Tracheal allotransplantation maintaining cartilage viability with long-term cryopreserved allografts

BACKGROUND: Cartilage viability of a cryopreserved tracheal allograft seems to affect graft function and durability. We previously reported the influence of warm ischemia and cryopreservation on cartilage viability of tracheal allografts. For the clinical application of tracheal allotransplantation, it is essential to preserve grafts for a long time. In this study, we assessed cartilage viability of tracheal allografts after long-term cryopreservation in transplantation models. METHODS: The tracheas were harvested from Lewis rats. The grafts were frozen to -80 degrees C in a programmable freezer immediately after being harvested and were then stored in liquid nitrogen (-196 degrees C) for different lengths of preservation (1, 2, 6, 9, 12, 18, and 24 months; n for each group = 8). Cartilage viability was evaluated by estimating proteoglycan synthesis. After harvest or thawing of the tracheas, the cartilage was labeled with 4 muCi/mL of Na2 35SO4. Specimens were then hydrolyzed in 0.5 mol/L NaOH, and a solution of the extracts was then counted by a liquid scintillation counter. 35Sulfur incorporation before and after cryopreservation was examined in each group. Tracheal allotransplantation was performed using Lewis rats as donors and Brown Norway rats as recipients. RESULTS: The average 35S incorporation in the cartilage before cryopreservation was 224 +/- 17 disintegrations per minute per milligram of tissue protein. The average 35S incorporation in the cartilage after cryopreservation decreased to 67% to 76% compared with that before cryopreservation. There were no significant differences among the groups in 35S incorporations after cryopreservation. Histologic examination after transplantation revealed normal tracheal cartilage in all groups. CONCLUSIONS: The viability of tracheal cartilage after cryopreservation decreased to 67% to 76%. There were no significant differences in viability of cartilage among the tracheas after different lengths of cryopreservation. Tracheal allotransplantation after long-term cryopreservation can be safely performed in the rat model.     

Preservation of brain tumor specimens for drug sensitivity testing

To examine the feasibility of shipment of brain tumor specimens to a central laboratory for drug sensitivity testing, an experimental gliosarcoma (9L) was grown subcutaneously in rats, harvested, stored, and disaggregated. Growth of the disaggregated tumor cells in monolayer culture was evaluated after storage for various times at 2 to 6 degrees C and 37 degrees C in saline and minimum essential medium. Growth potential was maintained for 24 hours when tumor specimens were stored under refrigerated conditions and was best maintained when specimens were stored in saline. Specimens stored at 37 degrees C grew best when stored in minimum essential medium, but growth potential was lost after 12 hours unless the specimens were refrigerated. Shipment of tumor specimens to a central laboratory for drug sensitivity testing appears to be feasible, since under most circumstances, specimens should reach the laboratory for processing within 24 hours of their removal. Storage and transport of specimens in saline on wet ice appears to be optimal.     

Cryopreservation of vascularized ovary: an evaluation of histology and function in rats

Cryopreservation of organs has been investigated to sustain the reproductive function of patients undergoing sterilizing chemotherapy and radiotherapy or reproductive surgery. A modified protocol for whole organ cryopreservation was described and the outcome of cryopreservative ovaries was evaluated, and apoptosis of cryopreservative cells stored for different time period and the viability of cryopreserved cells stored at different temperature was examined in rats. Lewis rat ovarian grafts were perfused for 30 min at 0.35 ml/min with M2 medium containing 0.1M fructose and increasing concentrations of 0-1.5M dimethylsulfoxide, cooled to -140 degrees C controlled by a computerized program, and stored in liquid nitrogen (-196 degrees C) for 24 hours. After being thawed, ovaries were transplanted to syngeneic recipients after bilateral oophorectomy. Graft functions were monitored postoperatively. The major findings were that: 1) A 100% survival rate of rat ovaries was achieved in this study. Ovarian hormone secretion recovered in 80% rats which had received cryopreservative ovarian grafts. Postoperative serum estradiol levels in the cryopreservative graft group were lower than in the sham surgery control, but much higher than in the bilateral oophorectomy group. 2) Histological examination of cryopreservative ovarian grafts showed preantral and antral follicles. Two gestations were obtained. 3) Estradiol levels remained low in ovariectomized rats while in the oophorectomized rats given cryopreservative ovarian grafts levels started to rise after 14 +/- 3 days. 4) The average viability in the cells from cryopreservative ovary organ (-196 degrees C) was about 71 +/- 18% compared to 90 +/- 9% of fresh cells. This success should encourage further improvement of cryopreservative techniques for large organs.     

Prolonged-fresh preservation of intact whole canine femoral condyles for the potential use as osteochondral allografts.

Defects in articular cartilage are often repaired with fresh osteochondral grafts. While fresh allografts provide viable chondrocytes, logistic limitations require surgical implantation within seven days of graft harvest. Here, we provide information on cold preservation of whole intact osteochondral materials that retains cartilage cell viability and function, and histologic and biochemical integrity for 28 days. Canine femoral condyles were obtained and stored at 4 degrees C for 14, 21 or 28 days. At the end of the storage period, cartilage was assessed for cell viability, 35S uptake, proteoglycan content and histologic parameters. The most noticeable histologic change was reduced Safranin-O near the cartilage surface with 14 days of cold preservation, but had recovered with 21 and 28 days. Cartilage thicknesses did not vary significantly. Cell viability was >95% at 14 days, 75-98% at 21 days and reduced to 65-90% at 28 days. Cell function measures showed that the level of 35SO4 incorporation was suppressed in samples stored at 4 degrees C. However, no significant differences were seen among groups at 14, 21 or 28 days of cold preservation. This data has implications for tissue banking protocols for osteochondral allograft material obtained for transplantation suggesting that cold preserved allograft material be implanted within 28 days.     

Limit of warm ischemia time before cryopreservation in rat tracheal isografts

Background. The viability of cadaveric tracheal grafts undergoing cryopreservation is still unclear. We evaluated the limit of warm ischemia time before cryopreservation in rat tracheal isografts.

Methods. Each isograft was harvested from donor rats 0 to 48 hours (0, 6, 12, 18, 24, and 48 hours) after circulatory arrest, immersed in the preservative solution, and stored in a deep freezer until reaching −80°C and then was kept in liquid nitrogen for 3 months. Heterotopic transplantation into the omentum was performed after the isografts were thawed. Graft morphology 3 months after transplantation was assessed.

Results. The stepwise increase of warm ischemia time significantly reduced graft survival. A prolonged period of warm ischemia had a degenerative effect on both the epithelium and cartilage. The morphology of the epithelium and cartilage in isografts undergoing warm ischemia for less than 18 hours was better preserved, whereas it deteriorated in isografts undergoing warm ischemia for more than 24 hours.

Conclusions. We thus conclude that the permissible period of warm ischemia before 3-month cryopreservation to maintain tracheal isograft viability is 18 hours in rats.     

Preservation of canine segmental pancreatic autografts: cold storage versus pulsatile machine perfusion

The reliability of cold storage and pulsatile machine perfusion for preservation of open-duct segmental pancreatic grafts for 24, 48, and 72 hours was determined in a canine autograft model. The tail (left limb) of the pancreas on a pedicle of splenic artery and vein was transplanted to the pelvis with vascular anastomoses to the iliac vessels and the remainder of the pancreas was excised. Twenty nonpreserved grafts functioned immediately (the recipients became normoglycemic, plasma glucose less than 150 mg/dl), 16 long term (80%). Collins solution (CS, osmolality 300 mosm/kg) and a silica gel filtered plasma solution (SGF-I, osmolality 430 mosm/kg) were compared for cold storage at 4 degrees C. The long-term functional survival rate for grafts stored for 24 hours in SGF-I was 9/12 (75%) and in CS was 8/12 (67%). When graft preservation time was extended to 48 hours, 9/12 (75%) stored in SGF-I and 4/10 (40%) in CS functional long term. The preservation failure rates were 0% in grafts stored in SGF for up to 48 hours, but were 20% and 50% for grafts stored in CS for 24 and 48 hours. Storage at 72 hours was not satisfactory; even when SGF-I was used, the preservation failure rate was 57%. SGF was also used as the perfusate for pancreas preservation on the Mox-100 machine at a pressure of 30 mm Hg (achieved by leaving the port opposite the connected pancreas open). Lower pressures gave insufficient perfusion and higher pressures led to severe edema of grafts. Mean (+/- S.E.) flow (ml/min) through the grafts were 4.5 +/- 0.3 initially, 6.5 +/- 0.7 at 24 hours and 6.3 +/- 0.9 at 48 hours. With SGF-I only 6/12 (50%) of grafts perfused for 24 hours and only 1/8 (12%) for 48 hours functioned long term. With a modified perfusate (SGF-II, osmolality 470-500 mosm/kg) the results were slightly improved; 7/12 (58%) grafts perfused for 24 hours and 5/10 (50%) for 48 hours functioned long term. The mean (+/- S.E.) flow rate (ml/min) was 5.0 +/- 0.6 in grafts that functioned long term and was 8.3 +/- 1.0 in those that failed due to preservation complications. Mean (+/- S.E.) peak serum amylase levels (I.U./L.) were similar in recipients of fresh grafts (3953 +/- 365) and those stored hypothermically in SGF (4226 +/- 327), but were significantly lower in recipients of machine perfused grafts (2988 +/- 228). Although the percentage of successful transplants varied according to the preservation techniques, the mean (+/- S.E.) IVGTT K-values in recipients with functioning grafts were similar between the groups (-1.44 +/- .19 to -1.82 +/- .17%). The pure pancreas preservation failure rates with machine perfusion were between 30% and 40% at 24 to 48 hours, compared to 0% in those simply stored in cold SGF for 24 to 48 hours. We conclude that pancreas preservation by cold storage in high osmolar silica gel filtered plasma is more reliable than pulsatile machine perfusion and provides sufficient time to complete the logistical maneuvers necessary for clinical pancreas transplantation from cadaver donors.     

Optimal conditions for the storage of split-thickness skin at 4C

Split-thickness human skin must, out of necessity, be stored for days to years prior to usage as a burn wound covering. This study was designed to utilized three different parameters of tissue viability in order to determine optimum conditions for skin survival at 4 degrees C for various periods of time in RPMI-1640 media. Viability was determined by 1) trypan blue dye exclusion of trypsinized basal cells (TBC), 2) 14C-leucine incorporation into TBC, and 3) in vitro skin explant growth. Electron microscopy was also done on selected samples. METHODS: Split-thickness (0.2-0.3 mm) cadaveric skin (obtained within 24 hours of death) was divided into 6 tubes containing RPMI-1640 (pH 7.3) supplemented with 10% Nu-serum and 25 mM Hepes. The ratio of skin to media varied from 0.2 to 8.0 cm2/ml. The samples were then stored at 4 degrees C for up to 4 weeks with weekly changes of media. RESULTS: Following 7 days storage, optimal viability (as determined by averaging the viabilities obtained from the above assays) was found for samples stored at a ratio of 2 cm2/ml (90.3 +/- 4.2%). Samples stored at ratios between 1.0 and 6.0 cm2/ml all maintained acceptable viabilities (i.e., greater than 78%), while samples at either 0.2 or 8.0 cm2/ml were both about 57% viable. EM demonstrated edema in all samples. The viability of skin stored for 4 weeks at 2-4 cm2/ml decreased dramatically after 2 weeks. CONCLUSION: RPMI-1640 is an excellent medium for the storage of split-thickness skin at 4 degrees C, 2-4 cm2/ml, for up to 7 days. Because of the high viability retained in skin stored under these conditions, further long term storage in liquid nitrogen should provide good quality tissue for transplantation.     

Effect of storage temperature on cell viability in cryopreserved canine aortic, pulmonic, mitral, and tricuspid valve homografts.

We determined how long cryopreserved aortic, pulmonic, mitral, and tricuspid valve homografts could be stored in a deep freezer (-80 degrees C) without compromising fibroblast viability. Valves harvested from 20 anesthetized mongrel dogs were grouped into nonfrozen control, frozen and stored in liquid nitrogen (-196 degrees C), and frozen and stored in a deep freezer (-80 degrees C). Frozen groups were divided into subgroups and stored for 2, 4, 8, or 12 weeks. A leaflet of each valve was divided into three fragments, and fibroblast viability was analyzed by flow cytometry. Cell viability was defined as staining by fluorescent diacetate but not by propidium iodide. The viability of untreated control valves from all four sites was about 70%, decreasing to about 50% when treated with low doses of antibiotics. The viability of frozen valves stored in liquid nitrogen was about 45% without a significant difference among storage periods. The viability of valves frozen and stored in a deep freezer was significantly lower than for the liquid nitrogen group at 2 weeks for the mitral valve and at 4 weeks for other valves. These results suggest that homografts can be stored in a deep freezer for up to 2 weeks without deterioration.     

Evaluation of methemoglobin formation during the storage of various hemoglobin solutions

Many researchers are trying to develop a blood substitute based on chemically modified human hemoglobin. In the process of making such solutions, we were faced with the problem of determining the best storage conditions to minimize oxidation of the solutions between the time of manufacture and use. Samples of stroma-free hemoglobin, purified A0 hemoglobin, and various cross-linked hemoglobins were stored for 8-12 months at +4 degrees C -20 degrees C, and -80 degrees C and were analyzed periodically for formation of methemoglobin (MetHb). Various suspending solutions were evaluated for their effects on the rate of MetHb formation, and the approximate rates of MetHb production per month were calculated. Short-term storage of hemoglobin solutions (< 14 days) can be done at +4 degrees C, but extended storage should be done at -80 degrees C with quick thawing. Salts minimize the hemoglobin oxidation during the stress of freeze-thaw operations. Storage at -20 degrees C. presents further problems and should be avoided.     

Postmortem degradation of demineralized bone matrix osteoinductive potential. Effect of time and storage temperature

The osteoinductive growth factors present in demineralized bone are degraded by tissue enzymes. Storage of rat limbs at low temperature (4 degrees C) before harvesting of bones preserves the osteoinductive potential of such factors. Storage at room temperature for more than 24 hours causes the recovered bone matrix to be biologically inactive, presumably as the result of biodegradation.     

Long-term preservation of segmental pancreas autografts

Clinical pancreas transplantation is logistically difficult because of uncertainty concerning how long pancreas grafts can be preserved. We studied the viability of canine segmental (tail) pancreatic autografts transplanted after 24 to 72 hours of hypothermic preservation in either modified Collins' solution or modified silica gel filtered dog plasma (SGF). All grafts stored for 24 to 48 hours functioned immediately (plasma glucose less than 140 mg/dl in recipients). Grafts that failed did so between 2 and 7 days, and five dogs died with functioning grafts. The long-term functional success rate was 80% for fresh transplants; 67% and 40% for grafts stored in Collins' solution for 24 and 48 hours, respectively; and 75%, 75%, and 30% for grafts stored in SGF for 24, 48, and 72 hours, respectively. If technical failures are excluded. 50% of grafts stored in Collins' solution and 100% of grafts stored in SGF for 48 hours functioned long term. K values for the intravenous glucose tolerance test at 2 weeks in dogs with functioning grafts ranged from -1.44% to -1.78% and were similar in all groups. In conclusion, pancreatic grafts can be preserved for 48 hours by simple cold storage, but SGF is more reliable than Collins' solution (P = 0.015). Four human pancreas grafts were stored in SGF for 7 to 22 hours, one with and three without prior warm ischemia, and all three of the latter functioned after transplantation.     

A study on factors influencing the stability of total nutrient admixture

To study the stability of total nutrient admixture, glucose, amino acid, fat emulsion, electrolyte, and other nutrients in different combinations and concentrations were admixed and infused into 16 3-liter polyvinyl chloride bags, which at last were stored at 4 degrees C, 22-25 degrees C, 32 degrees C, respectively. Samples were inspected at 0, 12, 24 and 48 hours. The results showed that the admixture was stable when total concentration of mono cation was lower than 150 mmol/L, Mg2+ less than 3.4 mmol/L, and Ca2+ less than 1.7 mmol/L, stored at 4 degrees C for 48 hours, or at 22-25 degrees C for 24 hours. We conclude that PVC 3L container is safe for storage of TNA at least within 24 hours.     

Comparative study on biologic and immunologic characteristics of the pancreas islet cell between 24 degrees C and 37 degrees C culture in the rat

The aim of this study was to investigate the effect of culture at 24 degrees C on cell viability, cellular function, immunogenicity, and cytokine profiles of rat pancreatic islets. Pancreatic islets were isolated from Lewis rats and cultured at either 24 degrees C or 37 degrees C for 14 days. Islet recovery was counted as islet equivalents; islet viability was examined with fluorescent vital staining. Islet function was measured with a glucose stimulation test. Annexin V, and MHC class I and II expression were measured using flow cytometric assay for apoptosis and immunogenicity, respectively. Lymphocyte cell proliferation was examined with WST-1 proliferation assay. Cytokine profiles were analyzed with quantitative real time RT-PCR. All these parameters were measured on 1, 3, 5, 7 and 14 culture days after islet isolation. Islet recovery was higher in islets cultured at 24 degrees C than 37 degrees C without a change in viability. Insulin secretion after glucose stimulation was more effective in 24 degrees C culture conditions. Decreased apoptotic cell death was demonstrated in 24 degrees C cultured islets. Both MHC class I and II expression on islets and lymphocyte proliferation upon coculture with islets were less prominent in 24 degrees C cultured islets. TNF-alpha expression was lower in islets cultured at 24 degrees C than in islets cultured at 37 degrees C. Both IL-1beta and IL-10 cytokine expressions were similar under both culture conditions. This study demonstrated that cell recovery and function are increased in islets cultured at 24 degrees C than those at 37 degrees C with decreased antigenicity and proinflammatory cytokine expression.     

Immunogenicity, Antigenicity, and Endothelial Viability of Aortic Valves Preserved at 4°C in a Nutrient Medium

In patients undergoing aortic valve replacement, allograft valves stored at 4 degrees C in a nutrient medium have been associated with excellent immediate and long-term results. The effects of this method of prolonged storage on the antigenic, immunological and cellular characteristics of these grafts are incompletely understood. This study was designed to study these phenomena in rat aortic valves subjected to antibiotic sterilization and stored for up to 3 weeks in RPMI containing 10% fetal calf serum. Selected valves from Brown Norway rats were implanted heterotopically into the abdominal aorta of Lewis rats. Other valves were studied prior to transplantation. Antigenicity was determined by immunocytochemical staining using monoclonal mouse antibodies directed at Class I and Class II rat antigens. Immunogenicity was determined by duration of second-set skin graft survival following heterotopic aortic valve implant. Endothelial cell viability was determined by flow cytometric analysis of endothelial cells harvested from aortic valve allografts by collagenase digestion. Only fresh valves and valves stored for 1 day were positive for Class I antigens; no valves were positive for Class II antigens. Duration of skin graft survival was prolonged with greater duration of storage, but grafts remained immunogenic after 21 days of storage. Endothelial cell viability declined from 95% in the fresh valves to 64% after 21 days of storage. With prolonged duration of allograft valve storage at 4 degrees C, there is an attenuation of antigenicity, immunogenicity, and endothelial cell viability. Loss of endothelial cells may contribute to the changes in immunological responses to the valve allografts. The expression of antigens on the endothelial surface is not a reliable predictor of immunological response.     

Effect of Cooling, Freezing and Thawing Rates and Storage Conditions on Preservation of Human Spermatozoa

Human spermatozoa was relatively resistant to cooling shock. However, when diluted semen was cooled faster than 10 degrees C per minute from room temperature (RT) to 5 degrees C and rewarmed to RT, percentage motility and percentage alive of spermatozoa decreased when compared to the slower cooling rates (less than 5 degrees C/min). The optimum cooling rate from RT to 5 degrees C resulting in maximum survival of human spermatozoa was found to be 0.5 to 1 degree per minute when cooled from RT to 5 degrees C and subsequently frozen-thawed in liquid nitrogen (LN2). The optimal freezing rate of 10 degrees C per minute, from 5 degrees to -80 degrees C, resulted in higher survival of human spermatozoa than slower (1.1 degrees C/min) or faster (87.1 degrees C/min) freezing rates. Slow thawing in 20 or 35 degrees C air, on a dry bench, resulted in better survival than the other slower or faster thawing methods used. The temperature at which human semen samples were transferred to LN2 significantly influenced spermatozoa survival. Survival was higher when transferred at -30 degrees C or lower when compared with samples transferred at -15 degrees C or higher. However, maximal spermatozoa survival was obtained when the samples were transferred at -80 degrees C or lower. Transfer of human semen from LN2 to -25 to -30 degrees C and storage for 24 hours significantly reduced spermatozoa viability when compared with storage at 196 degrees C or -80 to -85 degrees C. No significant differences were found between storage temperatures of -80 to -85 degrees C and -196 degrees C in the maintenance of spermatozoa viability for up to 90 days.     

Heterotopic transplantation of cryopreserved tracheae in a rat model.

INTRODUCTION: The successful use of cryopreserved tracheal allografts in canine models suggests their use in humans. The grade of genetic difference, the mechanism of revascularisation and the method of cryopreservation are not clearly defined. The purpose of our study was to investigate the rejection of tracheal transplants in a standardised heterotopic rat model using different forms of cryopreservation. METHODS: Tracheae from Brown Norway rats were implanted into the omentum from Brown Norway rats or Lewis rats. We transplanted fresh isografts or allografts and pretreated isografts or allografts. Cryopreservation was performed in a medium containing 10% dimethyl sulphoxide at -80 degrees C for 28 days (I) or -196 degrees C for 84 days (II) or without medium at -80 degrees C for 28 days (III). The transplants were excised after 7 and 21 days, respectively. RESULTS: Histological examinations revealed normal structure and function of isografts after 21 days. In the cryopreserved isograft, the epithelium had disappeared and the tracheal lumen was partially obstructed by a non-compact fibrous tissue. In the fresh allografts, the epithelium was replaced by aggressive fibrous tissue, infiltrating the membranous part of the trachea and occluding the tracheal lumen. The cartilage was vital without any sign of rejection. In the cryopreserved allografts, the tracheal lumen was obstructed by dense fibrous tissue with an inflammatory reaction. The cartilage of cryopreserved allografts (II) and (III) had lost the nuclei corresponding to non-vital tissue. Only in the cryopreserved allografts (I) did we find nodular regeneration at the edges of the cartilaginous bow. CONCLUSIONS: The heterotopic transplantation model allows the study of the mechanisms leading to tracheal obstruction. Cryopreservation was found to have no clear advantage in reducing transplant immunogenicity. Cryopreservation leads to significant damage to the cartilage, the intensity of which is dependent on the mode of cryopreservation.     

Enhancement of osteoinduction by vitamin D metabolites in rachitic host rats.

Diaphyseal bone from normal Sprague-Dawley rats was delipidated in chloroform-methanol and demineralized in 0.6 N HCl at 4 degrees C. The bones were then implanted for 7-28 days into rats made rachitic by a low-phosphate, vitamin D-deficient diet (VDP-) for 3 weeks. Bones from VDP- and normal rats were also implanted into normal hosts. When normal rats were used as the host environment, a consistent sequence of cartilage induction and bone formation was observed. Demineralized rachitic bone (RB) implanted into normal host rats resulted in cartilage and bone induction similar to that seen for normal bone (NB) implants. Transmission electron microscopy of RB in normal hosts revealed morphologically normal chondrocytes and cartilage matrix with normal mineralization. In contrast, implantation of NB in VDP- hosts resulted in delayed chondrogenesis and lack of calcification. Furthermore, similar results were observed when RB was implanted into VDP- hosts. Treatment of VDP- hosts with either 1 alpha-hydroxyvitamin D3 or 24,25-dihydroxyvitamin D3 did not accelerate the sequential appearance of precartilage or cartilage. However, 24,25-(OH)2D3 administered alone or in combination with 1 alpha-OHD3 significantly increased the amount of calcified cartilage observed at 2 weeks postimplantation compared to implants from either untreated VDP-hosts or those treated only with 1 alpha-OHD3. New bone formation was observed at 4 weeks postimplantation in all vitamin D-treated groups as determined by von Kossa staining or direct electron microscope examination. There was no apparent difference in the quantitative or qualitative bone formed within the various vitamin D-treated groups. Serum calcium and phosphorus levels were lower and alkaline phosphatase levels were higher in VDP- hosts compared with normal animals or those treated with vitamin D metabolites. The results of this study show a reduction in the capacity of progenitor cells in VDP- rat hosts to respond to osteoinductive factor(s). This impaired response appears to be corrected by vitamin D metabolites.     

Orthotopic transplantation of the canine heart after prolonged preservation by simple immersion

The viability of the heart was assessed following orthotopic transplantation. Following coronary vascular washout with cold potassium-verapamil cardioplegia, the heart was removed, immersed in the same solution, and stored at 4 degrees C for 24 hours in Group I (6 animals), 36 hours in Group II (5 animals), and 48 hours in Group III (3 animals). All six animals in Group I and four of five animals in Group II maintained a stable recipient circulation for the acute phase of 2 hours after transplantation, without cardiopulmonary bypass. For the dogs in Group III, cardiopulmonary bypass was vital. Contraction band injury after transplantation was more frequently observed in the Group II grafts than those of Group I. We conclude that the combination of coronary vascular washout with cold potassium-verapamil cardioplegia and storage at 4 degrees C in the same solution may preserve the canine heart for up to 24 to 36 hours, as demonstrated by post-orthotopic transplantation function.     

Long-term preservation of the mammalian myocardium. Effect of storage medium and temperature on the vulnerability to tissue injury

Human heart preservation for transplantation commonly involves infusion of cold cardioplegic solutions and subsequent immersion in the same solution. The objectives of the present study were (1) to establish the temporal relationship between storage time (at 10 degrees C) and the postischemic recovery of function in the isolated rat heart, (2) to assess, by metabolic and functional measurements, whether storing the heart in fluid as opposed to moist air had any effect on the viability of the preparation, and (3) to ascertain the optimal storage temperature. Isolated rat hearts (at least 6 in each group) were infused for 3 minutes with St. Thomas' Hospital cardioplegic solution No. 2 at 10 degrees C, stored at 10 degrees C for 6, 12, 18, or 24 hours, and then reperfused at 37 degrees C. Mechanical function, assessed by construction of pressure-volume curves (balloon volumes: 20, 40, 60, 80, 100, and 120 microliters), was measured before ischemia and storage and after 60 minutes of reperfusion. Function deteriorated in a time-dependent manner; thus at a balloon volume of 60 microliters the recovery of left ventricular developed pressure was 84.2% +/- 5.3% after 6 hours (p = not significant when compared with preischemic control); 69.1 +/- 3.3% after 12 hours (p less than 0.05); 55.6% +/- 4.4% after 18 hours (p less than 0.05), and 53.0% +/- 6.8% (p less than 0.05) after 24 hours of storage. Other indices of cardiac function, together with creatine kinase leakage and high-energy phosphate content, supported these observations. Since the recovery of the left ventricular developed pressure balloon volume curves were essentially flat after 18 and 24 hours of storage, either 6 or 12 hours of storage were therefore used in subsequent studies. Comparison of storage environment (hearts either immersed in St. Thomas Hospital cardioplegic solution No. 2 or suspended in moist air at 10 degrees C for 6 or 12 hours) revealed no significant differences in functional recovery between the groups. Thus hearts recovered 94.9% +/- 3.5% and 113.7% +/- 12.4%, respectively, after 6 hours of storage and 71.6% +/- 2.4% and 54.2% +/- 7.9%, respectively, after 12 hours of storage. Enzyme leakage and tissue water gain were also similar in both groups of hearts. Finally, hearts (n = 6 per group) were subjected to 12 hours' storage at 1.0 degree, 5.0 degrees, 7.5 degrees, 10.0 degrees, 12.5 degrees, 15.0 degrees, and 20.0 degrees C.(ABSTRACT TRUNCATED AT 400 WORDS)