Role of soluble factors and three-dimensional culture in in vitro differentiation of intestinal macrophages.

To examine the factor(s) involved in differentiation of intestinal macrophages (IMACs) using a recently established in vitro model. To test whether soluble or membrane bound factors induce IMAC-differentiation, freshly elutriated monocytes (MO) were incubated with conditioned media or cell membranes of intestinal epithelial cells (IEC) or cultured with IEC in transwell systems. To determine the importance of an active migration of MO, three-dimensional aggregates from a 1:1-mixture of MO and IEC were examined by immunohistochemistry and flow cytometry. Apoptosis was examined by caspase-3 Western blots. Extracellular matrix production in differentiation models was compared by immunohistochemistry. IMAC differentiation was observed in a complex three-dimensional co-culture model (multicellular spheroid, MCS) with IEC after migration of MO into the spheroids. By co-culture of MO with conditioned media or membrane preparations of IEC no IMAC differentiation was induced. Co-culture of MO with IEC in transwell-cultures, with the two cell populations separated by a membrane also did not result in intestinal-like differentiation of MO. In contrast to IEC-spheroids with immigrating MO in mixed MCS of IEC and MO only a small subpopulation of MO was able to survive the seven day culture period. Intestinal-like differentiation of MO in vitro is only induced in the complex three-dimensional MCS model after immigration of MO indicating a role of cell-matrix and/or cell-cell interactions during the differentiation of IMACs.