In vitro Self-organized Mouse Small Intestinal Epithelial Monolayer Protocol

Rat small intestinal epithelial cell lines have been established in vitro and subcultured serially for periods up to 6 mo. These cells have an epithelioid morphology, grow as monolayers of closely opposed polygonal cells, and during the logarithmic phase of growth have a population doubling time of 19--22 h. Ultrastructural studies revealed the presence of microvilli, tight junctions, an extensive Golgi complex, and the presence of extracellular amorphous material similar in appearance to isolated basement membrane. These cells exhibit a number of features characteristic of normal cells in culture; namely, a normal rat diploid karyotype, strong density inhibition of growth, lack of growth in soft agar, and a low plating efficiency when seeded at low density. They did not produce tumors when injected in syngeneic animals. Immunochemical studies were performed to determine their origin using antisera prepared against rat small intestinal crypt cell plasma membrane, brush border membrane of villus cells and isolated sucrase-isomaltase complex. Antigenic determinants specific for small intestinal epithelial (crypt and villus) cells were demonstrated on the surface of the epithelioid cells, but they lacked immunological determinants specific for differentiated villus cells. An antiserum specifically staining extracellular material surrounding the cells cultured in vitro demonstrated cross-reactivity to basement membrane in rat intestinal frozen sections. It is concluded that the cultured epithelioid cells have features of undifferentiated small intestinal crypt cells.

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Murine Norovirus Transcytosis across anIn VitroPolarized Murine Intestinal Epithelial Monolayer Is Mediated by M-Like Cells

Journal of Virology ◽

10.1128/jvi.02378-13 ◽

2013 ◽

Vol 87 (23) ◽

pp. 12685-12693 ◽

Cited By ~ 31

Author(s):

Mariam B. Gonzalez-Hernandez ◽

Thomas Liu ◽

Luz P. Blanco ◽

Heather Auble ◽

Hilary C. Payne ◽

...

Keyword(s):

Viral Entry ◽

Cell Monolayer ◽

Small Animal ◽

Intestinal Epithelial ◽

Murine Norovirus ◽

Small Animal Model ◽

Intestinal Epithelial Barrier ◽

M Cell ◽

Epithelial Monolayer

Noroviruses (NoVs) are the causative agent of the vast majority of nonbacterial gastroenteritis worldwide. Due to the inability to culture human NoVs and the inability to orally infect a small animal model, little is known about the initial steps of viral entry. One particular step that is not understood is how NoVs breach the intestinal epithelial barrier. Murine NoV (MNV) is the only NoV that can be propagatedin vitroby infecting murine macrophages and dendritic cells, making this virus an attractive model for studies of different aspects of NoV biology. Polarized murine intestinal epithelial mICcl2cells were used to investigate how MNV interacts with and crosses the intestinal epithelium. In thisin vitromodel of the follicle-associated epithelium (FAE), MNV is transported across the polarized cell monolayer in the absence of viral replication or disruption of tight junctions by a distinct epithelial cell with microfold (M) cell properties. In addition to transporting MNV, these M-like cells also transcytose microbeads and express an IgA receptor. Interestingly, B myeloma cells cultured in the basolateral compartment underlying the epithelial monolayer did not alter the number of M-like cells but increased their transcytotic activity. Our data demonstrate that MNV can cross an intact intestinal epithelial monolayerin vitroby hijacking the M-like cells' intrinsic transcytotic pathway and suggest a potential mechanism for MNV entry into the host.

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Bovine dairy complex lipids improve in vitro measures of small intestinal epithelial barrier integrity

PLoS ONE ◽

10.1371/journal.pone.0190839 ◽

2018 ◽

Vol 13 (1) ◽

pp. e0190839 ◽

Cited By ~ 9

Author(s):

Rachel C. Anderson ◽

Alastair K. H. MacGibbon ◽

Neill Haggarty ◽

Kelly M. Armstrong ◽

Nicole C. Roy

Keyword(s):

Epithelial Barrier ◽

Intestinal Epithelial ◽

Intestinal Epithelial Barrier ◽

Barrier Integrity ◽

Complex Lipids ◽

Small Intestinal

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Transcellular transport of calcium and inorganic phosphate in the small intestinal epithelium

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1981.240.6.g409 ◽

1981 ◽

Vol 240 (6) ◽

pp. G409-G416 ◽

Cited By ~ 5

Author(s):

H. Murer ◽

B. Hildmann

Keyword(s):

Brush Border ◽

Inorganic Phosphate ◽

Membrane Vesicles ◽

Calcium Flux ◽

Intestinal Epithelial ◽

Transcellular Transport ◽

Lateral Membrane ◽

Sodium Flux ◽

Small Intestinal

Transport mechanisms involved in the small intestinal handling of inorganic phosphate and calcium have been studied by different in vitro methods during the last few years. In concordance with studies on intact epithelial preparations, studies with brush-border and basal-lateral membrane vesicles isolated from the small intestinal epithelial cell revealed that transcellular calcium and inorganic phosphate fluxes are coupled to transcellular sodium flux, i.e., secondary active via coupling to the primary active sodium flux. A sodium-coupled mechanism in the brush-border membrane leads to cellular accumulation of inorganic phosphate. A sodium-coupled mechanism leads to extrusion of calcium from the cell into the serosal interstitium. A primary active transport mediated by the Ca-ATPase and located in the basal-lateral membrane also exists for calcium. Regulation of transcellular phosphate and calcium flux proceeds via altered influx rates at the luminal cell pole.

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Differentiation of rat small intestinal epithelial cells by extracellular matrix

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1988.254.3.g355 ◽

1988 ◽

Vol 254 (3) ◽

pp. G355-G360 ◽

Cited By ~ 5

Author(s):

K. M. Carroll ◽

T. T. Wong ◽

D. L. Drabik ◽

E. B. Chang

Keyword(s):

Extracellular Matrix ◽

Basement Membrane ◽

Glucose Absorption ◽

Cell Maturation ◽

Intestinal Cell ◽

Synthetic Activity ◽

Intestinal Epithelial ◽

Intestinal Cell Line ◽

Small Intestinal

The role of extracellular matrix as a determinant of intestinal cell maturation was explored by growing a normal, but immature, rat small intestinal cell line (IEC-6) on basement membrane extract from Engelbreth-Holm-Swarm (EHS) sarcoma cells (ECM). Grown on plastic or glass, these cells are relatively immature and proliferate rapidly. In contrast, cells on ECM attached more rapidly, stopped proliferating, and rapidly organized into multicellular complex structures. Ultrastructurally, cells grown on ECM displayed significantly more mitochondria, rough endoplasmic reticulum, apical microvilli, and complex golgi apparatus, consistent with greater maturity and synthetic activity. By indirect immunofluorescence, sucrase, alkaline phosphatase, and cellular apolipoprotein B were present in cells grown on ECM only. In contrast to cells grown on glass, these cells also demonstrated Na-dependent glucose absorption, a function unique to mature villus cells (7). We conclude that the basement membrane may be a key determinant of intestinal epithelial cell maturation. The development of a mature villuslike intestinal cell in vitro may have wide application for future studies of induction and regulation of intestinal maturation and function.

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Development of an endogenous epithelial Na+/H+exchanger (NHE3) in three clones of Caco-2 cells

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1999.277.2.g292 ◽

1999 ◽

Vol 277 (2) ◽

pp. G292-G305 ◽

Cited By ~ 14

Author(s):

Andrzej J. Janecki ◽

Marshall H. Montrose ◽

C. Ming Tse ◽

Fermin Sanchez de Medina ◽

Alain Zweibaum ◽

...

Keyword(s):

Membrane Domains ◽

Intestinal Epithelial ◽

Major Mechanism ◽

Kinase C ◽

Cell Clones ◽

Nhe1 Activity ◽

Epidermal Growth ◽

Small Intestinal ◽

Vitro Model

Expression of endogenous Na+/H+exchangers (NHEs) NHE3 and NHE1 at the apical (AP) and basolateral (BL) membrane domains was investigated in three clones (ATCC, PF-11, and TC-7) derived from the human adenocarcinoma cell line Caco-2. In all three clones, NHE1 was the only isoform detected at the BL domain during 3 to 22 postconfluent days (PCD). In clone PF-11, the BL NHE1 activity increased up to 7 PCD and remained stable thereafter. Both NHE1 and NHE3 were found at the AP domain at 3 PCD and contributed 67 and 33% to the total AP Na+/H+exchange, respectively. The AP NHE3 activity increased significantly from 3 to 22 PCD, from 93 to 450 μM H+/s, whereas AP NHE1 activity decreased from 192 to 18 μM H+/s during that time. Similar results were obtained with the ATCC clone, whereas very little AP NHE3 activity was observed in clone TC-7. Surface biotinylation and indirect immunofluorescence confirmed these results and also suggested an increase in the number of cells expressing NHE3 being the major mechanism of the observed overall increase in NHE3 activity in PF-11 and ATCC clones. Phorbol 12-myristate 13-acetate (PMA, 1 μM) acutely inhibited NHE3 activity by 28% of control, whereas epidermal growth factor (EGF, 200 ng/ml) stimulated the activity by 18%. The effect of PMA was abolished by the protein kinase C (PKC) inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, suggesting involvement of PKC in the PMA-induced inhibition of NHE3. Similar magnitude of inhibition by PMA and stimulation by EGF was observed at 7 and 17 PCD, suggesting the development of regulatory mechanisms in the early postconfluent period. Taken together, these data suggest a close similarity of membrane targeting and regulation of endogenous NHE3 between Caco-2 cells and native small intestinal epithelial cells and support the usefulness of some Caco-2 cell clones as an in vitro model for studies on physiology of NHE3 in the intestinal epithelium.

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A new in vitro model using small intestinal epithelial cells to enhance infection of Cryptosporidium parvum

Journal of Microbiological Methods ◽

10.1016/j.mimet.2014.07.017 ◽

2014 ◽

Vol 106 ◽

pp. 47-54 ◽

Cited By ~ 16

Author(s):

Eunice A. Varughese ◽

Christina L. Bennett-Stamper ◽

Larry J. Wymer ◽

Jagjit S. Yadav

Keyword(s):

Epithelial Cells ◽

Cryptosporidium Parvum ◽

In Vitro Model ◽

Intestinal Epithelial Cells ◽

Intestinal Epithelial ◽

Small Intestinal ◽

Vitro Model

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Primary Murine Small Intestinal Epithelial Cells, Maintained in Long-Term Culture, Are Susceptible to Rotavirus Infection

Journal of Virology ◽

10.1128/jvi.74.12.5597-5603.2000 ◽

2000 ◽

Vol 74 (12) ◽

pp. 5597-5603 ◽

Cited By ~ 40

Author(s):

Kristine K. Macartney ◽

Daniel C. Baumgart ◽

Simon R. Carding ◽

Jeffery O. Brubaker ◽

Paul A. Offit

Keyword(s):

Epithelial Cells ◽

Mhc Class Ii ◽

Intestinal Epithelial Cells ◽

Intestinal Cell ◽

Intestinal Epithelial ◽

Long Term Culture ◽

Intestinal Cell Line ◽

Small Intestinal

ABSTRACT We describe a method for long-term culture of primary small intestinal epithelial cells (IEC) from suckling mice. IEC were digested from intestinal fragments as small intact units of epithelium (organoids) by using collagenase and dispase. IEC proliferated from organoids on a basement-membrane-coated culture surface and remained viable for 3 weeks. Cultured IEC had the morphologic and functional characteristics of immature enterocytes, notably sustained expression of cytokeratin and alkaline phosphatase. Few mesenchymal cells were present in the IEC cultures. IEC were also cultured from adult BALB/c mice and expressed major histocompatibility complex (MHC) class II antigens for at least 48 h in vitro. Primary IEC supported the growth of rhesus rotavirus (RRV) to a greater extent than a murine small intestinal cell line, m-ICcl2. Cell-culture-adapted murine rotavirus strain EDIM infected primary IEC and m-ICcl2 cells to a lesser extent than RRV. Wild-type EDIM did not infect either cell type. Long-term culture of primary murine small intestinal epithelial cells provides a method to study (i) virus-cell interactions, (ii) the capacity of IEC to act as antigen-presenting cells using a wide variety of MHC haplotypes, and (iii) IEC biology.

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