In vivo isolation and characterization of telocytes using supplemented biomatrices

The leeches (Hirudo verbana) have proven to be a good model for deciphering basic biological processes for two main reasons: first of all, they have a reduced dimension and, despite a relative anatomical simplicity, share with vertebrates the complexity of immunological mechanisms and wound-healing processes; secondly, in animal kingdom there is a remarkable evolutionary conservation of biological responses, cell types, cellular mechanisms, and molecules. One of the most phylogenetically conserved system, from lower invertebrates to man, is the innate immune system that, strictly interacting with neuro-endocrine one, guarantee a powerful protection to organisms. In invertebrates, in addition to a plethora of cytokines, a wide range of immunocytes such as macrophages, NK cells, granulocytes and a new type of cells, the telocytes, are involved. Leech telocytes (TCs) are stromal cells engaged in surveillance and protection spread in various tissues and strategically localized among resident cells, nearby the capillaries and the nerve endings. These cells, contacting via gap junctions, are organized in an extensive three-dimensional network, and show cell-cell contacts with other cell types and interactions with collagen bundles of connective tissue. The interactions among these and the other cells is obtained in two ways: physically, by direct cell–cell contacts and, chemically, via the release of microvesicles and exosomes, which can transport a variety of soluble factors involved in the regulation of different physiological processes. Leech-TCs originate from circulating precursor cells and, once activated in response to chemical or physical stimuli, are able to change their morphology and behaviour, moving towards the injured area to participate in repair/regenerative processes. To better characterize leech-TCs we have isolated and cultured these cells. The injection of an appropriate combination of Matrigel biopolymer, supplemented with selected factors in the leech H. verbana, has allowed to recruit these cells. While few migrating cells are present in control Matrigel specimens lacking factors, an increased number of cells in relation to the time elapsed from the injection of the supplemented biomatrix, colonize Matrigel specimens containing: Monocyte Chemoattractant Protein-1 (MCP-1/ CCL2) or IL-8 or Ribonuclease T2 (RNASET2).