human intestinal follicle-associated epithelium to study nanoparticle transport by M cells.", "type" : "article-journal", "volume" : "30" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "[12]" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }[14]))Caco-2 cells were seeded in the normal orientation of the insert (as in Model B) and allowed to attach and grow for 4-6 days. Inserts were asceptically-transferred from the multi-well plates to a sterilized, glass petri dish filled with medium. Sterile silicone tubing was affixed to the basolateral end of each up-turned insert to create a sealed compartment, which was filled with media. Monolayers were cultivated in an inverted fashion until ~15-16 days, when inserts were asceptically separated into mono- and co-cultures. Co-cultures were initiated by addition of B cells to the upturned basolateral compartment and the inverted orientation was maintained for ~5 days, Control mono-cultures underwent an identical process in the absence of B cells (Fig. 1C). Characterization of co-cultures Papp values of [14C]-mannitol and TEER measurementsFor determination of the Papp across mono- and co-cultures on filters, medium on both sides was replaced with transport buffer (HBSS, buffered to pH 7.4 with 25 mM HEPES and supplemented with 5.5mM glucose). TEER was measured before and after buffer replacement to confirm monolayer integrity. The plate was equilibrated for 30 min in transport buffer before [14C]-mannitol was added to give a final concentration of 0.1 ?Ci/ml mannitol on the donor (apical) side. The Papp of [14C]-mannitol was determined over 120 min by sampling of the basolateral or receiver-side chamber and measurement using liquid scintillation (Packard Tricarb 2900 TR). Fluxes were calculated from the disintegrations per minute (dpm) using the equation: Papp = (dQ/dt) / (A.C0), where dQ/dt is the transport rate mannitol per unit time (dpm/sec), A is the surface area of the cell monolayer (1.12 cm2), and C0 is the initial concentration in the donor compartment (dpm/cm3) ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1038/nprot.2007.303", "ISSN" : "1750-2799", "PMID" : "17853866", "abstract" : "Permeability coefficients across monolayers of the human colon carcinoma cell line Caco-2, cultured on permeable supports, are commonly used to predict the absorption of orally administered drugs and other xenobiotics. This protocol describes our method for the cultivation, characterization and determination of permeability coefficients of xenobiotics (which are, typically, drug-like compounds) in the Caco-2 model. A few modifications that have been introduced over the years are incorporated in the protocol. The method can be used to trace the permeability of a test compound in two directions, from the apical to the basolateral side or vice versa, and both passive and active transport processes can be studied. The permeability assay can be completed within one working day, provided that the Caco-2 monolayers have been cultured and differentiated on the permeable supports 3 weeks in advance.", "author" : [ { "dropping-particle" : "", "family" : "Hubatsch", "given" : "Ina", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Ragnarsson", "given" : "Eva G E", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Artursson", "given" : "Per", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Nature protocols", "id" : "ITEM-1", "issue" : "9", "issued" : { "date-parts" : [ [ "2007", "1" ] ] }, "page" : "2111-9", "title" : "Determination of drug permeability and prediction of drug absorption in Caco-2 monolayers.", "type" : "article-journal", "volume" : "2" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "[13]", "manualFormatting" : "[15]", "plainTextFormattedCitation" : "[13]", "previouslyFormattedCitation" : "[14]" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }[15]. Monolayer integrity was monitored by TEER measurements throughout the culture period and for the duration of the experiment using EVOMTM, Epithelial Voltohmmeter, STX2 “chopstick” electrodes (World Precision Instruments, UK). TEER was calculated: TEER (Ω. cm2) = Measured resistance – blank insert (Ω) x surface area of insert (1.12 cm2).Transmission electron microscopy (TEM)Mono- and co-cultures grown on filters for the full cycle of ~21 days were fixed by adding 2.5% (w/v) glutaraldehyde in 0.1 M Sorensen’s phosphate buffer on both apical and basolateral sides, followed by incubation at room temperature for a minimum of 120 min at 4°C until further processing. Monolayers were then fixed with 1% osmium tetroxide in 0.1 M Sorensen’s buffer for 1 h. Using standard methods ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1016/j.ejpb.2015.05.013", "ISSN" : "09396411", "author" : [ { "dropping-particle" : "", "family" : "Brayden", "given" : "David J.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Maher", "given" : "Sam", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Bahar", "given" : "Bojlul", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Walsh", "given" : "Edwin", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "European Journal of Pharmaceutics and Biopharmaceutics", "id" : "ITEM-1", "issued" : { "date-parts" : [ [ "2015" ] ] }, "page" : "194-206", "publisher" : "Elsevier B.V.", "title" : "Sodium caprate-induced increases in intestinal permeability and epithelial damage are prevented by misoprostol", "type" : "article-journal", "volume" : "94" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "[14]", "manualFormatting" : "[16]", "plainTextFormattedCitation" : "[14]", "previouslyFormattedCitation" : "[15]" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }[16], ultrathin sections (80-100 nm) were cut using a Leica EM UC6 ultra-microtome (Leica Microsystems, Germany) and imaged by transmission electron microscopy (TEM), (Tecnai G2 12 BioTWIN, FEI Company, USA), using an accelerating voltage of 120 kV and an objective aperture of 20 μm and digital images recorded at a magnification of 20-1250X. Samples from each model were processed and analysed.Histochemical assessment of monolayersFilter-grown monolayers from mono- and co-cultures were fixed in 4% (w/v) paraformaldehyde for 20 min at room temperature. They were gently washed twice with HBSS and cut out from the Transwell? device with a sharp blade. Tissue-Tek? O.C.T. compound (Sakura? Finetek) was applied to both sides of the membrane and samples snap-frozen, labelled and stored for processing at -80°C. O.C.T. embedded samples were sectioned on a cryostat at -20°C to obtain 7 ?m thin sections of the monolayers. Sections were mounted on adhesive coated slides, and stained with hematoxylin and eosin (H & E). For each insert, 5-7 sections were obtained. The slides were visualized under a light microscope (Labophot-2A; Nikon, Japan) and images taken with a high-resolution camera (Micropublisher 3.3 RTV; Q Imaging, Canada) using Image-Pro? Plus (Media Cybernetics Inc., USA) acquisition software. Acquired images were checked for presence of double monolayers on filters. The total surface area and the area over which double monolayers were present was determined for each model using ImageJ? software. Results were expressed as percentage of area over which double monolayers were seen. The ratio of the height (thickness) of true monolayers to secondary monolayers was also determined.Measurement of functional phenotype of co-cultures Particle translocationTissue culture medium was replaced with transport buffer and monolayers incubated at 37°C for 30 min to equilibrate. Then the transport buffer on the donor (apical) side of the monolayer was replaced with pre-warmed fluorescent latex microparticle suspensions such that each monolayer was exposed to 2.5 x 108 particles/ml ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1006/bbrc.2000.4038", "ISSN" : "0006-291X", "PMID" : "11162433", "abstract" : "The aim of this work was to establish a new, simplified in vitro model of the human M-cell. Cocultures of physically separated human intestinal epithelial Caco-2 cells and B-cell lymphoma Raji cells were established. The cocultures were characterized under the criteria of morphology, integrity, expression of M-cell markers and cell adhesion molecules (CAMs), and altered particle transport. Using this construct, the epithelial cells were transformed to cells with an M-cell-like morphology and had altered expression of potential human M-cell markers (alkaline phosphatase down-regulation and Sialyl Lewis A antigen up-regulation). The expression of intercellular adhesion molecule-1 and vascular cell adhesion molecule was altered and there was an increased binding of lectins wheat germ agglutinin and peanut agglutinin with a 40-fold increase in microparticle transport. The particle transport was size-dependent and could be inhibited at 4 degrees C or by replacing the Raji B-cells with Jurkat T-cells. This new coculture model will enable controlled studies of M-cell development and function in vitro.", "author" : [ { "dropping-particle" : "", "family" : "Gullberg", "given" : "E", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Leonard", "given" : "M", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Karlsson", "given" : "J", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Hopkins", "given" : "A M", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Brayden", "given" : "D", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Baird", "given" : "A W", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Artursson", "given" : "P", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Biochemical and biophysical research communications", "id" : "ITEM-1", "issue" : "3", "issued" : { "date-parts" : [ [ "2000", "12", "29" ] ] }, "page" : "808-13", "title" : "Expression of specific markers and particle transport in a new human intestinal M-cell model.", "type" : "article-journal", "volume" : "279" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "[10]", "manualFormatting" : "[11]", "plainTextFormattedCitation" : "[10]", "previouslyFormattedCitation" : "[11]" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }[11] for each of the 0.5 and 1.0 ?m diameter particle types. Particle transport across monolayers to the basolateral chamber was studied over 120 min, with receiver chamber sampling only at this end point. TEER measurements were made before and after the transport experiment to ensure monolayer integrity. The number of particles translocated across monolayers was quantified by a Dako CyAn ADP flow cytometer (Beckman Coulter). HBSS was used to determine the background signal.Salmonella enterica translocation across monolayersSalmonella enterica serovar typhimurium (donated by the National Salmonella, Shigella and Listeria Reference Laboratory, NUIG, Ireland) was grown on Mueller Hinton (MH)-2 agar plates at 37°C. A single colony was used to inoculate 50 ml of MH-2 broth and grown overnight at 100 rpm at 37°C. 100 ?l of overnight culture was inoculated into 25 ml of MH-2 broth and grown for 180 min to obtain mid-log phase bacteria for invasion assay. 1 ml of mid-log phase bacteria was centrifuged at 15,000g for 2 min. The pellet was washed twice with HBSS and re-suspended in 1 ml HBSS. Filter-grown monolayers were infected apically (donor side) for 120 min by diluting bacteria in an appropriate volume of HBSS to yield OD600 values equivalent to the required multiplicity of infection (MOI) of 20. OD600 values were measured using a UVM 340 plate reader (ASYS Hitech, Austria). The number of translocated bacteria was determined by plating serial dilutions from the samples on MH-2 agar plates and calculating the number of colony forming units per ml (CFU/ml) [17]. Results were expressed as percentage of bacteria translocated across co-cultured monolayers relative to control mono-cultured monolayers. TEER measurements were made before and after the transport experiment to ensure monolayer integrity. Attempts to improve Model B (Gullberg) Learnings from the comparison of the three M cell models were then applied to Model B (Gullberg) architecture in attempts to further improve it. This is because this model is less labour intensive and by avoiding inversion protocols and use of silicone tubing, it is also easier to maintain sterility. Modified Transwell? filter coating protocolFilters were coated with an extracellular matrix as described previously ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1016/j.ejps.2006.12.006", "ISSN" : "0928-0987", "PMID" : "17291730", "abstract" : "An alternative in vitro model of human follicle-associated epithelium (FAE) to study nanoparticle transport mechanisms by M cells was developed and characterized. The previous in vitro model of human FAE has been improved by inverting inserts after Caco-2 cell seeding. Raji and M cells were identified only in inverted co-culture cell monolayers by immunohistochemistry, confocal microscopy, and electron microscopy. The M cell conversion rate evaluated by scanning electron microscopy ranged between 15 and 30% of cells. Transport of 200 nm carboxylated polystyrene nanoparticles was higher and more reproducible in the inverted model. Nanoparticle transport was temperature-dependent, not affected by the presence of EGTA or by potassium depletion, but inhibited by EIPA or nystatin, suggesting that it occurs most likely by macropinocytosis. The inverted model appears more physiologic, functional and reproducible than the normally oriented model.", "author" : [ { "dropping-particle" : "", "family" : "Rieux", "given" : "Anne", "non-dropping-particle" : "des", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Fievez", "given" : "Virginie", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Th\u00e9ate", "given" : "Ivan", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Mast", "given" : "Jan", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Pr\u00e9at", "given" : "V\u00e9ronique", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Schneider", "given" : "Yves-Jacques", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "European journal of pharmaceutical sciences", "id" : "ITEM-1", "issue" : "5", "issued" : { "date-parts" : [ [ "2007", "4" ] ] }, "page" : "380-91", "title" : "An improved in vitro model of human intestinal follicle-associated epithelium to study nanoparticle transport by M cells.", "type" : "article-journal", "volume" : "30" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "[12]" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }[11, 12] with some modifications. MatrigelTM was prepared in ice-cold serum-free DMEM and poured onto filters at a concentration of 30 μg/insert. Filters were incubated at room temperature overnight before aspiration and gently rinsing off unbound material with DMEM. Another set of filter inserts were coated twice with Matrigel? (45 μg) over a 24 hour period to produce (2X coated filters; i.e. 90 μg/insert) and unbound material removed by rinsing the following day before cell seeding. Filters coated once (1X) served as controls.Optimisation of a medium replenishment and conditioning protocolDuring the co-culture period for Model B (Gullberg), the apical media inside the well on which Caco-2 cells are grown is normally replenished every day, whereas the basolateral media supporting the Raji B cells is left unchanged for the 4-6 days. Media in the basolateral chamber provides nutrition to both cell populations, and this exerts pressure on the media in terms of nutritional and buffering capacity. Un-replenished basolateral media can undergo dramatic pH changes over this period and can have adverse effects on cells. To offset this, a method of replenishment on the basolateral side was developed during the co-culture period. In addition, a medium-conditioning strategy was also instituted with the aim of reducing intra-experimental variation. Medium replacement and conditioning on the apical side was carried out every day as follows: spent media from all replicates of a group was pooled in a sterile tube. Half of the spent media was discarded and fresh supplemented DMEM added as replacement. 0.5 ml of this mixture was added back to the apical side of inserts. Media replacement and conditioning on the basolateral side was carried out every 40 h (thrice over the co-culture period): spent media from mono-cultures was aspirated and pooled in a tube. One-third of this spent medium was discarded and replaced with fresh supplemented DMEM. 1.5 ml of this mixture was then added back to the basolateral side. In the case of co-cultures, one-third of spent medium (0.5 ml) was individually removed from the basolateral side of each filter and discarded. Care was taken to not aspirate the settled Raji B cells. Next, the remaining basolateral medium from each insert including the Raji B cells was aspirated and pooled in a tube. Fresh media (RPMI:DMEM 1:2 mixture) was added to this tube to compensate for the discarded volume. The tube was mixed by inversion and 1.5 ml of the mixture (containing Raji B cells) was added back to the basolateral compartment. This new methodology was expected to equalise the concentration of soluble B cell mediators in the basolateral compartment between replicates. Viability and proliferation of B cells was monitored in the basolateral chamber using Vi-CELL? Analyzer (Section 2.1).Passage of Raji B cellsThe effect of passaging Raji B cells on M-like cell formation was examined. After resuscitation from frozen state, cells were grown for two passages before being used in experiments. Caco-2 monolayers grown for 16 days were co-cultured with Raji B cells from 8 successive passages (starting from passage 6). Co-cultures created with each passage of Raji B cells were examined for latex particle translocation using flow cytometry. Results were expressed relative to the first passage of Raji B cells used.StatisticsAt a minimum, each treatment group contained an n=5, and experiments were repeated on three separate occasions (N=3). Differences between groups were analysed using ANOVA or unpaired t-test where applicable. P values less than 0.05 were considered significant. Results are expressed as mean ± standard error of mean (SEM).ResultsAssessment of three M cell model constructsMonolayer integrityAn increase in the Papp value of [14C]-mannitol and a decrease in the TEER provides indirect evidence of M like cell formation ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1006/bbrc.2000.4038", "ISSN" : "0006-291X", "PMID" : "11162433", "abstract" : "The aim of this work was to establish a new, simplified in vitro model of the human M-cell. Cocultures of physically separated human intestinal epithelial Caco-2 cells and B-cell lymphoma Raji cells were established. The cocultures were characterized under the criteria of morphology, integrity, expression of M-cell markers and cell adhesion molecules (CAMs), and altered particle transport. Using this construct, the epithelial cells were transformed to cells with an M-cell-like morphology and had altered expression of potential human M-cell markers (alkaline phosphatase down-regulation and Sialyl Lewis A antigen up-regulation). The expression of intercellular adhesion molecule-1 and vascular cell adhesion molecule was altered and there was an increased binding of lectins wheat germ agglutinin and peanut agglutinin with a 40-fold increase in microparticle transport. The particle transport was size-dependent and could be inhibited at 4 degrees C or by replacing the Raji B-cells with Jurkat T-cells. This new coculture model will enable controlled studies of M-cell development and function in vitro.", "author" : [ { "dropping-particle" : "", "family" : "Gullberg", "given" : "E", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Leonard", "given" : "M", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Karlsson", "given" : "J", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Hopkins", "given" : "A M", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Brayden", "given" : "D", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Baird", "given" : "A W", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Artursson", "given" : "P", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Biochemical and biophysical research communications", "id" : "ITEM-1", "issue" : "3", "issued" : { "date-parts" : [ [ "2000", "12", "29" ] ] }, "page" : "808-13", "title" : "Expression of specific markers and particle transport in a new human intestinal M-cell model.", "type" : "article-journal", "volume" : "279" }, "uris" : [ "" ] }, { "id" : "ITEM-2", "itemData" : { "DOI" : "10.1016/j.ejps.2006.12.006", "ISSN" : "0928-0987", "PMID" : "17291730", "abstract" : "An alternative in vitro model of human follicle-associated epithelium (FAE) to study nanoparticle transport mechanisms by M cells was developed and characterized. The previous in vitro model of human FAE has been improved by inverting inserts after Caco-2 cell seeding. Raji and M cells were identified only in inverted co-culture cell monolayers by immunohistochemistry, confocal microscopy, and electron microscopy. The M cell conversion rate evaluated by scanning electron microscopy ranged between 15 and 30% of cells. Transport of 200 nm carboxylated polystyrene nanoparticles was higher and more reproducible in the inverted model. Nanoparticle transport was temperature-dependent, not affected by the presence of EGTA or by potassium depletion, but inhibited by EIPA or nystatin, suggesting that it occurs most likely by macropinocytosis. The inverted model appears more physiologic, functional and reproducible than the normally oriented model.", "author" : [ { "dropping-particle" : "", "family" : "Rieux", "given" : "Anne", "non-dropping-particle" : "des", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Fievez", "given" : "Virginie", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Th\u00e9ate", "given" : "Ivan", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Mast", "given" : "Jan", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Pr\u00e9at", "given" : "V\u00e9ronique", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Schneider", "given" : "Yves-Jacques", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "European journal of pharmaceutical sciences", "id" : "ITEM-2", "issue" : "5", "issued" : { "date-parts" : [ [ "2007", "4" ] ] }, "page" : "380-91", "title" : "An improved in vitro model of human intestinal follicle-associated epithelium to study nanoparticle transport by M cells.", "type" : "article-journal", "volume" : "30" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "[10,12]", "manualFormatting" : "[11,14]", "plainTextFormattedCitation" : "[10,12]", "previouslyFormattedCitation" : "[11,13]" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }[17]. A higher Papp of co-cultures (2.3 x 10-6 cm/s) compared to mono-cultures was observed for Model C (P < 0.001; Fig. 2A). While a similar trend was also observed for Models A and B co-cultures, values did not reach significance. The basal Papp values for mono-cultures across the three models were in a similar range (~10-7 cm/s), in agreement with published values for Caco-2 monolayers ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1038/nprot.2007.303", "ISSN" : "1750-2799", "PMID" : "17853866", "abstract" : "Permeability coefficients across monolayers of the human colon carcinoma cell line Caco-2, cultured on permeable supports, are commonly used to predict the absorption of orally administered drugs and other xenobiotics. This protocol describes our method for the cultivation, characterization and determination of permeability coefficients of xenobiotics (which are, typically, drug-like compounds) in the Caco-2 model. A few modifications that have been introduced over the years are incorporated in the protocol. The method can be used to trace the permeability of a test compound in two directions, from the apical to the basolateral side or vice versa, and both passive and active transport processes can be studied. The permeability assay can be completed within one working day, provided that the Caco-2 monolayers have been cultured and differentiated on the permeable supports 3 weeks in advance.", "author" : [ { "dropping-particle" : "", "family" : "Hubatsch", "given" : "Ina", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Ragnarsson", "given" : "Eva G E", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Artursson", "given" : "Per", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Nature protocols", "id" : "ITEM-1", "issue" : "9", "issued" : { "date-parts" : [ [ "2007", "1" ] ] }, "page" : "2111-9", "title" : "Determination of drug permeability and prediction of drug absorption in Caco-2 monolayers.", "type" : "article-journal", "volume" : "2" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "[13]", "manualFormatting" : "[e.g. 15]", "plainTextFormattedCitation" : "[13]", "previouslyFormattedCitation" : "[14]" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }[e.g. 15]. The Papp across Model C co-cultures was 5- and 3-fold higher than Model A and B co-cultures respectively, and the values did not exceed those in the original publication for Model C ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1016/j.ejps.2006.12.006", "ISSN" : "0928-0987", "PMID" : "17291730", "abstract" : "An alternative in vitro model of human follicle-associated epithelium (FAE) to study nanoparticle transport mechanisms by M cells was developed and characterized. The previous in vitro model of human FAE has been improved by inverting inserts after Caco-2 cell seeding. Raji and M cells were identified only in inverted co-culture cell monolayers by immunohistochemistry, confocal microscopy, and electron microscopy. The M cell conversion rate evaluated by scanning electron microscopy ranged between 15 and 30% of cells. Transport of 200 nm carboxylated polystyrene nanoparticles was higher and more reproducible in the inverted model. Nanoparticle transport was temperature-dependent, not affected by the presence of EGTA or by potassium depletion, but inhibited by EIPA or nystatin, suggesting that it occurs most likely by macropinocytosis. The inverted model appears more physiologic, functional and reproducible than the normally oriented model.", "author" : [ { "dropping-particle" : "", "family" : "Rieux", "given" : "Anne", "non-dropping-particle" : "des", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Fievez", "given" : "Virginie", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Th\u00e9ate", "given" : "Ivan", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Mast", "given" : "Jan", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Pr\u00e9at", "given" : "V\u00e9ronique", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Schneider", "given" : "Yves-Jacques", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "European journal of pharmaceutical sciences", "id" : "ITEM-1", "issue" : "5", "issued" : { "date-parts" : [ [ "2007", "4" ] ] }, "page" : "380-91", "title" : "An improved in vitro model of human intestinal follicle-associated epithelium to study nanoparticle transport by M cells.", "type" : "article-journal", "volume" : "30" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "[12]", "manualFormatting" : "[14]", "plainTextFormattedCitation" : "[12]", "previouslyFormattedCitation" : "[13]" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }[14]. The Papp value for Model B co-cultures (1x10-6 cm/s) was also similar to that obtained in the original publication for that model ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1006/bbrc.2000.4038", "ISSN" : "0006-291X", "PMID" : "11162433", "abstract" : "The aim of this work was to establish a new, simplified in vitro model of the human M-cell. Cocultures of physically separated human intestinal epithelial Caco-2 cells and B-cell lymphoma Raji cells were established. The cocultures were characterized under the criteria of morphology, integrity, expression of M-cell markers and cell adhesion molecules (CAMs), and altered particle transport. Using this construct, the epithelial cells were transformed to cells with an M-cell-like morphology and had altered expression of potential human M-cell markers (alkaline phosphatase down-regulation and Sialyl Lewis A antigen up-regulation). The expression of intercellular adhesion molecule-1 and vascular cell adhesion molecule was altered and there was an increased binding of lectins wheat germ agglutinin and peanut agglutinin with a 40-fold increase in microparticle transport. The particle transport was size-dependent and could be inhibited at 4 degrees C or by replacing the Raji B-cells with Jurkat T-cells. This new coculture model will enable controlled studies of M-cell development and function in vitro.", "author" : [ { "dropping-particle" : "", "family" : "Gullberg", "given" : "E", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Leonard", "given" : "M", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Karlsson", "given" : "J", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Hopkins", "given" : "A M", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Brayden", "given" : "D", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Baird", "given" : "A W", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Artursson", "given" : "P", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Biochemical and biophysical research communications", "id" : "ITEM-1", "issue" : "3", "issued" : { "date-parts" : [ [ "2000", "12", "29" ] ] }, "page" : "808-13", "title" : "Expression of specific markers and particle transport in a new human intestinal M-cell model.", "type" : "article-journal", "volume" : "279" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "[10]", "manualFormatting" : "[11]", "plainTextFormattedCitation" : "[10]", "previouslyFormattedCitation" : "[11]" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }[11]. In contrast, the Papp values across mono- and co-cultures in Model A configuration were comparatively lower (1.8 x10-7 cm/s and 4.8 x10-7 cm/s respectively) than counterparts in the other two models. Absence of published [14C]-mannitol Papp data in Model A prevented a direct comparison.There was a concern that high Papp values across co-cultures may simply reflect a loss of monolayer integrity, however measurements of TEER revealed acceptable values in the three co-culture models. In Model C, following 16 days of mono-culture, mean TEER values were 777 ± 296 Ω. cm2), but after a further 5 days in co-culture, the mean values were373 ± 38 Ω. cm2, a mean reduction 42 ± 12 % (P< 0.05). Mean mono-culture TEER values for Models A and B at Day 16 were 1210 ± 321 Ω. cm2 and 926 ± 162 Ω. cm2 respectively. Corresponding reductions in Model A and B were 16% and 22% respectively (Fig. 2B), but these differences were not significant. These data show that the highest Papp and lowest TEER values were achieved in Model C co-cultures. Moreover, the data suggest that a reduction in TEER was not consistently present in all the models, but this does not mean that phenotypic changes will be absent in Models A and B.Particle translocationTranslocation of latex particles was enhanced in each co-culture configuration compared to respective mono-cultures (Fig. 3). Model C co-cultures demonstrated statistically-increased translocation for both particle diameter sizes (0.5 ?m and 1.0 ?m) with mean increases of 74- and 40-fold respectively compared to mono-cultures. 13- and 8 fold increases were recorded for 0.5 ?m and 1.0 ?m particles respectively across Model B, whereas just 2 and 1.8 fold increases were obtained for Model A. Compared to Models A and B, the fold increase in particle translocation was therefore higher in Model C for both size ranges of particles compared to monocultures and this was also confirmed for the absolute numbers translocated. Translocation across Model C co-cultures was 5.5 and 3.6 fold higher for 0.5 ?m particles, and 3 and 1.3 fold higher for 1.0 ?m particles compared to Model A and B co-cultures respectively. Particle translocation was also size-dependent in each of the three co-culture models, with 0.5 ?m particles crossing to a greater extent than 1.0 ?m particles. In co-cultures, the difference was greatest in Model C, with 74% more 0.5 ?m diameter particles crossing than the 1.0 ?m ones (P < 0.001), while the values were 50% and 43% in Models A and B, but these were not statistically different. Furthermore, standard deviations for particles translocated across co-cultures were lower in Model C compared to other two models, indicating superior reproducibility. Model C co-cultures again had the more convincing phenotype compared other two models under this criterion. S. typhimurium translocation We compared S. typhimurium translocation patterns over 120 min across the three models. Results are expressed as fold increase in translocated bacteria relative to mono-cultures (Fig. 4). Translocation across the three models was higher in co-cultures compared to mono-cultures. Total mean bacterial transport across co-cultures was highest in Model C at 25 fold higher (compared to its mono-cultures), followed by Models B and A (16.5 and 15.7 fold respectively). Although co-cultures of all models displayed a trend for an increase in bacterial translocation, this difference was only statistically significant for Model B. The original studies for each of the models did not have any quantitative bacterial transport data to compare. Secondary epithelial cell layer formation on underside of filters.Build-up of secondary epithelial cell layers on the opposite side of Transwells? to which Caco-2 cells were seeded was investigated in the three models. Their presence can lead to confounding data. Secondary layers were present in representative images from each model, however this seemed to occur to a lesser extent in Model C compared to the others (Fig. 5A). Model A was most susceptible to secondary layers: 79% of the surface area was covered by secondary layers compared to 32% in Model C (P < 0.01). However, there was no statistical difference between Model B (50%) and Model C (32%). H & E assessments of cellular growth on each side of the filter revealed contrast between the ample epithelial cell growth on the basolateral side of a Model A co-culture compared to a reduced layer for a Model B co-culture and just occasional spotty patches evident for Model C (Fig. 5B). The ratio of the height (at the thickest point in each section) of the true monolayer on the intended side was also calculated for each model. The ratio was the highest for Model C (2.8 times the height of secondary cell layer), followed by Model B (2.5), and Model A (1.02). Overall, Model C co-cultures had fewer cells migrating through to the underside of the filters than the other models.Ultrastructural analysis M-like cell morphology The loss and/or re-arrangement of microvilli on the apical surface of epithelial cell monolayers provides visual evidence of the formation of in vitro M-like cells ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1016/j.ejps.2006.12.006", "ISSN" : "0928-0987", "PMID" : "17291730", "abstract" : "An alternative in vitro model of human follicle-associated epithelium (FAE) to study nanoparticle transport mechanisms by M cells was developed and characterized. The previous in vitro model of human FAE has been improved by inverting inserts after Caco-2 cell seeding. Raji and M cells were identified only in inverted co-culture cell monolayers by immunohistochemistry, confocal microscopy, and electron microscopy. The M cell conversion rate evaluated by scanning electron microscopy ranged between 15 and 30% of cells. Transport of 200 nm carboxylated polystyrene nanoparticles was higher and more reproducible in the inverted model. Nanoparticle transport was temperature-dependent, not affected by the presence of EGTA or by potassium depletion, but inhibited by EIPA or nystatin, suggesting that it occurs most likely by macropinocytosis. The inverted model appears more physiologic, functional and reproducible than the normally oriented model.", "author" : [ { "dropping-particle" : "", "family" : "Rieux", "given" : "Anne", "non-dropping-particle" : "des", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Fievez", "given" : "Virginie", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Th\u00e9ate", "given" : "Ivan", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Mast", "given" : "Jan", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Pr\u00e9at", "given" : "V\u00e9ronique", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Schneider", "given" : "Yves-Jacques", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "European journal of pharmaceutical sciences", "id" : "ITEM-1", "issue" : "5", "issued" : { "date-parts" : [ [ "2007", "4" ] ] }, "page" : "380-91", "title" : "An improved in vitro model of human intestinal follicle-associated epithelium to study nanoparticle transport by M cells.", "type" : "article-journal", "volume" : "30" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "[12]", "manualFormatting" : "[14]", "plainTextFormattedCitation" : "[12]", "previouslyFormattedCitation" : "[13]" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }[17]. TEM of mono-cultures from each model displayed typical brush border apical microvilli (Fig. 6). In contrast, co-cultures from all models showed evidence of varying degree of loss of microvilli from epithelial surfaces. The M-like cells observed in Models A and C in general displayed similar apical re-arrangement of microvilli. The majority of M-like cells in these two models exhibited microvilli that were significantly reduced in height. On the other hand, the majority of M-like cells observed in Model B exhibited shortened and irregularly arranged microvilli. Overall, TEM revealed evidence of conversion of at least some enterocytes to an M-like cell morphology in each model. Table 2 compares the data from each model. Based on a semi-quantitative histological analysis, the estimates of M cell conversion for each model were 7.5% for Model A, 13% for Model B and 8% for Model C, noting that values of 15-30% have recently been reported for C [17].Improvement of Model B (Gullberg) by varying culture parametersEffect of MatrigelTM filter-coating The impact of the modified coating on TEER, Papp of [14C]-mannitol and prevention of secondary Caco-2 epithelial cell layers on the underside of the filter was studied in Model B. Filters coated with a single MatrigelTM layer (1X) using the current protocol were less efficient at preventing cell migration to the opposite side compared to filters with two MatrigelTM (2X) coatings (Fig.7). A 10-20% decrease in surface area covered by epithelia on the filter underside was observed using a double coating. Although the formation of secondary cell layers was diminished, MatrigelTM double coating did not preclude it. Co-cultures with double-coated filters had a 1.7-1.8 fold lower TEER values compared to single coated co-cultures after the 21 days cycle, but the Papp of [14C]-mannitol was not altered across co-cultures by the process. The mean TEER for MatrigelTM coated blank inserts was 136 Ω..cm2 which was not different from uncoated inserts (128 Ω..cm2). Overall, double coating with MatrigelTM improved histological parameters and lowered TEER in Model B (Gullberg) co-cultures compared to the original coating method. Influence of modified media conditioning and replenishment protocolDuring the co-culture period in Model B, the impact of a modified feeding protocol on Raji B cell viability and proliferation was examined. Media conditioning along with controlled media replenishment produced a notable improvement in proliferation and the survival of Raji B cells in the basolateral compartment (Fig. 8). The proportion of viable Raji B cells increased ~6 fold during co-culture. In unconditioned cultures a 4 fold increase was recorded. In all cultures, Raji B cell viability initially declined. The rate of decline was more pronounced in co-cultures that were not subjected to altered media replenishment/conditioning. Following this, Raji B cells underwent proliferation and as a result viability in both groups improved. After 5 days of co-culture, Raji B cells from co-cultures subjected to media-conditioning had better viability (86%) compared to 65% in the unconditioned group Passage of Raji B cells The effect of passaging Raji B cells on their capacity to transform differentiated Caco-2 monolayers into the M-like cell phenotype was examined functionally by studying the level of particle translocation across co-cultures (Fig. 9). Translocation of particles (0.5 μm and 1.0 μm diameter) in Model B co-cultures is shown across eight passages, from starting passage # 6. Relative to passage 6, the data shows a decline in the number of particles translocated in consecutive passages. The impact of passage was different for the particle diameters. Overall, the effect of passage of Raji B cells was significant, especially in later passages where the number of particles translocated declined to 61% (for 0.5 μm) and to 24% (for 1.0 μm) compared to the values seen at passage 6. This suggests that better conversion occurs at earlier Raji passages and that Raji B cells lose efficacy in the capacity to convert Caco-2 cells the more they are passaged. Combined parameters to improve the Model B phenotypeThe modified MatrigelTM double coating coupled with apical medium conditioning and basolateral medium replenishment resulted in reduced variation between replicates within experiments in terms of particle translocation across Model B co-cultures. Additionally, inter-experimental variation was also reduced by confining the range of Raji B cell passages used. While no differences were observed between mono-cultures using either the standard method or the improved method, particle translocation was enhanced several fold across co-cultures of the improved co-culture Model B compared to the standard Gullberg model (Fig. 10).DiscussionWe compared the three published in vitro M-like cell models for the first time in a head-to-head quantitative study by standardising protocols. The Model A (Kerneis et al., [9]) construct produced a phenotype indicative of a relatively inefficient conversion of Caco-2 cells to M-like cells arising from interactions with intercalated B cells or from B cell released cytokines and growth factors. Although this was the first M-like model to be created in 1997, it has a reputation for being difficult to transfer to other laboratories and this contributed to the impetus for the development of alternative models. The data in the current study would support the view that Model A remains somewhat problematic. Even with the modification in using a silicone tubing on the upturned filter to contain well dispersed Caco-2 cells in high concentrations in preference to the step of pipetting cells onto the upturned filter in a very low volume of medium, variability in the phenotype is a feature. The use of silicone tubing in Model A was identical to that used for Model C except that in this instance it was intended to contain B cells. The low Papp of [14C]-mannitol across Model A co-cultures correlated with relatively high TEER values and these values were lower and higher respectively compared to models B and C. Histochemical analysis of the three models confirmed the presence of secondary cell layers on the opposite side of the filter to which Caco-2 cells were seeded, and this was especially prevalent in Model A. Superfluous layers can introduce errors in electrophysiological data and inaccurate determination of vectorial transport of molecules and particles across the co-culture . In the case of Model A, formation of well differentiated secondary cell layers was perhaps a result of migration through the filter pores to the opposite side at the beginning of the culture period. In contrast, the occurrence of secondary cell layers was less evident for Model C (Des Rieux et al., ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1016/j.ejps.2006.12.006", "ISSN" : "0928-0987", "PMID" : "17291730", "abstract" : "An alternative in vitro model of human follicle-associated epithelium (FAE) to study nanoparticle transport mechanisms by M cells was developed and characterized. The previous in vitro model of human FAE has been improved by inverting inserts after Caco-2 cell seeding. Raji and M cells were identified only in inverted co-culture cell monolayers by immunohistochemistry, confocal microscopy, and electron microscopy. The M cell conversion rate evaluated by scanning electron microscopy ranged between 15 and 30% of cells. Transport of 200 nm carboxylated polystyrene nanoparticles was higher and more reproducible in the inverted model. Nanoparticle transport was temperature-dependent, not affected by the presence of EGTA or by potassium depletion, but inhibited by EIPA or nystatin, suggesting that it occurs most likely by macropinocytosis. The inverted model appears more physiologic, functional and reproducible than the normally oriented model.", "author" : [ { "dropping-particle" : "", "family" : "Rieux", "given" : "Anne", "non-dropping-particle" : "des", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Fievez", "given" : "Virginie", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Th\u00e9ate", "given" : "Ivan", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Mast", "given" : "Jan", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Pr\u00e9at", "given" : "V\u00e9ronique", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Schneider", "given" : "Yves-Jacques", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "European journal of pharmaceutical sciences", "id" : "ITEM-1", "issue" : "5", "issued" : { "date-parts" : [ [ "2007", "4" ] ] }, "page" : "380-91", "title" : "An improved in vitro model of human intestinal follicle-associated epithelium to study nanoparticle transport by M cells.", "type" : "article-journal", "volume" : "30" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "[12]", "plainTextFormattedCitation" : "[12]", "previouslyFormattedCitation" : "[13]" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }[14]. Perhaps upturning the inserts with established Caco-2 monolayers (in contrast to Model A) does not encourage cells to subsequently migrate through the pores to the opposite side. Model B (Gullberg et al., [11]) also displayed evidence of secondary monolayers and occasional thickening and differentiation to a degree that was in between that of Models A and C. Microparticle translocation across each model was examined by comparing the fold increase in co-cultures versus respective mono-cultures. By this criterion, Model C was superior to A and B, whereas there was no difference between the latter two. Secondly, in terms of numbers of particles translocated, Model C performed better than the other two models. Higher fold differences in particle translocation across co-cultures versus mono-cultures in Model C compared to Model B has been previously documented ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1006/bbrc.2000.4038", "ISSN" : "0006-291X", "PMID" : "11162433", "abstract" : "The aim of this work was to establish a new, simplified in vitro model of the human M-cell. Cocultures of physically separated human intestinal epithelial Caco-2 cells and B-cell lymphoma Raji cells were established. The cocultures were characterized under the criteria of morphology, integrity, expression of M-cell markers and cell adhesion molecules (CAMs), and altered particle transport. Using this construct, the epithelial cells were transformed to cells with an M-cell-like morphology and had altered expression of potential human M-cell markers (alkaline phosphatase down-regulation and Sialyl Lewis A antigen up-regulation). The expression of intercellular adhesion molecule-1 and vascular cell adhesion molecule was altered and there was an increased binding of lectins wheat germ agglutinin and peanut agglutinin with a 40-fold increase in microparticle transport. The particle transport was size-dependent and could be inhibited at 4 degrees C or by replacing the Raji B-cells with Jurkat T-cells. 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The previous in vitro model of human FAE has been improved by inverting inserts after Caco-2 cell seeding. Raji and M cells were identified only in inverted co-culture cell monolayers by immunohistochemistry, confocal microscopy, and electron microscopy. The M cell conversion rate evaluated by scanning electron microscopy ranged between 15 and 30% of cells. Transport of 200 nm carboxylated polystyrene nanoparticles was higher and more reproducible in the inverted model. Nanoparticle transport was temperature-dependent, not affected by the presence of EGTA or by potassium depletion, but inhibited by EIPA or nystatin, suggesting that it occurs most likely by macropinocytosis. 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For Model C, we observed a 40-74 fold increase in particle translocation, similar to previous values (50 fold increase) [11]. On the other hand, Model B displayed a 8-13 fold increase in the current study, while des Rieux et al. only detected a 3 fold increase for the same model ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1016/j.ejps.2006.12.006", "ISSN" : "0928-0987", "PMID" : "17291730", "abstract" : "An alternative in vitro model of human follicle-associated epithelium (FAE) to study nanoparticle transport mechanisms by M cells was developed and characterized. The previous in vitro model of human FAE has been improved by inverting inserts after Caco-2 cell seeding. Raji and M cells were identified only in inverted co-culture cell monolayers by immunohistochemistry, confocal microscopy, and electron microscopy. The M cell conversion rate evaluated by scanning electron microscopy ranged between 15 and 30% of cells. Transport of 200 nm carboxylated polystyrene nanoparticles was higher and more reproducible in the inverted model. Nanoparticle transport was temperature-dependent, not affected by the presence of EGTA or by potassium depletion, but inhibited by EIPA or nystatin, suggesting that it occurs most likely by macropinocytosis. 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There was no published quantitative data on particle translocation to make a comparison with Model A. To further interpret the microparticle translocation data, the geometry and orientation of the models must be considered as potential confounding factors. Even though transport experiments were carried out with continuous agitation to ensure homogeneity particle suspensions on the donor side of co-cultures, particles still tended to settle under gravitational influence at the apical pole of monolayers in Models B and C. However, in the reverse orientation of Model A, particles on the donor side do not have the gravitational pull. Therefore, it is likely that M-like cells of Model B and C co-cultures were in closer physical contact with particles compared to those of Model A. Although this can partially explain the observations of very low particle transport across co-cultures in Model A, it is debatable which orientation is more physiologic. Luminal contents in the GI tract are subject to shear stress of peristalsis, which prevents sustained contact of particulate matter with intestinal mucosae or with M cells in the FAE of PP. Comparative data collected from the three models suggests that Model C was overall more reproducible with lower intra- and inter-experimental variation than the other models in terms of fluxes and particle translocation. While this study was under review, an extensive and detailed protocol of Model C (des Rieux) was published [17]. Using microscopy for assessment, the proportion of M-like cells in Model C was estimated at 15-30%, less than we estimated here. Possible reasons are that the low TEER C2BBBe clone from the American Type Culture Collection (ATCC) was the source in [17] in contrast to the high TEER Caco-2 line sourced from the European Collection of Authenticated Cell Cultures (ECACC) in the current study. Moreover, caution must be expressed in terms of predictions of particle uptake by M cells in vivo since the estimate of M cells in the human FAE is <5% [18]. In the case of Salmonella translocation however, while an increase was seen in all three models, Model B displayed more consistent data. Perhaps this explains why the Gullberg model B has been used widely to probe M-like cell uptake and translocation of both Salmonella typhiumurium [19], and more recently, vibrio parahaemolyticus [20]. Ultrastructurally, each model showed evidence of truncated microvilli and it was not possible to discriminate them. A unique feature of Model C is that several Transwells? with cultured Caco-2 monolayers share apical-side medium in a petri dish for the majority of the 21 day culture period and this promotes internal consistency. Despite this, of the three models, Model C is the most technically-challenging and requires several aseptic manipulations, thereby making it more susceptible to contamination. As several inserts are cultured in a communal petri dish, contamination would result in loss of all the inserts. By contrast in the other two models, inserts are physically segregated as individual units. Model B is the easiest to set up as it requires no filter inversion nor silicone tubing. It was originally promoted as an improvement over the Model A construct ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1006/bbrc.2000.4038", "ISSN" : "0006-291X", "PMID" : "11162433", "abstract" : "The aim of this work was to establish a new, simplified in vitro model of the human M-cell. Cocultures of physically separated human intestinal epithelial Caco-2 cells and B-cell lymphoma Raji cells were established. The cocultures were characterized under the criteria of morphology, integrity, expression of M-cell markers and cell adhesion molecules (CAMs), and altered particle transport. Using this construct, the epithelial cells were transformed to cells with an M-cell-like morphology and had altered expression of potential human M-cell markers (alkaline phosphatase down-regulation and Sialyl Lewis A antigen up-regulation). The expression of intercellular adhesion molecule-1 and vascular cell adhesion molecule was altered and there was an increased binding of lectins wheat germ agglutinin and peanut agglutinin with a 40-fold increase in microparticle transport. The particle transport was size-dependent and could be inhibited at 4 degrees C or by replacing the Raji B-cells with Jurkat T-cells. This new coculture model will enable controlled studies of M-cell development and function in vitro.", "author" : [ { "dropping-particle" : "", "family" : "Gullberg", "given" : "E", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Leonard", "given" : "M", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Karlsson", "given" : "J", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Hopkins", "given" : "A M", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Brayden", "given" : "D", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Baird", "given" : "A W", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Artursson", "given" : "P", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Biochemical and biophysical research communications", "id" : "ITEM-1", "issue" : "3", "issued" : { "date-parts" : [ [ "2000", "12", "29" ] ] }, "page" : "808-13", "title" : "Expression of specific markers and particle transport in a new human intestinal M-cell model.", "type" : "article-journal", "volume" : "279" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "[10]", "plainTextFormattedCitation" : "[10]", "previouslyFormattedCitation" : "[11]" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }[10] because it is suitable for gene expression studies where physical interaction and intercalation between Caco-2 and B cells would be a disadvantage [13]. Moreover, since Model B relies on release of a cocktail of cytokines and pro-inflammatory molecules from B cells [11], this model has potential to be developed further in respect of using a fully-defined tissue culture medium. This led us therefore, to attempt to achieve a phenotype closer to Model C by further manipulating aspects of the Model B protocol.The first challenge was to reduce the secondary cell layer coverage on the opposite side of the filters in model B. To resolve this, a modified Matrigel? coating protocol was tested and the best results were to use two coats on filters over a 24 h period. The success of the 2X coating methodology could possibly be due to better cross-linking of the basement membrane protein, which in turn better prevents cell migration through pores. Induction of M-like cells in these individual monolayers during co-culture with Raji B cells in Model B format is dependent on the proportion of Caco-2 cells that had not undergone terminal differentiation and hence can be converted. This can potentially lead to variations in the number of M-like cells between replicates within an experiment. Observations from experiments conducted with Model C however suggested that sharing of the apical media between inserts reduced this variation observed in particle translocation. We therefore designed a protocol that allowed sharing of apical media between replicates to ensure homogenous exposure to soluble factors that could influence M-like cell differentiation. We also ensured sharing of basolateral media along with replenishment during the co-culture period. This policy of basolateral conditioning and replenishment had a positive impact on the viability of Raji B cells. In addition to media conditioning and replenishment, we also controlled the number of passages Raji B cells spend in routine culture. This was due to our observation of Raji B cells gradually losing efficiency in inducing M-like cell differentiation in Caco-2 monolayers following several passages in cell culture. We therefore combined the above strategies (i.e. double coating with MatrigelTM, media conditioning and replacement, and using defined passages of Raji B cells) to produce an improved Model B with reduced variability. Recently, attempts have been made by others to further improve the Gullberg et al. (Model B) by supplementing the basolateral medium supporting B cell differentiation ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1016/j.xphs.2016.03.043", "ISSN" : "1520-6017", "PMID" : "27262206", "abstract" : "The aim of this study was to investigate whether the pro-inflammatory cytokines improved the function of the cell monolayer model of the human follicle-associated epithelium (FAE) of co-culture of Caco-2 cells on permeable filters with Raji B-cells underneath from the viewpoint of particle transport. Exposure to tumor necrosis factor-\u03b1 resulted in an almost maintained epithelial integrity/paracellular permeability combined with an increased nanoparticle transport in a dose-dependent manner while the effects of interleukin (IL)-1\u03b2 were limited. Exposure to IL-6 significantly enhanced the nanoparticle transport with the limited disruption of the cell monolayer integrity. The addition of IL-6 or tumor necrosis factor-\u03b1 to Caco-2 monolayers without Raji B-cells did not enhance nanoparticle transport. In our IL-6 treated FAE model, the nanoparticle transport almost disappeared at 4\u00b0C or after the addition of 5-(N-ethyl-N-isopropyl) amiloride, an inhibitor of macropinocytosis. Furthermore, IgA binding, presumably by a secretory IgA receptor, a marker of M-cells was observed on the apical side of our model FAE. These results indicate that the combined effect of IL-6 with unknown factors from Raji-B cells made the FAE model more functional with regard to nanoparticle transport. The IL-6 enhanced FAE model will be a useful platform for nanoparticle drug delivery research across the intestinal epithelium.", "author" : [ { "dropping-particle" : "", "family" : "Miyake", "given" : "Masateru", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Ragnarsson", "given" : "Eva", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Nakai", "given" : "Daisuke", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Artursson", "given" : "Per", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Journal of pharmaceutical sciences", "id" : "ITEM-1", "issue" : "7", "issued" : { "date-parts" : [ [ "2016", "7" ] ] }, "page" : "2099-104", "publisher" : "Elsevier", "title" : "The Pro-inflammatory Cytokine Interleukin-6 Regulates Nanoparticle Transport Across Model Follicle-Associated Epithelium Cells.", "type" : "article-journal", "volume" : "105" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "[16]", "plainTextFormattedCitation" : "[16]", "previouslyFormattedCitation" : "[16]" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }[21]. When B cells were added to create a co-culture at Day 14, Miyake et al. examined the effects of co-addition of the pro-inflammatory molecules, Tumour Necrosis Factor (TNF)-α, IL-6, or IL-1β. Of these agents, both TNF- and IL-6 induced and increase in the Papp of [14C]-mannitol, the flux of polystyrene nanoparticles, along with apical expression of the M cell marker, β1 integrin. IL-6 was especially effective over a concentration range of 1-100ng/ml. It is likely that use of IL-6 supplemented co-culture medium in combination with double coating of filters and a revised feeding regime for the basolateral side of co-cultures will further improve the model. Recently, another team has produced another variation of the Gullberg model by simply mixing Caco-2 and Raji B cell suspensions in 1:1 ratios before seeding on filters. When they pre-treated the Caco-2 cells with 50 ng/mL epidermal growth factor for 24 hours, conversion was improved further, as detected by expression of an M cell-specific marker (clusterin), stem cell markers, and particle translocation [22]. Finally, the three models examined here are not the only in vitro options for investigating M-like cell function in an FAE. Others have recently further modified the Caco-2/Raji B combination to include mucus-secreting human HT29-MTX cells in attempts to factor in the influence of mucus on particle uptake and translocation by M cells [23, 24]. It is interesting to note that both of these groups derived the triple culture concept using the original Gullberg (Model B) normally-oriented design, as it is the simplest and most flexible of the three models. In conclusion, this study provides data from a single laboratory that compares all three M-like cell co-culture models under set criteria using the same cell types throughout. While Model C (des Rieux) provided the most consistent overall phenotype, Model B was more suitable for bacterial interaction studies. Moreover, modifications of the Model B protocol conferred improvements that may eventually lead to wider adoption since it is the most straightforward of the models due to its normal orientation and is less likely to be contaminated over the 21 day period. AcknowledgmentsThis study was funded by Science Foundation Ireland (SFI) Strategic Research Cluster Grant 07/SRC/B1154 (Irish Drug Delivery Network)ReferencesADDIN Mendeley Bibliography CSL_BIBLIOGRAPHY [1]A. Reboldi, J.G. Cyster, Peyer’s patches: Organizing B-cell responses at the intestinal frontier, Immunol. Rev. 271 (2016) 230–245. [2]M.H. Jang, M.-N. Kweon, K. Iwatani, M. Yamamoto, K. Terahara, C. Sasakawa, T. Suzuki, T. Nochi, Y. Yokota, P.D. Rennert, T. Hiroi, H. Tamagawa, H. Iijima, J. Kunisawa, Y. Yuki, H. Kiyono, Intestinal villous M cells: an antigen entry site in the mucosal epithelium., Proc. Natl. Acad. Sci. U. S. A. 101 (2004) 6110–6115. [3]N.A. Mabbott, D.S. Donaldson, H. Ohno, I.R. Williams, Microfold ( M ) cells?: important immunosurveillance posts in the intestinal epithelium, 6 (2013) 666–677. [4]D.E. Katz, A.J. DeLorimier, M.K. Wolf, E.R. Hall, F.J. Cassels, J.E. Van Hamont, R.L. Newcomer, M.A. Davachi, D.N. Taylor, C.E. McQueen, Oral immunization of adult volunteers with microencapsulated enterotoxigenic Escherichia coli (ETEC) CS6 antigen, Vaccine. 21 (2003) 341–346.[5] B. Singh, S. Maharjan, T. Jiang, S.K. Kang, Y.J. Choi, C.S. Cho, Combinatorial approach of antigen delivery using M cell-homing peptide and mucoadhesive vehicle to enhance the efficacy of oral vaccine, Mol. Pharm. 12 (2015) 3816–3828.[6]D. Brayden, M. Jepson, A. Baird, Keynote review: intestinal Peyer’s patch M cells and oral vaccine targeting, Drug Discov. Today. 10 (2005) 1145–1157. [7]C.A. Cuvelier, J. Quatacker, H. Mielants, M. De Vos, E. Veys, H.J. Roels, M-cells are damaged and increased in number in inflamed human ileal mucosa., Histopathology. 24 (1994) 417-426. [8]P.J. Giannasca, K.T. Giannasca, A.M. Leichtner, M.R. Neutra, Human intestinal M cells display the sialyl Lewis A antigen., Infect. Immun. 67 (1999) 946–953. [9]S. Kernéis, A. Bogdanova, J. Kraehenbuhl, E. Pringault, Conversion by Peyer’s Patch lymphocytes of human enterocytes into M Cells that transport bacteria, Science (80-. ). 277 (1997) 949–952. [10]S. Kernéis, E. Caliot, H. Stubbe, A. Bogdanova, J. Kraehenbuhl, E. Pringault, Molecular studies of the intestinal mucosal barrier physiopathology using cocultures of epithelial and immune cells: a technical update., Microbes Infect. 2 (2000) 1119–1124. [11]E. Gullberg, M. Leonard, J. Karlsson, A.M. Hopkins, D. Brayden, A.W. Baird, P. Artursson, Expression of specific markers and particle transport in a new human intestinal M-cell model, Biochem. Biophys. Res. Commun. 279 (2000) 808-813. [12]A. des Rieux, E. G. Ragnarsson, E. Gullberg, V. Préat, Y. J. Schneider, P. Artursson, Transport of nanoparticles across an in vitro model of the human intestinal follicle associated epithelium, Eur J Pharm Sci. 25 (2005) 455-465.[13]D. Lo, W. Tynan, J. Dickerson, M. Scharf, J. Cooper, D. Byrne, D. Brayden, L. Higgins, C. Evans, D.J. O’Mahony, Cell culture modeling of specialized tissue: identification of genes expressed specifically by follicle-associated epithelium of Peyer’s patch by expression profiling of Caco-2/Raji co-cultures, Int. Immunol. 16 (2004) 91–99. [14]A. des Rieux, V. Fievez, I. Théate, J. Mast, V. Préat, Y.-J. Schneider, An improved in vitro model of human intestinal follicle-associated epithelium to study nanoparticle transport by M cells., Eur. J. Pharm. Sci. 30 (2007) 380-391. [15]I. Hubatsch, E.G.E. Ragnarsson, P. Artursson, Determination of drug permeability and prediction of drug absorption in Caco-2 monolayers., Nat. Protoc. 2 (2007) 2111–2119.[16]D.J. Brayden, S. Maher, B. Bahar, E. Walsh, Sodium caprate-induced increases in intestinal permeability and epithelial damage are prevented by misoprostol, Eur. J. Pharm. Biopharm. 94 (2015) 194–206. [17]A. Beloqui A, D. J. Brayden, P. Artursson, V. Préat, A. des Rieux,A human intestinal M-cell-like model for investigating particle, antigen and microorganism translocation. Nat Protoc. 12 (2017) 1387-1399.[18]A. Buda, C. Sands, M. A. Jepson, Use of fluorescence imaging to investigate the structure and function of intestinal M cells. Adv Drug Deliv. Rev. 57 (2005)123-134.[19]I. Martinez-Argudo, M. A. Jepson, Salmonella translocates across an in vitro?M?cell model independently of SPI-1 and SPI-2, Microbiology, 154 (2008) 3887-3394.[20] R. Finn, T. Ahmad, E. T. Coffey, D. J. Brayden, A. W. Baird,, A. Boyd, Translocation of vibrio parahaemolyticus across an in vitro M cell model. FEMS Microbiology Letters. 350 (2014) 65-71.[21]M. Miyake, E. Ragnarsson, D. Nakai, P. Artursson, The pro-inflammatory cytokine interleukin-6 regulates nanoparticle transport across model follicle-associated epithelium cells, J. Pharm. Sci. 105 (2016) 2099-2104. [22] P. Chaikhumwang, D. Nilubol, A. Tantituvanont, P. Chanvorachote,A new cell-to-cell interaction model for epithelial microfold cell formation and the enhancing effect of epidermal growth factor. Eur. J. Pharm. Sci. 106 (2017) 49-61. [23] F. Araujo, C. Pereira, J. Costa, C. Barrias, P. L. Granja, B. Sarmento, In vitro M-like cells genesis through a tissue-engineered triple-culture intestinal model. J. Biomed. Mater. Res. B Appl. Biomater. 104 (2015) 782-788 (2015).[24]C. Schimpel, B. Teubl, M. Absenger, C. Meindl, E. Fr?hlich, G. Leitinger, A. Zimmer, E. Roblegg, Development of an advanced intestinal in vitro triple culture permeability model to study transport of nanoparticles. Mol. Pharm. 11 (2014) 808-818.Figure legendsFig. 1. Design of the three M cell models. (A) In Model A, adapted from [9], Caco-2 cells were seeded on the underside (ap) of inverted membrane inserts with a silicone wrap (black) for 12 h and then re-oriented to the normal configuration; Raji B cells were added to the inner cup (bl) after 15-16 days and co-cultures were grown for a further 5 days. (B) In Model B [11] Caco-2 cells were seeded in the inner cup (ap) and grown for 15-16 days, whereupon Raji B cells were added to the outer basolateral compartment (bl) and grown for a further 5 days. (C) In Model C [14], Caco-2 cells were seeded in the normally-oriented cup side of the filter (ap) and grown for 4-6 days before being inverted and placed in a large petri dish with internal medium levels at the height of the filter for a further 8-9 days. Raji B cells added to the compartment formed with silicone tubing (bl) on the up-turned basolateral side and co-cultured for a further 5 days.Fig. 2. (A) Papp of [14C]-mannitol was measured after 5 days of co-culture (Day 21 over 120 min for each model. (B) TEER of co-cultures at Day 21 compared to respective Day 16 mono-culture values before B cell addition for each model. Results are expressed as mean ± SEM of three independent studies, each group in quintuplicate. *P < 0.05 compared to respective Day 16 TEER values of monocultures before B cells addition; ***P < 0.001 for Model C co-cultures versus matched monocultures at Day 21.Fig. 3. Latex particle translocation across co-cultures of the three models over 120 min. (A) 0.5 ?m (B) 1.0 μm. Data is presented as fold increase in particle translocation in co-cultured monolayers over mono-cultures of respective models. (C) Inter-model comparison of particle numbers translocated across co-cultures. Results are expressed as mean ± SEM of three independent studies, each in quintuplicate. *P < 0.05, **P < 0.01, ***P < 0.0001, NS = not significant.Fig 4. Translocation of S. typhimurium across co-cultures of the three models. Mono- and co cultures were apically-infected with S. typhimurium at an MOI of 20-25 for 120 min. Data is presented as the fold increase of bacterial translocation in co-cultured monolayers over mono-cultures of respective models over 120 min.*P < 0.05 compared to respective monoculture.?Data presented is from three independent studies with N = 3-5 per group.Fig 5. Secondary cell layers on the opposite side of filters in co-cultures. (A) The surface area covered by secondary monolayers was quantified for each M cell model (in both mono- and co-cultures). A significantly high proportion of the surface of filter membrane in Model A (79%) was covered by secondary cell layers (P < 0.01). (B) Representative H & E stained sections of M cell models show the presence of normal monolayers on the apical side (ap) of the filter (f) and secondary cell layers on the basolateral side (bl). Arrows depict position of maximum height of monolayer used to calculate ratio of true: secondary cell layers. For every insert, 5-7 sections were obtained. Scale bar = 10 ?m.Fig 6. Representative micrographs of ultrastructural morphology of co-cultures by TEM at Day 21. Co-cultures (left and middle column panels for low and high power). M-like cells (arrows) show loss and/or re-arrangement of microvilli. Mono-cultures from each model (right columns): apical membranes with microvilli displaying typical brush-border morphology.Fig. 7. Prevention of secondary cell layers on filters double-coated with Matrigel? and seeded with Caco-2 cells (Model B) Transwell? were coated once (1X) or twice (2X) prior to seeding Caco-2 cells. (A) TEER values of mono-cultures and co-cultures grown on single- or double-coated membranes. *P < 0.05, **P < 0.01 and ***P < 0.0001. Mean ± SEM (N=3). (B) Representative images after 5 days of co-culture. Membranes were fixed and stained with H & E and depict apical side (ap) of the filter (f) and secondary cell layers on the basolateral side (bl). Scale bar = 25 ?m. Fig 8. Effect of media conditioning on proliferation and viability of Raji B cells used in Model B. Apical and basolateral media of Caco-2 monolayers co-cultured with Raji B cells was replenished and conditioned according to a modified protocol. (A) Proliferation and (B) viability of Raji B cells during co-culture. **P < 0.01 and ***P < 0.0001 for conditioned vs non-conditioned. Mean ± SEM (N=3).Fig 9. Impact of Raji B passage on M-like cell function during co-culture. The influence of successive passages of Raji B was determined by the degree of particle translocation across co-cultures. Results were expressed as a relative percentage of the first passage, passage 6 (P6). The fifth passage was not determined due to insufficient N numbers. *P < 0.05 and ***P < 0.0001. Mean ± SEM (N=3).Fig 10. Particle transport across mono- and co-cultures of Model B with standard or improved culture conditions. The double coated filter protocol and the media-conditioning regimen (improved) was compared to the standard method. Particles translocated across the improved co-cultures were increased compared to co-cultures created using standard conditions. (A) 0.5 ?m (B) 1.0 μm. **P < 0.01, NS = not significant Mean ± SEM (N=3).Table 1. Features of the three M-like cell co-culture models from data in source publications. M cell modelsCaco-2 Transwell? Duration of mono-culture/ orientationDuration of co-cultureCo-culture conditionsKerneis, (Model A [9])Caco-2,(clone 1) Polycarbonate, 6.5mm diameter, 3.0 ?m pores (Costar-Corning cat. number 3415)14 days (monolayer grown on underside of filter, normal orientation)1-7 daysFreshly isolated murine PP lymphocytes* (60% B cells, 40% CD3 T cells); 106 cells; (added on cup side of insert, facing basolateral side of Caco-2 monolayer)Gullberg, (Model B, [11])Caco-2, passage 30-40 (ECACC)Polycarbonate, 12mm diameter, 3.0 ?m pores (Costar-Corning cat. number 3402) 14 days (monolayer grown in normal orientation on normal side of filter)4-6 daysRaji B cells (ATCC); 5x105 cells added to basolateral bathDes Rieux, (Model C, [14])Caco-2 (clone 1) passage 12-30Polycarbonate, diameter 12mm, 3.0 ?m pores (Costar-Corning cat. number 3402)Monolayer grown in normal orientation on the cup side for 3-5 days; then for 9-11 days inverted in petri dish.5 daysRaji B cells (ATCC); 5x105 cells added to inverted basolateral side with silicone wrap ECACC: European Collection of Animal Cell Cultures, ATCC: American Type Culture Collection. *Raji B cells were used in Model A in the current study instead of murine PP lymphocytes in order to help standardize comparisons.Table 2. Summary comparison of M cell models with a qualitative attempt to rank orderModelTEER Papp [14C]-mannitol Nanoparticle translocationSalmonella translocationHistology (TEM)Kerneis(Model A)Reducing trend but no statistical changeIncreasing trend but no statistical changeStatistical increase over mono-culturesTrend for an increase over mono-culturesTruncated microvilli(qualitative)Gullberg, (Model B)Reducing trend but no statistical changeIncreasing trend but no statistical changeStatistical increase over mono-culturesStatistical increase over mono-culturesTruncated microvilli (qualitative)Des Rieux, (Model C)Statistical decrease over mono-culturesStatistical increase over mono-culturesStatistical increase over mono-culturesTrend for an increase over mono-culturesTruncated microvilli (qualitative)Rank order C > A=BC > A=BC > B > AB > A = CA = B = CThe most significant changes in models were in particle translocation. Significant changes are marked by italics. ................
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