Mucus Producing Cell Model of the Intestinal Epithelium: Robustness toward Simulated Intestinal
Media
L. Saaby1, J. Liu2, M. Fano1, A. Mullertz3
1
Bioneer A/S, 2University of Copenhagen, 3Bioneer:FARMA
Purpose
The human epithelial colorectal adenocarcinoma cell line, Caco-2, is widely used as a model of the absorptive intestinal epithelium.
One shortcoming of the Caco-2 cell cultures, is the lack of mucus secretion. Previously a co-culture model of Caco-2 cells and the
mucus-secreting goblet cell line HT29-H was established, retaining barrier properties similar to Caco-2 cell monolayers while also
secreting a visible mucus layer [1].
Often, transport experiments across in vitro cell monolayers are performed in Hanks Buffered Salt Solution (HBSS) based buffer
systems. Due to the simple nature of these buffer systems they poorly mimic the complexity of human intestinal fluids. However,
simulated intestinal fluids are known to be incompatible with in vitro cellular systems. The purpose of the present work was therefore
to investigate and compare the robustness of a Caco-2/HT29-MTX co-culture model Caco-2 cell monolayers towards different
simulated fed-state intestinal media.
Methods
Using a Design of Experiment approach seven fed state simulated intestinal media were prepared in Hank’s buffered saline solution
(HBSS) supplemented with bovine serum albumin (BSA, 0.05 %) and MES (10 mM) (Figure 1).
For exposure studies, Caco-2 cells and HT29-MTX-E12 cells at a ratio of 1:1, or Caco-2 cells alone, were seeded onto Transwell®
inserts and cultured for 21 days. Cell monolayers were incubated with 7 fed state media and a control HBSS based buffer on the apical
side for 2 hours at 37 °C and circular rotation. The basolateral compartments contained HBSS buffer. Transepithelial electrical
resistance (TEER) was measured prior to and after exposure to the media. Subsequently, the cells were washed in HBSS and an apical
to basolateral transport experiment with 14C-mannitol (1 μCi) was completed. Samples were taken from the basolateral compartment
at t=15, 30, 45 and 60 minutes and counted in a liquid scintillation counter. All experiments were done in triplicate in three passages.
For visualization of mucus, cell monolayers were incubated with 1 % Alcian Blue 8GX (pH 2.5) on the apical side for 5 minutes.
Subsequently, cells were washed twice with PBS and investigated by microscopy.
Results
Three (N1, N3 and N5) of the 7 fed state media, containing the lowest amounts of taurocholate (TC), did not affect the barrier
properties of both monolayers independent of the monoglyceride level (Figure 2). In contrast, incubation with media N4, N6 and N7,
containing the highest amounts of TC, caused a significant change in the barrier properties of the monolayers of both co-culture and
Caco-2 cells seen as a significant increase in mannitol transport and drop in TEER-values after exposure. Incubation with media N2
only caused a slight increase in mannitol transport and had no effect on TEER values across both types of monolayers. In general, the
effect of incubation with the different fed state media was similar between monolayers of the co-culture model and monolayers of
Caco-2 cells. Thus, the results indicate that the mucus layer on the co-culture model does not provide a protective barrier for the cells.
Visualization of the mucus layer on co-culture monolayers showed that the secreted mucus does not cover the entire monolayer, but
are rather found as “islands” of different sizes scattered throughout the monolayers.
Conclusion
In the present study, incubation of the monolayers with the different fed state simulated intestinal media had similar effects on the
barrier properties of the co-culture model and Conclusions
In the present study, incubation of the monolayers with the different fed state simulated intestinal media had similar effects on the
barrier properties of the co-culture model and Caco-2 cell cultures. This indicates that the mucin secreted on the co-culture model does
not provide a protective barrier. In accordance, Alcian Blue staining and microscopy showed that the secreted mucus did not cover the
entire monolayer.