Tomotake Koizumi

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.

Genetic polymorphism of the human organic solute carrier protein 1 (hOSCP1) gene in Japanese patients with non-viral liver carcinoma.

Human organic solute carrier protein 1 (hOSCP1) is a Na+-independent multispecific organic solute transporter. To date, several studies have revealed that gene mutations of the transporters are likely to be associated with some diseases; however, there are no data concerning the genetic polymorphism of the hOSCP1 gene in Japanese patients with non-viral liver carcinoma (LC). In the present study, we isolated genomic DNA from a normal portion of LC, and analyzed 41 single nucleotide polymorphisms (SNPs) chosen from a database of SNPs (dbSNPs). We found genotype frequencies for 2 non-synonymous SNPs [rs34409118 (Thr131 → Ala) and rs1416840 (Ile219 → Thr)] and 1 synonymous SNP [rs16822954 (Ser193 → Ser)] to be statistically significant when compared with dbSNPs. No statistical significance was observed in rs2275477 (Gly307 → Arg) in the hOSCP1 gene. With respect to the allele frequency, we also observed rs34409118 to be statistically significant. Interestingly, we found that non-viral LC patients do not carry heterozygous mutations in rs1416840 (A/G) and rs16822954 (A/G), suggesting that a non-carrier of heterozygous mutations in these two SNPs might be a biomarker for susceptibility for non-viral LC in Japanese. Further analyses of patients with hOSCP1 variants may elucidate the relationship between the hOSCP1 gene and susceptibility of non-viral LC in Japanese patients.

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.

Skeletonization and Isolation of the Glissonean and Venous Branches in Liver Surgery With an Ultrasonic Scalpel Technology.

This study describes a novel technique for skeletonization and isolation of Glissonean and venous branches during liver surgery using a harmonic scalpel (HS). Hepatic resections with HS were performed with the skeletonization and isolation technique in 50 patients (HS group). Variables evaluated were blood loss, operative time, biliary leak, and morbidity. The results were compared with 50 hepatic resections that were performed using a previously established technique: Cavitron ultrasonic surgical aspirator with electric cautery, ligatures, and hemoclips (NHS group). The HS group had shorter total operative times (285 versus 358 minutes; P = 0.01), less blood loss (389 versus 871 mL; P = 0.034), and less crystalloid infusion (2744 versus 3299 mL; P = 0.027) compared with the NHS group. Postoperative liver function and complication rates were similar when comparing the two groups. These data demonstrate that HS is a simple, easy, and effective instrument for the skeletonization and isolation of vessels during liver transection.

Preoperative Tattooing for Precise and Expedient Localization of Landmark in Laparoscopic Liver Resection

Laparoscopic liver surgery is a safe and effective approach for the management of surgical liver disease in the hands of trained surgeons who are experienced in hepatobiliary and laparoscopic surgery. Intraoperative ultrasound (IOUS) has become an important pillar of modern surgery with diagnostic and therapeutic value; it has become an almost indispensable procedure for the intraoperative diagnosis of liver lesions. It is also beneficial for guidance to the parenchymal transection plane with immediate feedback on any changes that might occur during surgery in not only open hepatic surgery, but also laparoscopic hepatic surgery. However, during laparoscopic hepatectomy, the reliability of laparoscopic IOUS has been poor when evaluating the entire liver because interpretation of the ultrasound image is challenging. It is often difficult to localize liver lesions during laparoscopy with IOUS or palpation via a laparoscopic approach, particularly if they are small or on the deep side of an intraparenchymal lesion; precise localization of a lesion or a vessel landmark is critical to achieving adequate surgical margins. To address this issue, we have demonstrated that preoperative tattooing allows the surgeon to determine the precise and expedient localization of a landmark or tumor during laparoscopic liver resection.

A long-term survival case after surgical resection of skeletal muscle metastasis following esophagectomy for squamous cell carcinoma of the esophagus

Abstract Cases of skeletal muscle metastasis of esophageal carcinoma are very rare, with few reports of long-term survival. We report a case of long-term survival after surgical resection of skeletal muscle metastasis. A 56-year-old man with advanced esophageal cancer and early gastric cancer underwent thoracoscopic esophagectomy, 2-field lymph node dissection, partial gastrectomy and gastric tube reconstruction. Six months later, cervical lymph node metastasis and mediastinal lymph node recurrence were found. Therefore, the patient underwent cervical lymph node dissection and adjuvant chemoradiotherapy. Two years and 3 months after the esophagectomy, a muscle metastasis was found in the left shoulder, and he underwent tumor dissection, followed by adjuvant chemotherapy for a year. There has been no sign of recurrence since, even 13 years after the esophagectomy. We believe our aggressive surgical treatment might have led to long-term survival.

One-stage laparoscopy-assisted colectomy for synchronous double colorectal cancers

Synchronous multiple malignant colorectal lesions are rare, and there have been very few studies about one‐stage laparoscopic operations in these cases. Here, we evaluated the short‐term outcomes of laparoscopy‐assisted colectomy (LAC) for synchronous double colorectal cancers. Seven patients underwent one‐stage LAC that required two resections and anastomoses in our hospital from 2010 to 2014. We retrospectively examined each patients background and subsequent surgical outcomes. The median age of patients was 78 years, and the median BMI was 19.8 kg/m2. The median operative time was 190 min, and blood loss was minimal. All resected specimens were extracted through a transumbilical incision. A radical operation was performed safely without procedural accidents or postoperative complications in all cases. The median postoperative hospital stay was 12.5 days. One‐stage LAC is considered a safe and viable procedure for resecting synchronous double colorectal cancers. It involves minimal invasiveness and is similar to standard LAC.