Daisuke Yasuda

Sentinel node mapping guided by indocyanine green fluorescence imaging in gastric cancer.

Objective:In this study, we determined the possible usefulness of sentinel node (SN) mapping guided by indocyanine green (ICG) fluorescence imaging in the management of gastric cancer. Summary Background Data:ICG fluorescence imaging system has recently been developed for obtaining biochemical information from living tissues. Methods:Our series consisted of 56 patients with gastric cancer who underwent standard gastrectomy with lymphadenectomy. Two milliliters of ICG solution (0.5%) was injected into the submucosa around the tumor endoscopically before the operation or into the subserosa intraoperatively. ICG fluorescence imaging was conducted using a charge-coupled device camera with a light-emitting diode having a wavelength of 760 nm as the light source and a cut filter to filter out light with wavelengths below 820 nm as the detector. Results:SNs were detected in 54 (96.4%) of the 56 patients, and the mean number of SNs was 7.2 ± 7.0. Even SNs that were not green in color could be easily and clearly visualized by ICG fluorescence imaging. cT1-stage cancers were associated with a significantly higher accuracy rate (97.2% vs. 72.2%, P = 0.0127) than cT2-or cT3-stage cancers. Preoperative ICG injection was associated with a significantly higher incidence of cT1-stage cancers (87.1% vs. 40.0%, P = 0.0004), a larger mean number of SNs (9.9 ± 7.5 vs. 4.1 ± 5.0, P < 0.0001), a higher accuracy rate (100% vs. 73.9%, P = 0.0039), and a lower false negative rate (0% vs. 60.0%, P = 0.0345) as compared with intraoperative ICG injection. Conclusions:This study shows that ICG fluorescence imaging allows highly sensitive image-guided intraoperative SN mapping in cases of gastric cancer. Our data suggest that SN mapping guided by ICG fluorescence imaging might be useful for predicting the metastatic status in lymph nodes in cases of gastric cancer, especially those with cT1-stage cancer.

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°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.

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 6β-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.

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.

Elevation of Serum Albumin Levels in Nagase Analbuminemic Rats by Allogeneic Bone Marrow Cell Transplantation

We investigated the feasibility of correcting the congenital absence of albumin in Nagase analbuminemic rats (NARs) by allogeneic bone marrow cell transplantation (BMT). Seven-week-old male NARs were used as recipients, and 6- to 8-week-old male Sprague-Dawley (SD) rats were used as allograft donors. NARs were divided into three groups: a BMT group (n = 10) in which bone marrow cells were infused into the liver; a hepatocyte transplantation (HCT) group (n = 8) in which hepatocytes were transplanted into the liver, and a control group (n = 8) in which PBS was injected into the portal vein. Serum albumin levels were measured as an indicator of the function of the grafted cells, and the phenotypic characteristics of the engrafted cells in the recipient’s liver were assessed with immunohistochemical and immunofluorescence techniques. At 8 weeks after cell transplantation, the serum albumin levels of the BMT group and HCT group were significantly higher than in the control group. The hepatocyte-like cells derived from bone marrow cells expressed albumin in liver of the NARs. According to this result, bone marrow cells can differentiate into hepatocyte-like cells in vivo. The results show that BMT is an effective treatment for congenital analbuminemia in a rat model and suggest that allogeneic BMT can be used as an efficient therapy for hereditary metabolic diseases.

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.

Cryopreservation of hepatocytes -Is it effective to cryopreserved hepatocytes using a new cryopreservation procedure combining a bioartificial approach? -

The number of patients with severe liver disease and needing whole organ transplantation or living related split liver transplantation has been increasing. However, the shortage of donor organs is particularly problematic and still awaits resolution.