A novel microfluidic device and analytical approach for migration studies of heterogeneous cell populations under interstitial flow.

Distinct anti-inflammatory macrophage (M2) subtypes, namely M2a and M2c, are reported to modulate the tissue repair process tightly and chronologically by modulating fibroblast differentiation state and functions. To establish a well-defined three-dimensional (3D) cell culture model to mimic the tissue repair process, we utilized THP-1 human monocytic cells and a 3D collagen matrix as a biomimetic tissue model. THP-1 cells were differentiated into macrophages, and activated using IL-4/IL-13 (M_IL-4/IL-13) and IL-10 (M_IL-10). Both activated macrophages were characterized by both their cell surface marker expression and cytokine secretion profile. Our cell characterization suggested that M_IL-4/IL-13 and M_IL-10 demonstrate M2a- and M2c-like subtypes, respectively. To mimic the initial and resolution phases during the tissue repair, both activated macrophages were co-cultured with fibroblasts and myofibroblasts. We showed that M_IL-4/IL-13 were able to promote matrix synthesis and remodeling by induction of myofibroblast differentiation via transforming growth factor beta-1 (TGF-β1). On the contrary, M_IL-10 demonstrated the ability to resolve the tissue repair process by dedifferentiation of myofibroblast via IL-10 secretion. Overall, our study demonstrated the importance and the exact roles of M2a and M2c-like macrophage subtypes in coordinating tissue repair in a biomimetic model. The established model can be applied for high-throughput platforms for improving tissue healing and anti-fibrotic drugs testing, as well as other biomedical studies.

Visualizing the dynamic behaviors of immune cells in living tissue has dramatically increased our understanding of how cells interact with their surroundings, contributing important insights into mechanisms of leukocyte trafficking, tumor cell invasion, and T cell education by dendritic cells, among others. Despite substantial advances with various intravital imaging techniques including two-photon microscopy and the generation of multitudes of reporter mice, there is a growing need to assess cell interactions in the context of specific extracellular matrix composition and microvascular functions, and as well, simpler and more widely accessible methods are needed to image cell behaviors in the context of living tissue physiology. Here we present an antibody-based method for intravital imaging of cell interactions with the blood, lymphatic, and the extracellular matrix compartments of the living dermis while simultaneously assessing capillary permeability and lymphatic drainage function. Using the exposed dorsal ear of the anesthetized mouse and a fluorescence stereomicroscope, such events can be imaged in the context of specific extracellular matrix proteins, or matrix-bound chemokine stores. We developed and optimized the method to minimize tissue damage to the ear, rapidly immunostain for multiple extracellular or cell surface receptors of interest, minimize immunotoxicity with pre-blocking Fcγ receptors and phototoxicity with extracellular antioxidants, and highlight the major dermal tissue structures with basement membrane markers. We demonstrate differential migration behaviors of bone marrow-derived dendritic cells, blood-circulating leukocytes, and dermal dendritic cells, with the latter entering sparse CCL21-positive areas of pre-collecting lymphatic vessels. This new method allows simultaneous imaging of cells and tissue structures, microvascular function, and extracellular microenvironment in multiple skin locations for 12 hours or more, with the flexibility of immunolabeling in addition to genetic-based fluorescent reporters.

Collagen alignment is one of the key microarchitectural signatures of many pathological conditions, including scarring and fibrosis. Investigating how collagen alignment modulates cellular functions will pave the way for understanding tissue scarring and regeneration and new therapeutic strategies. However, current approaches for the fabrication of three‐dimensional (3D) aligned collagen matrices are low‐throughput and require special devices. To overcome these limitations, a simple approach to reconstitute homogeneous 3D collagen matrices with adjustable degree of fibril alignment using 3D printed inclined surfaces is developed. By characterizing the mechanical properties of reconstituted matrices, it is found that the elastic modulus of collagen matrices is enhanced with an increase in the alignment degree. The reconstituted matrices are used to study fibroblast behavior to reveal the progression of scar formation where a gradual enhancement of collagen alignment can be observed. It is found that matrices with aligned fibrils trigger fibroblast differentiation into myofibroblasts via cell contractility, while collagen stiffening through a crosslinker does not. The results suggest the impact of collagen fibril organization on the regulation of fibroblast differentiation. Overall, this approach to reconstitute 3D collagen matrices with fibril alignment opens opportunities for biomimetic pathological‐relevant tissue in vitro, which can be applied for other biomedical research.

Adipose tissue is contemplated as a dynamic organ that plays key roles in the human body. Adipogenesis is the process by which adipocytes develop from adipose-derived stem cells to form the adipose tissue. Adipose-derived stem cells’ differentiation serves well beyond the simple goal of producing new adipocytes. Indeed, with the current immense biotechnological advances, the most critical role of adipose-derived stem cells remains their tremendous potential in the field of regenerative medicine. This review focuses on examining the physiological importance of adipogenesis, the current approaches that are employed to model this tightly controlled phenomenon, and the crucial role of adipogenesis in elucidating the pathophysiology and potential treatment modalities of human diseases. The future of adipogenesis is centered around its crucial role in regenerative and personalized medicine.