Evidence that temporary complete occlusion of splenic vessels prevents massive embolization and sudden death associated with intrasplenic hepatocellular transplantation

It has been reported elsewhere that liver cell suspensions injected at several locations retain some proper hepatic functions, significantly improve the survival rate of rats with different models of acute fulminant hepatic injury, correct some congenital enzyme deficiency diseases, and improve liver function in cirrhotic animals. Among several locations, the splenic parenchyma has been shown to be the most suitable place for hepatocellular transplantation. Unfortunately, infusion of cells into the splenic pulp is not without risk. In fact, portal hypertension and hepatic embolizations have been described after intrasplenic transplantation of hepatocytes or pancreatic islets or fragments. In addition, pulmonary hepatocyte embolizations have been observed in rats with spontaneous (unpublished observations) or surgically induced portosystemic shunts. In this work, we evaluate the efficacy of temporary occlusion of splenic vessels to prevent hepatic and pulmonary embolizations after liver cell transplantation into the spleen in portal hypertension cirrhotic rats with portosystemic shunts.     

Transplantation of Hepatocytes for Prevention of Intracranial Hypertension in Pigs With Ischemic Liver Failure

Intracranial hypertension leading to brain stem herniation is a major cause of death in fulminant hepatic failure (FHF). Mannitol, barbiturates, and hyperventilation have been used to treat brain swelling, but most patients are either refractory to medical management or cannot be treated because of concurrent medical problems or side effects. In this study, we examined whether allogeneic hepatocellular transplantation may prevent development of intracranial hypertension in pigs with experimentally induced liver failure. Of the two preparations tested—total hepatectomy (n = 47), and liver devascularization (n = 16)—only pigs with liver ischemia developed brain edema provided, however, that animals were maintained normothermic throughout the postoperative period. This model was then used in transplantation studies, in which six pigs received intrasplenic injection of allogeneic hepatocytes (2.5 × 10⁹ cells/pig) and 3 days later acute liver failure was induced. In both models (anhepatic state, liver devascularization), pigs allowed to become hypothermic had significantly longer survival compared to those maintained normothermic. Normothermic pigs with liver ischemia had, at all time points studied, ICP greater than 20 mmHg. Pigs that received hepatocellular transplants had ICP below 15 mmHg until death; at the same time, cerebral perfusion pressure (CPP) in transplanted pigs was consistently higher than in controls (45 ± 11 mmHg vs. 16 ± 18 mmHg; p < 0.05). Spleens of transplanted pigs contained clusters of viable hepatocytes (hematoxylin-eosin, CAM 5.2). It was concluded that removal of the liver does not result in intracranial hypertension; hypothermia prolongs survival time in both anhepatic pigs and pigs with liver devascularization, and intrasplenic transplantation of allogeneic hepatocytes prevents development of intracranial hypertension in pigs with acute ischemic liver failure.     

Monitoring of Intrasplenic Hepatocyte Transplantation for Acute-on-Chronic Liver Failure: A Prospective Five-Year Follow-Up Study

Background

Acute-on-chronic liver failure (ACLF) is defined as an acute deterioration of chronic liver disease. Intrasplenic hepatocyte transplantation is increasingly recognized as a treatment for liver failure and genetic metabolic liver diseases. We describe our experience of intrasplenic hepatocyte transplantation in a small cohort of patients as bridge therapy or as an alternative to orthotopic liver transplantation (OLT).

Methods

Seven patients with ACLF with an expected survival of less than 8 weeks were enrolled into the study. The donor hepatocytes were collected from 2 healthy males and cryopreserved. Donor hepatocytes were transplanted into the spleen of recipients via catheterization of the femoral artery. All patients were followed up for 5 years or to death.

Results

A total of (4.2–6.0) × 10¹⁰ hepatocytes were harvested from the 2 donors' livers and their survival after recovery from the frozen stock was 63% ± 2.8% and 73.5% ± 3.2%, respectively. Following intrasplenic hepatocyte transplantation, 3 patients fully recovered from liver failure, 1 survived and subsequently underwent OLT, and the remaining 3 patients died between 2.5 and 12 months after intrasplenic hepatocyte transplantation. At month 48 post–intrasplenic hepatocyte transplantation, living hepatocyte signals were observed in the spleen using magnetic resonance imaging (MRI) with gadobenate dimeglumine (Gd-BOPTA).

Conclusions

Intrasplenic hepatocyte transplantation is a promising therapy for liver failure that may reduce mortality rates among patients with end-stage liver disease awaiting OLT. Conceivably, intrasplenic hepatocyte transplantation may be considered an alternative to OLT for patients with acute liver failure. MRI (Gd-BOPTA) is a useful tool for detecting living hepatocytes in the spleen after intrasplenic hepatocyte transplantation.     

Auxiliary canine hepatic transplantation without portal blood flow

Auxiliary liver transplantation for acute reversible hepatic failure has been suggested as a way to provide temporary hepatic support. The major technical difficulty in such an operation is providing portal blood flow, although it is unclear if this is necessary for short-term function of such a graft. Three groups of five dogs each underwent common duct ligation and cholecystectomy. One week later all animals underwent an end-to-side portocaval shunt. In Group I dogs only a "sham" transplant was done while those in Group II also received an auxiliary hepatic graft without portal blood flow which had been perfused with iced Collins solution and immediately grafted. Group III received similar auxiliary hepatic grafts with 22 hours of cold ischemia time. Reductions in mean total serum bilirubin values to near normal, usually lasting for one week (Group I, 12.02 mg% +/- 1.23 SEM vs. Group II, 3.58 mg% +/- 0.52 SEM, p = 0.01) were observed. Although several livers stored for 22 hours demonstrated function, there was considerable evidence of ischemic injury.     

Technical aspects of segmental pancreatic autotransplantation in dogs

Heterotopic segmental pancreatic autotransplantation in dogs is more appropriate than allograft models for the investigation of several problems associated with transplantation. We have defined the anatomic variations of blood supply in the pancreatic tail and designed various modifications of vascular anastomosis to the iliac vessels in order to eliminate technical failures, such as thrombosis, as much as possible. In 187 of 240 dogs (77.9 percent), the pancreatic artery originated from the splenic artery and the pancreatic vein entered the splenic vein (normal anatomy). The main venous variation was direct confluence of the pancreatic and portal veins (12.1 percent), and the main arterial variation was origin of the pancreatic artery from the superior mesenteric artery (10 percent). Ninety-seven animals with normal anatomy qualified for a comparative study of seven methods of segmental pancreatic autotransplantation. Venous anastomoses were always performed in an end-to-side fashion between the splenic and external iliac veins. Arterial anastomosis techniques follow. Group I: interposition of the splenic artery into the external iliac artery; (14 days, failure rate 50 percent), Group II: end-to-end arterial anastomosis of the splenic artery to a long external iliac artery segment with the graft directed caudad, resulting in an acute curve to the vessel loop (8 dogs, failure rate 38 percent); Group III: end-to-end arterial anastomosis to a long external iliac artery with the graft directed cephalad, resulting in a gentle curve to the vessel loop (11 dogs, failure rate 36 percent); Group IV: end-to-end arterial anastomosis to a short external iliac artery stump (20 dogs, failure rate 20 percent); Group V: same as in Group IV with the addition of a distal splenic arteriovenous fistula (12 dogs, failure rate 17 percent); Group VI: end-to-side anastomosis of the splenic artery to the external iliac artery (31 dogs, failure rate 6 percent); Group VII: same as in Group VI but with the addition of an arteriovenous fistula of the distal splenic vessels (1 dog, failure rate 0). The end-to-side technique proved to be straight-forward and reliable. The low failure rate with this method allows metabolic preservation and other aspects of pancreatic transplantation to be studied and the results to be interpreted without the influence of a high complication rate from the operation itself.     

Route of hepatocyte delivery affects hepatocyte engraftment in the spleen

In laboratory animals, intrasplenic hepatocyte transplantation corrects the physiologic abnormalities associated with decompensated liver disease. The clinical experience with hepatocyte transplantation for cirrhosis has been disappointing when compared with laboratory experience. The route of hepatocyte delivery may influence hepatocyte engraftment and function. Outbred pigs were recipients of allogeneic pig hepatocytes. Donor hepatocytes were isolated by collagenase perfusion and labeled using 5(6)-carboxyfluorescein diacetate succinimidyl-ester (CMFSE). Cells were introduced into pig spleens by infusion through the splenic artery or by direct splenic puncture. Direct intrasplenic injection produced engraftment that was far superior to that obtained using splenic artery infusion. Splenic artery infusion produced a gastric erosion and large areas of splenic necrosis secondary to vascular occlusion with hepatocytes, whereas direct splenic injection was associated with clinically insignificant intraabdominal hemorrhage. The route of hepatocyte delivery may influence hepatocyte engraftment and explain the disparity in efficacy of hepatocyte transplantation between the laboratory and clinic.     

Intrasplenic transplantation of encapsulated hepatocytes decreases mortality and improves liver functions in fulminant hepatic failure from 90% partial hepatectomy in rats

BACKGROUND: Encapsulated cell therapy might be a promising approach to enable cell transplantation without immunosuppression. This study investigates the viability and hepatic function of hepatocytes encapsulated with alginate/poly-L-lysine in vitro and the effect of the intrasplenic transplantation of cultured encapsulated hepatocytes on survival in 90% hepatectomized rats as a preliminary step toward allogeneic hepatocyte transplantation without immunosuppression. MATERIALS AND METHODS: Rat hepatocytes were isolated and encapsulated using alginate/poly-L-lysine. Encapsulated hepatocytes were cultured for 28 days to measure cell viability, liver function, and morphology. Rats were treated with a 90% partial hepatectomy and then immediately underwent the intrasplenic transplantation of the cultured encapsulated hepatocytes, the capsule alone, or the allogeneic hepatocytes without the capsule. The survival rate, liver function, and cell morphology were assessed after transplantation. RESULTS: The cultured encapsulated hepatocytes maintained their viability and showed better metabolic activity than day 0 cultured encapsulated hepatocytes. The encapsulated cells strongly expressed albumin and were positive for periodic acid-Schiff staining. Electron microscopy demonstrated that the microencapsulated hepatocytes retained the structural elements of hepatic cytoplasm and nuclei. Intrasplenic transplantation of the encapsulated hepatocytes increased the survival rate and improved the hepatic function. Encapsulated hepatocytes transplanted into rat spleen survived well and retained their hepatic function. Moreover, dramatic liver regeneration was observed 48 hr after transplantation in the group that received intrasplenic transplantations of encapsulated hepatocytes. CONCLUSIONS: The intrasplenic transplantation of cultured encapsulated hepatocytes improved the survival rate of an acute liver failure rat model induced by a 90% partial hepatectomy.     

Transplantation of hepatocytes for treatment of surgically induced acute hepatic failure in the rat

Efficacy of transplanted hepatocytes was evaluated in rats with a surgically induced acute hepatic failure. After 75% liver resection and portacaval shunt, the intrasplenic or intraperitoneal injection of 20 million isolated fresh hepatocytes was shown to significantly reduce the mortality rate. These results confirm that the transplantation of isolated hepatocytes may prevent death in rats with acute hepatic failure, and suggest that hepatocyte transplantation acts by a mechanism of hepatic support.     

Role of activated carbon hemoperfusion in the recovery of livers exposed to ischemic damage

Adequate hepatic assistance by artificial liver support systems after ischemic damage would allow for the possibility of liver regeneration. This study assessed the efficacy of activated carbon hemoperfusion (ACH) for artificial liver support after ischemically induced hepatic failure. Severe liver failure was induced in 18 adult mongrel dogs by cross clamping of the portal vein and hepatic artery for 40 minutes after creation of a portacaval shunt. Group 1 (n = 8) animals served as untreated controls and survived for only two to 22 hours after the procedure (10.6 +/- 8.8 hours, mean +/- SD). A significant improvement in survival was seen in group 2 animals (n =10), which received one ACH treatment after ischemic injury (range of survival, two to more than seven days). These results encourage further development of this procedure for possible clinical application.     

Reversal of ischemic liver failure by intrasplenic liver cell transplantation

A model of ischemic hepatic failure in Sprague-Dawley rats (SD) with 100% mortality has been developed by one-hour occlusion of the right portal vein and hepatic artery followed by left 70% hepatectomy. The intrasplenic injection of 40 x 10(6) syngeneic adult or three-day neonatal single liver cell suspensions decreased the mortality from 100% to 50% and 36%, respectively. Mortality decreased with increasing time from the intrasplenic injection of neonatal liver cells to the time of acute hepatic ischemia. Mortality also decreased with increasing interval between hepatic ischemia and removal of the transplanted liver cells by splenectomy. Intrasplenic injection of graded doses of neonatal liver cells decreased mortality from 75% at a dose of 10 x 10(6) cells, to 36% at 160 x 10(6) cells. Treatment of neonatal liver cells with metabolic inhibitors did not significantly affect their ability to reverse acute hepatic ischemia.     

Extracorporeal liver perfusion system for successful hepatic support pending liver regeneration or liver transplantation: a pre-clinical controlled trial.

BACKGROUND: There is a well recognized need for a system capable of providing effective support for patients with hepatic failure pending liver regeneration or liver transplantation. Recent attempts of using bioartificial liver containing encapsulated porcine hepatocytes, the deployment of emergency whole liver, or hepatocyte transplantation are complex and not consistently successful. The technique of ex vivo hepatic perfusion developed and used clinically by Abouna in the 1970s, has now been redesigned in a perfusion circuitry that mimics the physiological conditions of a normal liver. Before clinical application of this system, a preclinical trial was carried out in dogs with induced hepatic failure. METHODS: Acute hepatic failure was induced in dogs by an end-to-side porto caval shunt, followed 24 hr later, by a 2-hr occlusion of the hepatic artery. All animals (n=18) were medically supported and were divided into three groups. In the control group (n=6) only medical support was used. In the experimental group (n=12) the animals were connected to the ex vivo liver support apparatus during acute hepatic failure via an AV shunt using a dog liver (n=6) or calf liver (n=6) (after a temporary extracorporeal bovine kidney transplant to remove preformed xeno antibody). Hepatic perfusion was carried out at 37 degrees C through the hepatic artery and portal vein at physiological pressures, and blood flow rate for 6-8 hr. RESULTS: All control animals died of progressive hepatic failure at 14-19 hr after clamping the hepatic artery. The animals treated with ex vivo liver showed remarkable clinical and biochemical improvement. Five animals survived for 36-60 hr. Another seven animals recovered completely and became long-term survivors with biochemical and histological evidence of regeneration of their own liver. Biopsy of the allogeneic ex vivo liver at the end of perfusion showed some interstitial edema. Similar biopsy of the xenogeneic calf liver showed only mild and delayed xenograft rejection, which was most likely due to removal of preformed xeno antibody by temporary transplantation of the calf kidney before liver perfusion. CONCLUSIONS: The observations and results obtained in this trial strongly confirm that extracorporeal perfusion through a whole liver, using the system described, is very successful and cost effective for the treatment of acute, but reversible hepatic failure, as well as serving as a bridge to liver transplantation. The time has come for this form of liver support technology to be reintroduced and widely used.     

犬辅助性原位肝左叶移植模型的建立

目的建立稳定可靠的辅助性肝移植动物模型。方法杂交犬14条8～25kg随机分两组。供体组肝脏均采用尸体肝左叶；受体组行标准左叶切除，供肝左叶原位移植于受体体内。结果供肝热缺血时间为零，冷缺血时间平均36．3min；灌注液的量平均2．96L，切取修剪肝左叶时间为23～40min。受体手术平均时间5．3h，平均出血量140ml，肝脏血管重建后红润柔软，5～11min内即见胆汁从胆管中溢出。受体组术后全部存活，围手术期未用任何血管活性药物、抗生素及免疫抑制剂。术后存活超过6h者5例．最长存活者达5．7d。结论犬是建立辅助性肝移植模型的理想动物。     

Liver transplantation after organ preservation with normothermic extracorporeal perfusion

OBJECTIVE: To study normothermic extracorporeal liver perfusion (NELP) as a means to preserve livers for transplantation and to reverse warm ischemic injury. SUMMARY BACKGROUND DATA: The authors provide experimental evidence that successful transplantation after 4 hours of normothermic extracorporeal liver perfusion is possible and as reliable as 4 hours of cold preservation in University of Wisconsin solution. NELP preserves liver function completely and can reverse 60 minutes of warm ischemic injury in non-heart-beating donors. METHODS: Thirty-six German Landrace pigs received transplants in six groups. Group 1 animals received direct transplantation. Group 2 received transplants after 4 hours of cold preservation with University of Wisconsin solution and Group 3 animals after 4 hours of NELP. Group 4 animals sustained 1 hour of warm ischemia before transplantation. Group 5 animals received transplants after 1 hour of warm ischemia and 4 hours of cold preservation and Group 6 animals after 1 hour of warm ischemia and 4 hours of NELP. RESULTS: All animals receiving livers treated by NELP survived more than 7 days after the transplant (Groups 3 and 6). In contrast, all animals in Group 5 developed primary graft nonfunction within 24 hours after transplantation. CONCLUSION: The technique of NELP holds the potential to keep a mammalian liver outside the body completely functional, possibly for more than 4 hours. NELP can be used for liver preservation before transplantation or for the use of organs from non-heart-beating donors.     

Techniques for intrasplenic hepatocyte transplantation in the large animal model

Background: The preferred therapy for acute and chronic liver insufficiency and severe heritable disorders of liver metabolism is whole-organ transplantation. However, due to the shortage of organ doproposed, including transplantation of normal allogeneic hepatocytes. Recently, it has been reported that many hepatocytes transplanted into the spleen migrated to the liver. We therefore carried out a series of large-animal experiments to reexamine the intrasplenic route and to develop a method for large-scale hepatocellular transplantation in pigs. Methods: Allogeneic porcine hepatocytes were transplanted using the following routes: (1) retrograde injection of cells via the splenic vein, (2) intraarterial injection of cells, (3) direct intrasplenic injection of cells after laparotomy, (4) percutaneous intrasplenic injection of cells under laparoscopic control, (5) laparoscopic intrasplenic injection of cells. The number of cells injected varied from 2 × 10⁹ to 10 × 10⁹ cells. Results: Of all the methods tested, only direct intrasplenic injection of 2 bln of cells was found to be compatible with survival. However, even with this ``small' number of cells (2% original liver mass), there was a significant risk of spleen infarction, perisplenic adhesion formation, and portal vein thrombosis. The laparoscopic approach was found to be reliable, simple, and safe. Conclusion: Even though the spleen is considered by many authors the optimal site for hepatocellular transplantation, transplantation of cells in a number needed to support the failing liver may be associated with significant complications, morbidity, and mortality. Received: 2 March 1996/Accepted 17 May 1996     

Therapeutic efficacy of the transplantation of isolated hepatocytes in rats with surgically induced acute hepatic failure: a study of the mechanism

In this study, the beneficial effect of intrasplenic transplantation of hepatocytes or splenocytes was shown in animals with 75% hepatectomy and portacaval shunt but not in animals with total dehepatization by hepatic vascular exclusion. No enhancement of the phagocytic activity was observed in the animals with 75% hepatectomy and portacaval shunt after injection of hepatocytes or splenocytes. This study confirms the efficacy of hepatocytes for the treatment of experimental liver failure but shows that nonhepatic cells may be equally as effective. Metabolic activity of the transplanted cells and stimulation of the phagocytic activity of the reticuloendothelial system probably do not explain the therapeutic effect of the transplanted cells.     

Auxiliary transplantation of part of the liver improves survival and provides metabolic support in pigs with acute liver failure

In pigs subtotal ischemic liver cell necrosis was induced 4 days after auxiliary transplantation of 60% of the liver of an MLC-compatible donor (ATPL group, n = 13). In control animals (n = 14) temporary liver ischemia was preceded by division of the hepatic ligaments and creation of an end-to-side portacaval shunt. In the ATPL group six animals died of gastric hemorrhage, intestinal strangulation, or sepsis. The remaining seven animals survived in excellent condition until sacrifice 26 days after the induction of liver ischemia. Excellent graft function was demonstrated by uptake and excretion of 99mTc-HIDA at cholescintigraphy, ammonia detoxification, synthesis of clotting factors and glucohomeostasis. EEG recordings in the animals that underwent transplantation did not change from preischemic levels. Evidence of hepatic regeneration was found in the transplanted livers but could not be demonstrated in the damaged host livers. The control animals died in coma within 72 hours. These results indicate that auxiliary transplantation of a partial liver provides metabolic support and improves survival in animals with induced acute liver failure.     

肝动脉和门静脉高位分支双重处理对犬肝脏结构和功能的影响

目的 观察肝动脉和门静脉分支双重处理后犬肝脏结构和功能的改变。方法 杂种犬25只。随机分成假手术对照组和实验组,实验组进行肝动脉左支结扎和门静脉左支缩窄70％。分别于术后1,2,3,7和14d处死动物,观察肝脏大体结构的变化,分别称取左右半肝脏的质量,检测肝脏功能。光学显微镜下观察肝脏显微结构改变,电镜下观察肝脏超微结构改变。结果 肝动脉左支结扎和门静脉左支缩窄70％后,左半肝脏呈进行性萎缩变小,右半肝脏则成比例地代偿性增生,在整个观察过程中,全肝的总质量维持恒定。肝脏功能在术后2-3d时有轻度异常,很快即恢复正常。光镜下左半肝脏在早期出现少量的点、片状坏死。坏死周围出现大量的肝细胞凋亡,右半肝脏的肝小叶结构保持完整,术后肝细胞的增殖明显,有较多的分裂相存在。电镜下左半肝脏可见到典型的肝细胞凋亡,晚期呈明显纤维化改变。结论 肝动脉左支结扎和门静脉左支缩窄70％后。左半肝脏由于肝细胞大量凋亡呈进行性萎缩变小,右半肝脏则成比例地代偿性增生肥大,全肝的总质量和肝脏功能保持正常。     

The effect of nifedipine,a calcium channel blocker,on liver ischemia in dogs

This study was undertaken in order to determine whether the administration of nifedipine, a calcium channel blocker, could protect the liver from ischemic damage and to investigate its effect on the hepatic cellular energy status and cardio-vascular system after 60 minutes of hepatic ischemia in dogs. The ischemia was induced by temporarily clamping the portal vein and hepatic artery. One group of animals (n=17) received nifedipine (5 μg/kg body weight) intravenously 15 minutes before the induction of liver ischemia, which was continued at a dose of 0.2 μg/kg body weight/min throughout the ischemic period, and for an additional 30 minutes afterwards. Control dogs (n=16) were not given nifedipine and survival was observed over seven days. The survival rate was 83 per cent in the nifedipine treated animals and 0 per cent in the control animals. Serum glutamic oxaloacetic transaminase levels were greatly increased following ischemia, and they were significantly lowered with the nifedipine treatment. The hepatic energy charge decreased remarkably during the hepatic ischemia, however it increased gradually after declamping but did not returned to its preoperative value in either group until one hour later and then it was higher in the nifedipine treated animals than in the control animals. Cardiac index and portal venous blood flow ratio remained higher in the nifedipine treated animals than in the control animals, after the ischemic period. These results suggest that nifedipine may have a powerful cytoprotective effect and that the period of warm hepatic ischemia could be prolonged with its use.     

In Vivo Identification,Survival, and Functional Efficacy of Transplanted Hepatocytes in Acute Liver Failure Mice Model by FISH Using Y-Chromosome Probe

Hepatocyte transplantation has excited much interest in lending temporary metabolic support to a failing liver following acute liver injury. The exact site from which they act and the clinical, biochemical, and histological changes in the recipient body following hepatocyte transplantation is yet to be worked out. The present study is an attempt to delineate location and function of transplanted hepatocytes and also the overall survival of these cells with a fluorescent in situ hybridization (FISH) technique using a Y-chromosome–specific probe in a carbon tetrachloride (CCl₄)-induced mice model of fulminant hepatic failure. Fifty-five syngenic adult Swiss female mice of approximately the same age and body weight were divided into three groups. Group-1 (n = 15), which received mineral oil, served as a negative control. Group-II (n = 15) received CCl₄ (3 mL/kg) 40% vol/vol in mineral oil, by gavage served as positive control for hepatic failure. Group-III (n = 25) received intrasplenic transplantation of syngenic single cell suspension of hepatocytes in Hanks medium, after 30 h of CCl₄ administration. Male Swiss adult mice (n = 15) served as donors of hepatocytes. The overall survival of animals in groups I to III was 100, 0, and 70%, respectively, by 2 wk of the study period. Transplanted hepatocytes were identified by Periodic Acid Schiff (PAS) staining and confirmed with a FISH technique using the Y-chromosome probe. The majority of exogenously transplanted hepatocytes were found in the liver and spleen sections even after 1 wk of hepatocyte transplantation. Transplanted cells were mostly found to be translocated into the sinusoids of the liver. Transplanted hepatocytes were found to be beneficial as a temporary liver support in a failing liver, significantly improving the survival of the animals. In the present study, the FISH technique was used to unequivocally distinguish the transplanted cells from the host, and thus describes a model for studying the distribution and survival of the transplanted cells.     

Multiple intrasplenic hepatocyte transplantations in the dalmatian dog

BACKGROUND: Hepatocyte transplantation is an attractive potential treatment for liver-based inborn errors of metabolism and for fulminant hepatic failure. Dalmatian dogs have a metabolic error that results in hyperuricosuria. This report focuses on the effect of multiple, sequential intrasplenic transplants of fresh and cryopreserved hepatocytes in dalmatians. METHODS: Dalmatians underwent intrasplenic hepatocyte transplantation with hepatocytes taken from healthy mongrels. Dalmatian urinary uric acid excretion was measured preoperatively, and this served as the control value. Three hepatocyte transplantations were performed at 30-day intervals--the first with freshly isolated cells, and both the second and the third with cryopreserved hepatocytes from the same donor. Urinary uric acid excretion was measured postoperatively twice per week. RESULTS: The urinary uric acid excretion decreased an average of 54% after the first hepatocyte transplantation. The effect was transient and lasted an average of 22 days (range, 19-50 days). Subsequent intrasplenic hepatocyte transplantation with cryopreserved hepatocytes resulted in similar decreases in urinary uric acid excretion. Each transplant resulted in a significant decrease in urinary uric acid excretion when compared with baseline values (P = < .001). CONCLUSIONS: Sequential intrasplenic hepatocyte transplantation is feasible in this model. This method provided a significant, but transient, correction in urinary uric acid excretion that was similar with either fresh or cryopreserved hepatocytes. A substantial biologic effect provided by cryopreserved hepatocytes has important implications in clinical hepatocyte transplantation.