Organ Culture of Arterial Conduits from Heart-Beating Donors

Background: The organ culture is an in vitro method for studying the vascular wall biology. The value of organ culture is twofold; it retains the in vivo structural tissue architecture while preserving the interaction between vascular cell subtypes and extracellular matrix components. Objectives: The present study is aimed at establishing whether putative resident vascular progenitors i) remain in niche niched within human vascular segments after collection in tissues banking facilities and ii) are able to develop an intimal lining in long-term cultures without VEGF support. Materials and methods: Human fresh arterial vascular conduits were collected from heart-beating donors (n. 3) following 72 hrs maintenance in an antibiotic mixture; before culturing, samples were processed for light microscopy (LM) and transmission electron microscopy (TEM); TUNEL assay and ultrastructural investigation were used to establish the degree of cell preservation; the remaining tissue was cut in 3 x 3 mm slices and cultured for up to 70 days in medium containing 5% of FBS. The samples were processed for LM and TEM weekly. Results: Before culturing, TUNEL assay revealed that the vast majority of the vascular wall cells had significant nuclear damage; this feature was confirmed by TEM. At 4 day of culture, TEM showed the presence of a few structurally preserved cells in correspondence with the adventitia layer; after 42 days, several undifferentiated cells with a high N/C ratio and prominent nucleoli were seen at this same location. LM revealed that spindle-shaped, epithelioid cells migrated near the inner and outer surfaces of the arterial segments; an almost continuous endothelial-like lining became evident starting after 56 days of culture. Immunostaining for CD34 surface molecule was negative in the adventitial lining and focally positive in the intimal side. Conclusion: This study demonstrates that human vascular conduits, after collection in tissues banking facilities, still contain a resident cell population which is particularly resistant to physical and chemical stresses; under plain tissue culture conditions these cells are able to expand and migrate toward the arterial surfaces and give origin to a pseudo-intimal lining. Noteworthy is the fact that we can exclude any contribute of hematopoietic cells to this phenomenon that, in this case, is dependent only on putative resident progenitors.