Autogenous endothelial seeding (AES) of vascular prostheses (VP) in dogs creates an endothelial cell (EC) lining, reduces VP-platelet interactions, and increases patent rates in small caliber VP. But a clinical AES method requiring long lengths of patients' peripheral veins for use as EC sources would be unacceptable. Whether AES can be used in humans may depend on how many EC capable of growth can be harvested from small vein segments. The goal of this study is to develop a harvest method which obtains over 100,000 EC per cm2 of vein area in a state capable of immediate initiation of growth. Adult human venous EC have a growth capacity of 14+ doublings theoretically adequate for AES, but present harvest methods using crude bacterial collagenases (CBC) are unreliable and only obtain about 21% of available EC and obtain these in a damaged state causing delayed initition of growth. Both EC loss from venospasm during surgical dissection and EC damage from undefined proteolytic components of impure commercial CBC probably cause this low efficiency. Harvesting other vascular wall cells with partially purified bacterial collagenase (PBC) gives higher yields of viable cells with better preservation of receptor-mediated responses. Our hypothesis is that EC harvest efficiency for AES cna be similarly improved by using PBC for harvest to selectively digest basement membranes together with use of papaverine treatment of the vein to prevent venospasm and EC loss during dissection. To do this new methods for complete purification of the components of CBC and an assay to set CBC and PBC solutions to equal levels of activity in lysis of basement membranes will be used to create EC harvest solutions. The study will then: i)Compare EC harvest efficiency using PBC versus CBC harvest solutions at concentrations equal in basement membrane lysis activity, ii)Compare the efficiency of harvests done with and without papaverine treatment of the vein before surgical dissection, and iii)Utilize the best harvest solution and vein preparation technique in design of a kit for rapid and efficient performance of EC harvest in a clinical or operating room setting with a minimum of extra personnel and apparatus.
