Characterisation of tissue microenvironment by the direct culture of mesenchymal stem cells on tissue sections

One ultimate goal of tissue engineering is to construct functional tissues and organs from isolated cells and fabricated materials. To achieve this, it is important to understand how extracellular environments affect and control cell behaviours. Although many studies have characterised the contributions of physical and biochemical factors in extracellular environments to cell physiology, these works often fail to mimic the complexity of the native tissue microenvironment. We have developed a novel experimental system to evaluate the interactions of cells and extracellular microenvironment. We discovered that cryosections of Bovine Achilles tendon display intricate textural details on the surface, and hypothesised that these surfaces represent the internal tissue environments to which endogenous cells are exposed. By manipulating the orientation of sectioning, we could create section surfaces with highly different ultrastructures. Since these sections were carved from the same piece of tendon, their biochemical compositions were identical. To assess how cells responded to these surfaces, we directly cultured mammalian cancer cell lines and human mesenchymal stem cells (MSC), on these sections. Morphology, adhesion and proliferation of cells were highly dependent on the tissue architecture they were exposed to. In particular, cells seeded on the longitudinal sections (LS) of tendon, but not cross sections (CS), adopted a highly elongated and aligned morphology. Time-lapse microscopy revealed that MSC on both LS and CS initially projected filopodia to all directions, but only cells on LS spread along one orientation. Remarkably, MSC cultured on LS, but not CS, expressed protein biomarkers characteristic of tenocytes, suggesting that LS contained biological cues that instructed MSC to commit to the tenogenic lineage. To delineate the nature of this signal, we prepared PDMS replicas using tendon sections as the mould. The resulting replicas, which faithfully copied the physical shape of tendon sections, but not the biochemical composition, promoted a small degree of cell elongation, but failed to induce MSC differentiation into tenocyte-like cells. This suggests that although the physical structure of tissue microenvironment may be enough to modulate cell shape and morphology, specific biochemical molecules, when presented in a correct orientation, are required to drive stem cell differentiation. This study demonstrated how biophysical and biochemical information in the extracellular microenvironment intertwined to form a unique cell type specific niche that influences the fate of cells within a tissue. Direct culture of cells on sectioned tissues is a simple and useful model system for the study of this process.