A Gelatin Hydrogel Nonwoven Fabric Combined With Adipose Tissue-Derived Stem Cells Enhances Subcutaneous Islet Engraftment.

Subcutaneous islet transplantation is a promising treatment for severe diabetes; however, poor engraftment hinders its prevalence. We previously revealed that a gelatin hydrogel nonwoven fabric (GHNF) markedly improved subcutaneous islet engraftment. We herein investigated whether the addition of adipose tissue-derived stem cells (ADSCs) to GHNF affected the outcome. A silicone spacer sandwiched between two GHNFs with (AG group) or without (GHNF group) ADSCs, or a silicone spacer alone (Silicone group) was implanted into the subcutaneous space of healthy mice at 6 weeks before transplantation, then diabetes was induced 7 days before transplantation. Syngeneic islets were transplanted into the pretreated space. Intraportal transplantation (IPO group) was also performed to compare the transplant efficiency. Blood glucose, intraperitoneal glucose tolerance, immunohistochemistry, and inflammatory mediators were evaluated. The results in the subcutaneous transplantation were compared using the Silicone group as a control. The results of the IPO group were also compared with those of the AG group. The AG group showed significantly better blood glucose changes than the Silicone and the IPO groups. The cure rate of AG group (72.7%) was the highest among the groups (GHNF; 40.0%, IPO; 40.0%, Silicone; 0%). The number of vWF-positive vessels in the subcutaneous space of the AG group was significantly higher than that in other groups before transplantation (P < 0.01). Lectin angiography also showed that the same results (P < 0.05). According to the results of the ADSCs tracing, ADSCs did not exist at the transplant site (6 weeks after implantation). The positive rates for laminin and collagen III constructed around the transplanted islets did not differ among groups. Inflammatory mediators were higher in the Silicone group, followed by the AG and GHNF groups. Pretreatment using bioabsorbable scaffolds combined with ADSCs enhanced neovascularization in subcutaneous space, and subcutaneous islet transplantation using GHNF with ADSCs was superior to intraportal islet transplantation.

A Recombinant Peptide Device Combined with Adipose Tissue-Derived Stem Cells Enhances Subcutaneous Islet Engraftment.

Subcutaneous space has been considered an attractive site for islet graft transplantation; however, the oxygen tension and vascularization are insufficient for islet graft survival. We investigated whether subcutaneous pre-implantation of a recombinant peptide (RCP) device with adipose tissue-derived stem cells (ADSCs) enhanced subcutaneous islet engraftment. RCP devices with/without syngeneic ADSCs were pre-implanted into the subcutaneous space of C57BL/6 mice. Syngeneic islets (300 or 120 islet equivalents (IEQs)) were transplanted into the pre-treated space after diabetes induction using streptozotocin. The cure rates of groups in which RCP devices were implanted four weeks before transplantation were significantly better than the intraportal transplantation group when 300 IEQs of islets were transplanted (p < 0.01). The blood glucose changes in the RCP+ADSCs-4w group was significantly ameliorated in comparison to the RCP-4w group when 120 IEQs of islets were transplanted (p < 0.01). Immunohistochemical analyses showed the collagen III expression in the islet capsule of the RCP+ADSCs-4w group was significantly enhanced in comparison to the RCP-4w and RCP+ADSCs-d10 groups (p < 0.01, p < 0.01). In addition, the number of von Willebrand factor-positive vessels within islets in the RCP+ADSCs-4w group was significantly higher than the RCP-4w group. These results suggest that using ADSCs in combination with an RCP device could enhance the restoration of the extracellular matrices, induce more efficient prevascularization within islets, and improve the graft function.

Functional Assessment of Cardiac Arrest Hepatocytes and Effect of Mechanical Perfusion on Function in a Rat Model.

BACKGROUND: Hepatocyte transplantation has been reported to be useful for metabolic diseases and acute liver failure. However, the shortage of donors limits its widespread use. The use of livers from donors after circulatory death, which are currently unavailable for liver transplantation, may alleviate donor shortage. In this study, we investigated the effects of mechanical perfusion on cardiac arrest hepatocytes in a rat model using cardiac arrest donor livers, and we evaluated the function of cardiac arrest hepatocytes. METHODS: F344 rat hepatocytes isolated from livers removed during cardiac pulsation were compared with those isolated from livers removed after 30 minutes of warm ischemia after cardiac arrest. We then compared hepatocytes isolated from livers removed after 30 minutes of warm ischemia with those isolated after 30 minutes of mechanical perfusion before isolation. The yield per liver weight, ammonia removal capacity, and adenosine diphosphate/adenosine triphosphate ratio were evaluated. RESULTS: Thirty minutes of warm inhibition reduced hepatocyte yield but did not alter ammonia removal capacity and energy status. Mechanical perfusion increased hepatocyte yield and improved the adenosine diphosphate/adenosine triphosphate ratio after 30 minutes of warm inhibition. CONCLUSION: Thirty minutes of warm ischemic time may decrease isolated hepatocyte yield without degrading their function. If increased yields are obtained, livers from donors dying of cardiac arrest could be used for hepatocyte transplantation. The results also suggest that mechanical perfusion may positively affect the energy status of hepatocytes.

Development of Wireless Power-Transmission-Based Photodynamic Therapy for the Induction of Cell Death in Cancer Cells by Cyclometalated Iridium(III) Complexes.

Photodynamic therapy (PDT), a noninvasive method for cancer therapy, involves the generation of reactive oxygen species (ROS) by the photochemical excitation of photosensitizers (PSs) to induce cell death in cancer cells. A variety of PS including porphyrin derivatives and metal complexes such as iridium (Ir) complexes have been reported. In clinical trials, red-near infrared (NIR) light (650-900 nm) is preferred for the excitation of PSs due to its deeper penetration into tissues compared with visible light (400-500 nm). To overcome this limitation, we established a PDT system that uses cyclometalated iridium(III) (Ir(III)) complexes that are excited with blue light in the wireless power transmission (WPT) system. To achieve this, we developed a light-emitting diode (LED) light device equipped with a receiver coil that receives electricity from the transmitter coil through magnetic resonance coupling. The LEDs in the receiving device use blue light (470 nm) to irradiate a given Ir(III) complex and excite triplet oxygen (3O2) to singlet oxygen (1O2) which induces cell death in HeLa S3 cells (human cervical carcinoma cells). The results obtained in this study suggest that WPT-based PDT represents a potentially new method for the treatment of tumors by a non-battery LED, which are otherwise difficult to treat by previous PDT systems.

Cotransplantation With Adipose Tissue-derived Stem Cells Improves Engraftment of Transplanted Hepatocytes.

BACKGROUND: Hepatocyte transplantation is expected to be an alternative therapy to liver transplantation; however, poor engraftment is a severe obstacle to be overcome. The adipose tissue-derived stem cells (ADSCs) are known to improve engraftment of transplanted pancreatic islets, which have many similarities to the hepatocytes. Therefore, we examined the effects and underlying mechanisms of ADSC cotransplantation on hepatocyte engraftment. METHODS: Hepatocytes and ADSCs were cotransplanted into the renal subcapsular space and livers of syngeneic analbuminemic rats, and the serum albumin level was quantified to evaluate engraftment. Immunohistochemical staining and fluorescent staining to trace transplanted cells in the liver were also performed. To investigate the mechanisms, cocultured supernatants were analyzed by a multiplex assay and inhibition test using neutralizing antibodies for target factors. RESULTS: Hepatocyte engraftment at both transplant sites was significantly improved by ADSC cotransplantation ( P < 0.001, P < 0.001). In the renal subcapsular model, close proximity between hepatocytes and ADSCs was necessary to exert this effect. Unexpectedly, ≈50% of transplanted hepatocytes were attached by ADSCs in the liver. In an in vitro study, the hepatocyte function was significantly improved by ADSC coculture supernatant ( P < 0.001). The multiplex assay and inhibition test demonstrated that hepatocyte growth factor, vascular endothelial growth factor, and interleukin-6 may be key factors for the abovementioned effects of ADSCs. CONCLUSIONS: The present study revealed that ADSC cotransplantation can improve the engraftment of transplanted hepatocytes. This effect may be based on crucial factors, such as hepatocyte growth factor, vascular endothelial growth factor, and interleukin-6, which are secreted by ADSCs.

Optimization of dye solutions for detecting damaged pancreatic tissues during islet isolation procedures.

We previously reported that dye was effective to prevent the leakage of enzyme solutions from pancreatic glands during an islet isolation procedure. However, the dye used for islet isolation has not yet been optimized. In this study, we focused on pyoktanin blue (PB), diagnogreen (DG), and indigo carmine (IC) as potential candidates among clinically established dyes. A serial dilution assay was performed to determine minimal effective concentrations of each dye for detecting damaged pancreatic tissues. According to the outcome of serial dilution assays, double minimum effective concentrations of each dye were used for in vitro toxicity assays on islets and used in the isolation procedure to investigate whether they adversely affect islet isolation efficiency. The evaluations included islet yield, ADP/ATP, ATP/DNA, glucose stimulation test, and insulin/DNA assays. Islet viability cultured with PB contained medium was significantly lower than the other dyes. DG and IC appeared to be non-toxic to the islets. In isolation experiments, the islet yield in the DG group was considerably lower than that in the Control group, suggesting that DG might inhibit enzyme activity. The present study demonstrates that IC could be a promising candidate for an effective dye to detect damaged pancreatic tissues without affecting the enzyme activity and islet quality.

A Simple and Useful Predictive Assay for Evaluating the Quality of Isolated Hepatocytes for Hepatocyte Transplantation.

No optimal assay for assessing isolated hepatocytes before hepatocyte transplantation (HTx) has been established, therefore reliable and rapid assays are warranted. Isolated rat hepatocytes were dipped in a water bath (necrosis model), and were also cultured with Okadaic acid (apoptosis model) or vehicle, followed by cellular assessment including trypan blue exclusion (TBE) viability, ADP /ATP ratio, plating efficiency (PE), DNA quantity and ammonia elimination. Hepatocytes were transplanted into the liver of analbuminemic rats, subsequently engraftment was assessed by serum albumin and the histology of transplanted grafts. In the necrosis model, the ADP/ATP ratio was strongly and negatively correlated with the TBE (R2 = 0.559, P < 0.001). In the apoptosis model, the ADP/ATP ratio assay, PE, DNA quantification and an ammonia elimination test clearly distinguished the groups (P < 0.001, respectively). The ADP/ATP ratio, PE and DNA quantity were well-correlated and the ammonia elimination was slightly correlated with the transplant outcome. TBE could not distinguish the groups and was not correlated with the outcome. The ADP/ATP ratio assay predicted the transplant outcome. PE and DNA quantification may improve the accuracy of the retrospective (evaluations require several days) quality assessment of hepatocytes. The ADP/ATP ratio assay, alone or with a short-term metabolic assay could improve the efficiency of HTx.

Synergistic Effect of Neutral Protease and Clostripain on Rat Pancreatic Islet Isolation.

BACKGROUND: Islet isolation currently requires collagenase, neutral protease and other components. Thermolysin (TL) from Bacillus thermoproteolyticus is the gold standard neutral protease. However, we speculated that neutral protease derived from Clostridium histolyticum (Ch; ChNP) would be biologically superior for islet isolation. Tryptic-like activity has also been reported to be important. Therefore, we focused on clostripain (CP), since it is one of the main proteases in Clostridium histolyticum which possesses tryptic-like activity. We then examined the synergistic effects of highly purified ChNP and CP on rat islet isolation. METHODS: The same amount of collagenase was used in all four groups (TL, ChNP, TL+CP and ChNP+CP; n = 12/group). The efficiency was evaluated by the islet yield and function. An immunohistochemical analysis, in vitro digestion assay for each enzyme component and evaluation of the activation of endogenous exocrine proteases during islet isolation were also performed. RESULTS: The islet yield of the TL group was significantly higher than that of the ChNP group (P < 0.01). The islet yield was dose dependently increased in the ChNP+CP group, but was decreased in the TL + CP group. The islet yield in the ChNP + CP group was significantly higher than that in the TL group, but their islet function was similar. Different specificities for laminin, especially laminin-511, were observed in the TL, ChNP, and CP groups. CONCLUSIONS: Clostripain had a strong synergistic effect with ChNP, but not with TL. Therefore, ChNP and CP, in combination with collagenase derived from the same bacteria, may effectively increase the isolation efficiency without affecting the quality of islets.

Ductal injection does not increase the islet yield or function after cold storage in a vascular perfusion model.

Several studies have reported that pancreatic ductal preservation greatly improved the islet yield and function after cold storage. However, these studies were devoid of appropriate controls, such as vascular perfusion, which is routinely performed to preserve organs in the clinical setting. In this study, we created a vascular perfusion model using inbred rats, and investigated the effect of ductal injection on the islet yield and function after cold storage. Rat pancreases after 10 h cold ischemia were classified as follows: without ductal/vascular perfusion; with ductal injection; with vascular perfusion; and with ductal/vascular perfusion. The islet yield, function, viability, release of inflammatory mediators, and pathological changes in the exocrine tissues were assessed in the Hanks' Balanced Salt Solution (HBSS) model. The islet yield was also assesed by introducing University of Wisconsin Solution (UWS) and Histidine-Tryptophan-Ketoglutarate solution (HTK), which are the standard clinical preservation solutions. In the HBSS model, ductal injection and vascular perfusion significantly improved the islet yield compared with the control group. However, ductal injection showed no additional effects on the islet yield, function, viability and suppressing the release of inflammatory mediators when vascular perfusion was performed. Although ductal injection significantly decreased the apoptosis of exocrine cells, no beneficial effect on vacuolation was observed. In contrast, vascular perfusion significantly suppressed vacuolation in the exocrine tissues. Likewise, in the UWS and HTK model, ductal injection and vascular perfusion improved the islet yield compared with the control group. Nevertheless, the combination group showed no additional effects. These data suggest that ductal injection has no additional effect on islet yield and function after cold storage in a vascular perfusion model. We propose that ductal injection can be an effective and simple alternative for vascular perfusion prior to pancreas harvest, but is not necessary in most cases, since vascular perfusion is routinely performed.