Development of the rat mesentery culture model for the multi-system investigation of microvascular network growth
Models that mimic angiogenesis are extremely valuable for elucidating underlining mechanisms and pre-clinical development of therapies. Angiogenesis, defined as the growth of new blood vessels from preexisting vessels, is a multi-cellular process that involves the temporal and spatial coordination between endothelial cells, pericytes, nerves, growth factors, and macrophages. A need exists for biomimetic models that bridge the gap between the mechanistic control of in vitro constructs and the multi-system physiological environment of in vivo models. To meet this need our lab has introduced the rat mesentery culture model as top down approach with intact microvascular networks and a nearly two-dimensional view. Previous development of the model has proven its time-lapse, angiogenic, and lymphangiogenic capabilities. The goal of this work is to advance the model to include the maintenance of peripheral nerves in culture and develop it as a platform for aging and cell therapy studies. The first aim of this study was to expand the rat mesentery culture model to maintain nerves and the spatiotemporal relationship between nerves and blood vessels in culture. We developed a nerve culture media to prevent regression of nerves. Nerve alignment was maintained at the network feeding arteriole and capillary plexus levels. We further demonstrated the presence of neurotransmitter precursors was preserved. We demonstrate for the first time the ability to maintain adult peripheral nerves in an ex vivo model. For the second aim of this study, we developed an aging rat mesentery culture model as a basis for investigating differences in angiogenesis across age groups. We demonstrated that impaired angiogenesis associated with advanced age could be recovered to adult-like levels with serum and individual growth factor stimulation. The discovery of increased vascular island frequency in aged tissues reveals that the method of angiogenesis for older networks can differ. These results establish the rat mesentery culture model as a method for studying aging effect on angiogenesis. The objective of the third aim was to demonstrate the capability of the rat mesentery culture model to study stromal vascular fraction therapy. We developed a protocol to isolate the SVF from adipose tissue and transplant onto the mesentery. We identified unique patterns of vasculogenesis and increased vascular coverage. We confirmed that this increase in vascular area was a combination of the vasculogenesis of SVF, proangiogenic effect on host vessels, and incorporation of SVF into the growing host vessels. We used the aging model in developed in the second aim to show that adult SVF on adult tissue has the greatest therapeutic potential. These results display the investigative potential of the rat mesentery culture model in cell therapy research. This work establishes for the first time, to the best of our knowledge, an ex vivo model capable of maintaining adult peripheral nerves. We demonstrate that angiogenesis can be rescued in aging scenarios. The results, for the first time, reveal the effect that SVF therapy has on preexisting networks as well as how it integrates during microvascular remodeling.