Cold preservation of the liver with oxygenation by a two-layer method.

BACKGROUND: The two-layer method (TLM) has recently been found to be superior to simple cold storage in University of Wisconsin (UW) solution as a means of pancreas preservation for islet transplantation. In this study, we investigated whether TLM would result in better hepatocyte function over UW cold storage and if it could be applied to hepatocyte transplantation. MATERIALS AND METHODS: Hepatocytes from male Sprague Dawley rat livers were isolated and divided into three groups: a non-preservation group (group 1), a 10-h preservation group (group 2), and a 24-h preservation group (group 3). Groups 2 and 3 were then divided into three subgroups: a group preserved by the TLM (subgroup a), a group preserved in UW solution (subgroup b), and a group preserved in water (subgroup c). Isolated hepatocytes were evaluated for cell yield, viability, and adenosine triphosphate level after preservation. Hepatocytes were either cultured or transplanted. RESULTS: Although no differences in cell yield or morphological findings were observed between any of the groups, TLM significantly improved hepatocyte viability and adenosine triphosphate levels in comparison with UW cold storage. Albumin production or urea synthesis were significantly higher in subgroup 3a than in subgroup 3b at almost all time points. Surprisingly, after hepatocyte transplantation, the serum albumin level in subgroup 2a was significantly higher than in subgroup 2b at every time point. CONCLUSIONS: The results of this study demonstrated that liver preservation by the TLM before hepatocyte isolation might be beneficial and will be useful in the field of hepatotocyte transplantation.

Microencapsule technique protects hepatocytes from cryoinjury.

AIM: Hepatocyte transplantation is a potential alternative to whole organ liver transplantation. To realize this procedure, a hepatocyte bank system capable of supplying large numbers of hepatocytes must be established. We previously reported an easy method for cryopreserving hepatocytes using a microencapsulation technique. Here, we investigated how cryoinjury to microencapsulated hepatocytes could be avoided during cryopreservation. METHODS: Hepatocytes from Sprague-Dawley rats were harvested in situ using a two-step ethylenediaminetetraacetic acid (EDTA)/collagenase digestion protocol. The cells were microencapsulated using alginate-poly L-lysine. The microencapsulated hepatocytes were put into vials and immediately immersed in liquid nitrogen. The growth of ice crystals in the vials containing the microencapsulated hepatocytes was observed using cryomicroscopy. The microencapsulated hepatocytes were sectioned for ultrastructural examination to investigate their intracellular conditions. Finally, total RNA was isolated from the cryopreserved microencapsulated hepatocytes and analyzed for hepatocyte nuclear factor (HNF) using reverse transcriptase polymerase chain reaction (RT-PCR) analysis. RESULTS: Cryomicroscopy showed that the alginate microencapsulation technique protected the hepatocytes from physical damage caused by the growth of extracellular ice crystals. Ultrastructural examination revealed that the intracellular environment of the microencapsulated hepatocytes was maintained. The RT-PCR analysis additionally suggested that the alginate gel also maintained the HNF level. CONCLUSION: Our microencapsulation technique protects hepatocytes from cryoinjury. This novel technique could be utilized by hepatocyte banks.

Long-term maintenance of the drug transport activity in cryopreservation of microencapsulated rat hepatocytes.

Transplantation of isolated hepatocytes has been proposed to compensate for essential functions lacking in liver failure or for genetic defects that alter a specific liver metabolic pathway. Hepatocyte utilization for these purposes would be facilitated with a reliable, reproducible, and effective method of long-term hepatocyte storage. We have recently developed a simple new system for cryopreservation of hepatocytes that encapsulates alginate microspheres and maintains liver-specific function. The aim of this study was to elucidate the transport and drug-metabolizing enzyme activities of cryopreserved microencapsulated hepatocytes stored for a long time. Morphological examinations showed there is no apparent injury of the hepatocytes during cryopreservation processes. A drug-metabolizing enzyme (testosterone 6beta-hydroxylase, a specific probe for CYP3A2) and drug transport activities [salicylate, allopurinol, and prostaglandin E2 (PGE2), typical substrates of rOat2] in cryopreserved microencapsulated hepatocytes were maintained up to 120 days. Our results thus demonstrate for the first time that cryopreservation of primary rat hepatocytes by the encapsulation technique allows long-term retention of drug metabolism and drug transport activities.

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.

A novel method of cryopreservation of rat and human hepatocytes by using encapsulation technique and possible use for cell transplantation.

Encapsulated hepatocyte transplantation is a promising approach to cell transplantation without immunosuppression as an alternative to whole organ liver transplantation. However, the shortage of donor cells for hepatocyte transplantation has not been resolved, and at this critical point, it seems necessary to establish a method of hepatocyte cryopreservation to allow clinical application of hepatocyte transplantation and the development of a bioartificial liver system in the near future. In this study we demonstrated that cryopreserved microencapsulated rat and human hepatocytes can retain their hepatic function and that cryopreserved microencapsulated human hepatocytes transplanted into rat spleen remain viable without immunosuppression. Rat and human hepatocytes were isolated by a collagenase digestion method, and they were microencapsulated with poly-L-lysine. The microencapsulated rat hepatocytes were transferred to culture medium (DMEM containing 10% FBS and 10% DMSO) and immediately frozen in liquid nitrogen. A warm water bath (37 degrees C) was used to thaw the microencapsulated hepatocytes. Hepatic function, drug metabolism, and cell morphology were assessed after 90 days of cryopreservation. After 1 week of cryopreservation, microencapsulated hepatocytes were cultured for up to 2 weeks to assess their hepatic function and morphology. The morphology of human hepatocytes was assessed after 30 days of cryopreservation. Cryopreserved human hepatocytes were transplanted into rat spleen to assess their morphology. Cryopreserved microencapsulated hepatocytes retained their viability and were strongly positive for expression of albumin, OAT2, CYP3A2, and CYP3A9. Two weeks after cultivation, the cryopreserved microencapsulated rat hepatocytes had retained their hepatic function (urea synthesis). Cryopreserved microencapsulated human hepatocytes also mainly survived and retained their hepatic function for at least 30 days after cryopreservation. Moreover, entrapped cryopreserved human hepatocytes also survived and expressed albumin in rat spleen after transplantation. We demonstrated a novel method of long-term cryopreservation of rat and human hepatocytes by using an encapsulation technique, with retention of biological activity and excellent survival of the cryopreserved microencapsulated human hepatocytes transplanted into rat spleen. We believe that this novel approach to hepatocytes cryopreservation provides a new direction in encapsulated cell therapy with the goal of clinical application in the near future.

Surgical repair for late-onset hepatic venous outflow block after living-donor liver transplantation.

The incidence of hepatic venous complications in partial liver transplantation is more frequent than that in whole liver transplantation. There are no reports of a surgical strategy for hepatic venous outflow block (HVOB) after living-donor liver transplantation. HVOB was diagnosed when the pull-through pressure gradient across the anastomotic site was over 5 mm Hg. Reoperation for venous anastomosis was performed if the angioplasty was unsuccessful. After dissection around the hepatic venous anastomotic site, a patch venoplasty of the anastomosis was performed. When the inferior vena cava was constricted, venoatrial anastomosis was performed. In 6 years, 5 of 223 patients experienced HVOB. Balloon angioplasty was successfully performed in two patients, a patch venoplasty of the anastomosis in two, and venoatrial anastomosis in one. In all patients, the ascites stopped. HVOB must be diagnosed as soon as possible with Doppler ultrasound and venography. Prompt surgical revision can salvage the grafts.

Capacity of hepatic regeneration following a second partial hepatectomy in rats.

In hepatic surgery, the number of repeated hepatectomy has increased for recurrent tumors after the first hepatectomy. We examined here, whether or not hepatic regeneration after the second partial hepatectomy was similar to that of the remnant liver after the primary partial hepatectomy. Using a new model of a second partial hepatectomy in rats, three groups of rats were studied. Group I rats underwent a standard one-third partial hepatectomy (P-PHx group, n=30). Group II rats underwent a one-third hepatectomy 2 weeks after a two-thirds hepatectomy was performed (S-PHx group, n=30). As a control, Group III rats underwent sham surgery. In each group, the rats were sacrificed at different time points postoperatively to evaluate changes in blood chemistry and to estimate the liver regenerative response in the remnant liver (weight, immunohistochemistry; BrdU, MIB-5) as well as expression of the hepatocyte growth factor. In the S-PHx group, a significant decrease was detected in the restitution of the liver mass until 24 h postoperatively which was closely associated with the number of mitotic hepatocytes. The S-PHx group showed significant signs of liver dysfunction until 48 h and significantly increased serum hyaluronic acid levels until 72 h in comparison to the P-PHx group. In situ RT-PCR analysis indicated that in the P-PHx group HGF transcripts were expressed between 24 and 48 h in the remnant liver, while in the S-PHx group HGF transcripts were expressed only at 24 h postoperatively. Our findings suggested that second partial hepatectomized rats exhibit a retarded hepatic regeneration during the early postoperative phase due to a depressed HGFmRNA expression.

Natural course of the remnant hepatic functional reserve as estimated by technetium-99m-galactosyl human serum albumin scintigraphy after a hepatectomy.

BACKGROUND: Technetium-99m-galactosyl human serum albumin (GSA) scintigraphy provides an accurate estimation of the hepatic functional reserve but is not applied after a hepatectomy. The aim of this study was to elucidate the natural course of the remnant hepatic functional reserve (RHFR) after hepatectomy by GSA scintigraphy. METHODS: Eighty-six patients (partial hepatic resection, Hr0 = 46; sectionectomy, Hr1 = 21; bisectionectomy, Hr2 = 19) classified as Child-Pugh class A before the hepatectomy were enrolled, and GSA scintigraphy to detect HH15 (uptake ratio of the heart at 15 min to that at 3 min) and LHL15 (uptake ratio of the liver at 15 min to the liver plus the heart at 15 min) was performed periodically before and after the hepatectomy. HH15, LHL15, and the percentages of patients that recovered to the preoperative levels of these entities were estimated. In addition, hematobiochemical tests and the remnant liver volume were also periodically monitored. RESULTS: HH15 and LHL15 levels deteriorated until 2 months postoperatively (PO) after the procedure and subsequently recovered to the preoperative levels at 6 months PO in Hr0 patients. In Hr1 patients, but not in Hr2 patients, these levels also deteriorated until 3 months PO and had improved by 6 months after the surgery. Only 40% of the patients showed recovery to the preoperative levels by 6 months PO in the Hr0 group; furthermore, the percentage of patients who showed recovery to the preoperative levels by 6 months PO was under 40% in the Hr1 group and around 10% in the Hr2 group. However, the results of hematobiochemical tests and the remnant liver volume in all types of hepatectomies were rapidly normalized after the hepatectomy. CONCLUSIONS: Remnant hepatic functional reserve estimated by GSA scintigraphy revealed that a larger resected liver volume induced both more serious and continued remnant hepatic dysfunction in comparison to results shown by hematobiochemical tests, while the functional regeneration was also appreciably slower and more gradual in comparison to the volume regeneration.

Intraoperative fluorescent imaging using indocyanine green for liver mapping and cholangiography.

BACKGROUND: Preoperative imaging is widely used and extremely helpful in hepatobiliary surgery. However, transfer of preoperative data to a intraoperative situation is very difficult. Surgeons need intraoperative anatomical information using imaging data for safe and precise operation in the field of hepatobiliary surgery. We have developed a new system for mapping liver segments and cholangiograms using intraoperative indocyanine green (ICG) fluorescence under infrared light observation. METHOD: The imaging technique for mapping liver segments and cholangiogram based on ICG fluorescence used an infrared-based navigation system. Eighty one patients with liver tumors underwent hepatectomy from 2006, January to 2009, March. In liver surgery, 1 ml of ICG was injected via the portal vein under observation by the fluorescent imaging system. Fourteen patients were underwent laparoscopic cholecystectomy for chronic cholecystitis with gallstones. In laparoscopic cholecystectomy, 5 ml of ICG was administered intravenously just before operation and the bile duct was observed using the infrared-based navigation system. RESULT: This new technique successfully identified stained subsegments and segments of the liver in 73 of 81 patients (90.1%). Moreover, clear mapping of liver segments was obtained even against a background of liver cirrhosis. Fluorescent cholangiography clearly showed the common bile duct and cystic duct in 10 of 14 patients (71.4%). No adverse reactions to the ICG were encountered. CONCLUSION: Application of this technique allows intraoperative identification of anatomical landmark in hepatobiliary surgery.

Image-guided liver mapping using fluorescence navigation system with indocyanine green for anatomical hepatic resection.

BACKGROUND: In malignant hepatic neoplasm, anatomic resection could improve survival and limit complications from hepatectomy. Our purpose was to develop an intraoperative method for identifying segment and subsegment of the liver with high-sensitivity near-infrared fluorescence imaging. METHODS: The subjects were 35 patients with hepatic malignant liver disease who received hepatectomy in 2006. The segments of liver method of identification that used infrared observation camera system termed Photo Dynamic Eye-2 (PDE-2) with indocianine green (ICG) for the patient with malignant liver tumor (hepatocellular carcinoma: 13 cases; metastatic liver cancer: 18 cases; intrahepatic cholangio carcinoma: 4 cases) were performed before liver resection. RESULTS: Although greenish stain of the liver surface after the injection of ICG via portal vein is not visible clearly without infrared observation camera system PDE-2, 1 minute after injection of ICG with fluorescent using infrared observation camera system PDE-2, demarcation of liver segment and subsegment was clearly detected. Ten minutes after injection of ICG with fluorescent using infrared observation camera system PDE-2, fluorescence of liver subsegment remained. Stained subsegment and segment of liver were identifiable in 33 (94.3%) of the 35 patients. There were no complications or side-effects related to the injection of patent blue dye. CONCLUSION: We demonstrated here that near-infrared fluorescence imaging system is a novel and reliable intraoperative technique to identify hepatic segment and subsegment for anatomical hepatic resection.

Hepatocyte transplantation from steatotic liver in a rat model.

BACKGROUND: Hepatocyte transplantation (HTx) has progressed significantly, but widespread application remains slow because of the shortage of donor hepatocytes. Many sources of hepatic cells have been proposed as alternatives to isolated hepatocytes, but primary isolated hepatocytes continue to be the best source for liver cell-based therapy. To expand the donor pool, we focused on steatotic liver as a new cell source for HTx because numerous steatotic livers are discarded as unsuitable for orthotopic liver transplantation. This study investigated the efficacy of steatotic hepatocyte transplantation (SHTx) using steatotic liver in a rat model. MATERIALS AND METHODS: Hepatocytes were isolated from obese and lean Zucker rats. Hepatocytes from each group were cultured to analyze the function of steatotic hepatocytes. Hepatocytes from each group were also transplanted into the spleens of Nagase analbuminemic rats (NARs) to investigate the efficacy of SHTx. RESULTS: In the in vitro experiment, a real-time reverse-transcription polymerase chain reaction assay showed that albumin and several hepatocyte nuclear factors were highly expressed in both groups. Morphologically, the steatotic hepatocytes were positive for albumin, and an enzyme-linked immunosorbent assay showed no significant differences between the two groups except for albumin production after 5 d of culture. In the in vivo experiment, the transplanted steatotic hepatocytes in the spleens of Nagase analbuminemic rats were positive for albumin and periodic acid-Schiff staining. Surprisingly, an enzyme-linked immunosorbent assay showed no significant differences in the serum albumin levels between the two groups throughout the study period. CONCLUSIONS: We have demonstrated that steatotic hepatocytes are a potential new cell source for HTx therapy.

Resection of a pulmonary lesion after liver transplantation: report of a case.

A 44-year-old Chinese-Indonesian man who underwent living-donor liver transplantation with a right liver graft presented 4 months later with a cough and fever. Chest X-ray showed a nodular shadow in the apex of the left lung, which was diagnosed as pulmonary tuberculosis. After 1 week of antituberculous chemotherapy, we performed a left upper lobectomy. Postoperative antituberculous chemotherapy, consisting of isoniazid (300 mg/day) and rifampin (450 mg/day), was continued for 4 months, and there has been no sign of recurrence for 1 year since the thoracotomy. This case supports the feasibility of surgery for localized pulmonary tuberculosis soon after transplantation.

Volume regeneration after right liver donation.

After right hepatectomy with the middle hepatic vein trunk for a graft, the venous outflow in segment IV is disturbed. There are limited data, however, regarding the effect of middle hepatic vein deprivation on liver regeneration or functional recovery. Living donors who underwent right hepatectomy with preservation of the middle hepatic vein (Group A, n = 58) and those deprived of the middle hepatic vein (Group B, n = 13) were reviewed. When the donor was under 50 years old and the remnant left liver was estimated to be more than 35% of the whole liver, right liver graft harvesting with the middle hepatic vein trunk was considered. Volume regeneration of segments I-III, segment IV, and overall liver volume was assessed at the third postoperative month using computed tomography. The regeneration rate of segment IV was significantly impaired in Group B donors compared with that in Group A donors (125% vs. 45%, P = 0.008). In contrast, the regeneration rate of segments I -III was significantly higher than that in Group A (208% vs. 263%, P = 0.004). There was no significant difference in the regeneration rate of the whole left liver or functional recovery between groups. Multivariate analysis revealed that the resection type (group) was a significant predictive factor for the regeneration rate of segments I-III and segment IV. When deprived of the middle hepatic vein, liver regeneration of segment IV was impaired but was compensated for by the regeneration of segments I-III. In conclusion, extended right hepatectomy can be safely performed with careful preoperative consideration using these criteria.

Refinement of venous reconstruction using cryopreserved veins in right liver grafts.

Short and direct vein anastomosis is generally performed in living donor liver transplantation using a right liver graft. The graft will regenerate, however, and might thus compress the anastomosis. We formulated a strategy for outflow reconstruction in right liver graft. When reconstruction of multiple short hepatic veins was necessary, a cryopreserved inferior vena cava graft was anastomosed with the hepatic veins of the graft in a basin. When there were no major short hepatic veins in the graft, a rectangular-shaped vein graft was used to make a single orifice using the middle and right hepatic veins in the graft. When there were no tributaries of the middle hepatic vein to be reconstructed, a diamond-shaped vein patch was anastomosed on the anterior wall of the right hepatic vein orifice of the graft. These techniques were satisfactorily applied in 40 patients with no torsion or tension at the anastomotic site of the hepatic venous reconstruction or other complications in outflow. The present strategy seemed to be technically feasible for outflow reconstruction in a right liver graft.

Liver regeneration after portal vein plus hepatic artery ligation performed heterochronously in rats.

BACKGROUND/PURPOSE: Portal vein ligation (PVL) has been used clinically to decrease the amount of liver before surgical resection, consequently, minimizing postoperative dysfunction in the remaining hypertrophied liver lobes. To date, few reports in the literature have demonstrated the regenerative capacity of unaffected lobes following PVL plus hepatic artery ligation (HAL). This study was conducted in rats to determine a safe and efficacious method of PVL plus HAL, focusing on liver function, the MIB-5 labeling index, and the ratio of the weight of the nonligated lobes to the body weight. METHODS: Group I rats were subjected to PVL of the left lateral and median branches alone (corresponding to approximately 70% total liver volume). In group II, we performed PVL and HAL of the same branches simultaneously, while in group III, HAL was performed 48 h after PVL. A laparotomy without ligature was performed in the control group. Rats from each group were killed at 24, 48, 72, 96, and 168 h after surgery. Standard serum liver functions were tested. Proliferative activity in the nonligated liver was expressed using the Ki-67 antigen (MIB-5) labeling index. Body and nonligated lobe weights were measured. RESULTS: At 96 h post-surgery, the ratio of the weight of the nonligated lobe to body weight was significantly higher in group III than in group I and group II, and induction of the MIB-5 labeling index showed maximum levels in group III. However, quantitative determination of serum glutamic-oxaloacetate transaminase (GOT) showed peak levels in group II at 24 h after surgery. CONCLUSIONS: From these results, we conclude that the PVL plus HAL heterochronous procedure is safer and more efficacious than PVL only, or simultaneous PVL plus HAL. A better knowledge of the events following such heterochronous ligation should improve the clinical outcome of hepatic resection for liver diseases.