Histochemical detection of mucus secreting cells in a stabilized in vitro CACO2/HT-29 co-culture model

The interactions between nutrients and intestine have been studied up to now by assays performed on in vitro human cell cultures. The main limitations of this approach are represented by both the difficulty to handle and the differences/complications compared to the physiological condition. Recently, we optimized an innovative model in which two human intestinal cell lines, i.e. Caco2 and HT-29, were co-cultured. Caco2 cell line is mostly absorptive and do not secrete mucus, while HT-29 are a heterogeneous population, comprising scattered enterocyte elements and mucus secreting cells. To mimic the human intestinal epithelium, the co-culture was set up with a ratio of 70/30 Caco2 / HT-29 respectively, starting from the parental cell populations differentiated as previously described (1, 2). The present work was designed to study the time course of the mucus secretion in two different cell growth conditions: i) in a standard culture conditions and ii) in presence of an excess of nutrients. In both conditions, co-culture was harvested at confluence (T0) and at 3, 7, and 15 days post-confluence (T3, T7, and T15, respectively). In the standard group, the culture medium was changed every four days, while in the excess group on alternate days from T0. Cells were seeded in 24 well plate inserts and fixed in 4% paraformaldehyde. The membranes were removed from the inserts by means of forceps and placed in embedding cassettes. Samples were dehydrated through an ascending series of ethanol, embedded in paraffin, cut with a microtome, and PAS stained. In standard conditions, together with the non-mucus-secreting cell type, which are always present, the mucus-producing cells increased since T7, while at T15 these cells were scattered. These observations are compared with both the excess group and previously obtained ultrastructural results. As the mucus presence is relevant not only for the barrier function but also for the nutrient permeability, the present co-culture model enlighted the morpho-functional changes induced by the modifications of the cell growth conditions.