Date of Award
Doctor of Philosophy (PhD)
Lloyd Wolfinbarger, Jr.
Mark S. Elliot
Paul H. Ratz
The purpose of this research was to determine the effects of cryopreservation on the physical, biochemical and cellular aspects of heart valve tissue. The present study demonstrated that radiolabeled proline accumulation provides a quantitative estimate of total cellular metabolic viability. Radiolabeled inulin was demonstrated to be a useful marker for extracellular tissue space. Assessment of metabolic viability of the cellular population of leaflet tissue as a function of procurement and processing revealed that proline accumulation declined with increasing warm and cold ischemic times. Amphotericin B and streptomycin were toxic to the cells. Cefoxitin, lincomycin, polymyxin B, vancomycin and penicillin had no apparent effect on viability of cells in heart valve tissue. The study also revealed that antibiotic solution (amphotericin B, cefoxitin, polymyxin B, lincomycin, vancomycin) utilized to disinfect tissue was able to kill E. coli, S. aureus, S. epidermidis, S. pneumoniae, C. peifiingens, but not S. faecalis after these bacteria were incubated with antibiotic solution for 24 or 48 hrs at 4°C. The cryoprotectants, dimethyl sulfoxide and glycerol were shown to reduce cellular metabolic viability when heart valve leaflets were incubated in RPMI 1640 medium supplemented with 10% fetal calf serum and either 10% Me2SO or 15% glycerol over 180 minutes at both 4°C and 37°C. Incubation of tissue for 10 minutes in concentrations of Me2SO and glycerol greater than 5% resulted in a significant reduction in proline accumulation at both 4°C and 37°C. The results revealed that penetration rates of both Me2SO and glycerol at 37°C were faster than at 4°C. After step-wise dilution to remove the cryoprotectants, there was still 0.25 M Me2SO and 0.2 M glycerol in the porcine aortic conduit tissue, respectively. The results demonstrated that cryopreserved human heart valve tissue, plunged into liquid nitrogen for as little as 5 minutes, appears to have numerous microffactures over the surfaces of the tissue, penetrating into the collagen / proteoglycan matrix. Thawing at 37°C and 42°C resulted in low percentage of nonviable cells, whereas thawing at 4°C, 20°C, or 75°C resulted in much higher percentage of nonviable cells.
Assessment of the tendency of heart valve tissue to "calcify" was performed by measuring the ability of the tissue to induce alkaline phosphatase activities in fibroblast cell cultures. Porcine heart valve tissue with prolonged warm ischemic time, cold ischemic time and amphotericin B treatment induced significantly higher alkaline phosphatase activities than fresh porcine heart valve tissue.
"Optimization of Cryopreservation Protocols for Cardiovascular Tissue"
(1992). Doctor of Philosophy (PhD), dissertation, Biological Sciences, Old Dominion University, DOI: 10.25777/vf4w-tv77