Spontaneous activation of macrophages in response to inflammation is a key part of innate immunity and host defense. Macrophages represent a heterogeneous population of cells with different phenotypic profiles performing distinct functions in host defense. Although a spectrum of macrophage activation stages exists in an inflamed region, the effect of local physical conditions on the heterotypic activation of macrophages is unknown. Here, we introduce an in vivo fluid-matrix interface analogous culture platform, an asymmetric microenvironment, facilitating the formation of macrophage aggregates (MAs). Macrophages were self-assembled to form MAs of ∼100 μm diameter at the collagen matrix-medium interface upon phorbol-12-myristate-13-acetate treatment. The macrophages within the half-embedded MAs into the matrix were heterogeneously activated, resulting in inhomogeneous cell-cell and cell-matrix interactions within the aggregates. Our demonstration may aid in a better understanding of the acquisition of macrophage heterogeneity in response to tissue-specific microenvironments.