Search

Abstract Primary hepatocytes are essential cellular resources for drug screening and medical transplantation. While culture systems have already succeeded in reconstituting the biomimetic microenvironment of primary hepatocytes, acquiring additional capabilities to handle them easily as well as to expand them remains unmet needs. This paper describes a culture system for primary rat hepatocytes, based on cell fiber technology, that brings scalability and handleability. Cell fibers are cell-laden core–shell hydrogel microfibers; in the core regions, cells are embedded in extracellular matrix proteins, cultured three-dimensionally, and exposed to soluble growth factors in the culture medium via the hydrogel shells. By encapsulating primary rat hepatocytes within cell fibers, we first demonstrated their proliferation while maintaining their viability and their hepatic specific functions for up to thirty days of subsequent culture. We then demonstrated the efficiency of proliferating primary rat hepatocytes in cell fibers not only as cell-based sensors to detect drugs that damage hepatic functions and hepatocellular processes but also as transplants to improve the plasma albumin concentrations of congenital analbuminemia. Our culture system could therefore be included in innovative strategies and promising developments in applying primary hepatocytes to both pharmaceutical and medical fields.

BackgroundMobile health (mHealth) interventions, a more cost-effective approach compared with traditional methods of delivering lifestyle coaching in person, have been shown to improve physical parameters and lifestyle behavior among overweight populations. In Japan, the Specific Health Checkups and Specific Health Guidance (SHG) started in 2008 to treat obesity and abdominal obesity. However, the effectiveness of SHG is limited owing to its in-person counseling. The effect of mHealth on SHG has yet to be demonstrated. ObjectiveThis study aims to determine whether a mobile self-management app (DialBeticsLite) could make the SHG more beneficial among patients with abdominal obesity to achieve a reduction in visceral fat area (VFA). MethodsThis study was an open-label, 2-arm, parallel-design randomized controlled trial. We recruited 122 people in September 2017 and randomly assigned them into either the intervention or control group. All participants attended an educational group session that delivered information regarding diet and exercise. In addition, participants in the intervention group were asked to use DialBeticsLite for 3 months. DialBeticsLite facilitated the daily recording of several physical parameters and lifestyle behavior and provided feedback to encourage an improvement in behavior. The primary outcome was the change in VFA from baseline to the 3-month follow-up. Secondary outcomes included changes in both physical and metabolic parameters from baseline to the 3-month follow-up. The Welch 2-tailed t test was conducted to analyze the effects of DialBeticsLite on both the primary and secondary outcomes. ResultsOf the 122 participants recruited, 75 (61.5%) were analyzed because 47 (38.5%) were excluded: 37 (30.3%) because of ineligibility and 10 (8.2%) because of withdrawal of consent. The mean age was 49.3 (SD 6.1) years in the intervention group (41/75, 55%) and 48.5 (SD 5.3) years in the control group (34/75, 45%), and all participants were men, although unintentionally. The baseline characteristics did not differ significantly between the intervention and control groups, except for VFA. The average change of VFA was −23.5 (SD 20.6) cm2 in the intervention group and +1.9 (SD 16.2) cm2 in the control group (P

Diabetes mellitus (DM) is a complex disease with high prevalence of comorbidity and mortality. DM is predicted to reach more than 700 million people by 2045. In recent years, several advanced in vitro models and analytical tools were developed to investigate the pancreatic tissue response to pathological situations and identify therapeutic solutions. Of all the in vitro promising models, cell culture in microfluidic biochip allows the reproduction of in-vivo-like micro-environments. Here, we cultured rat islets of Langerhans using dynamic cultures in microfluidic biochips. The dynamic cultures were compared to static islets cultures in Petri. The islets’ exometabolomic signatures, with and without GLP1 and isradipine treatments, were characterized by GC-MS. Compared to Petri, biochip culture contributes to maintaining high secretions of insulin, C-peptide and glucagon. The exometabolomic profiling revealed 22 and 18 metabolites differentially expressed between Petri and biochip on Day 3 and 5. These metabolites illustrated the increase in lipid metabolism, the perturbation of the pentose phosphate pathway and the TCA cycle in biochip. After drug stimulations, the exometabolome of biochip culture appeared more perturbed than the Petri exometabolome. The GLP1 contributed to the increase in the levels of glycolysis, pentose phosphate and glutathione pathways intermediates, whereas isradipine led to reduced levels of lipids and carbohydrates.

Summary: Hydrogel glucose sensors with boronic acid-based fluorescence intensity theoretically hold promise to improve in vivo continuous glucose monitoring (CGM) by facilitating long-lasting accuracy. However, these sensors generally degrade after implantation and the fluorescence intensity decreases immediately over time. Herein, we describe a hydrogel glucose sensor with in vivo stability based on boronic acid-based fluorescence intensity, integrating two antioxidant enzymes, superoxide dismutase (SOD), and catalase. These protected the arylboronic acid from being degraded by hydrogen peroxide in vitro and preserved the boronic acid-based fluorescence intensity of the hydrogel glucose sensors in rats for 28 days. These antioxidant enzymes also allowed the hydrogel glucose sensor attached to a homemade semi-implantable CGM device to trace blood glucose concentrations in rats for 5 h with the accuracy required for clinical settings. Hydrogel glucose sensors with boronic acid-based fluorescence intensity containing SOD and catalase could comprise a new strategy for in vivo CGM.

The collagenase secreted by Grimontia hollisae strain 1706B is a 74 kDa protein that consists of two parts: the catalytic module and a C‐terminal segment that includes the bacterial pre‐peptidase C‐terminal domain. Here, we produced a recombinant C‐terminal segment protein and examined its ability to bind collagen and other characteristics as compared with collagen‐binding domains (CBDs) derived from Hathewaya histolytica (Clostridium histolyticum) collagenases; these CBDs are the only ones thus far identified in bacterial collagenases. We found that the C‐terminal segment binds to collagen only when the collagen is in its triple‐helical conformation. Moreover, the C‐terminal segment and the CBDs from H. histolytica have comparable characteristics, including binding affinity to type I collagen, substrate spectrum, and binding conditions with respect to salt concentration and pH. However, the C‐terminal segment has a completely different primary structure from those of the CBDs from H. histolytica. As regards secondary structure, in silico prediction indicates that the C‐terminal segment may be homologous to those in CBDs from H. histolytica. Furthermore, we performed collagenase assays using fluorescein isothiocyanate‐labeled type I collagen to show that the C‐terminal segment positively contributes to the collagenolytic activity of the 74 kDa collagenase from G. hollisae.

Abstract Human pluripotent stem cells are a potentially powerful cellular resource for application in regenerative medicine. Because such applications require large numbers of human pluripotent stem cell-derived cells, a scalable culture system of human pluripotent stem cell needs to be developed. Several suspension culture systems for human pluripotent stem cell expansion exist; however, it is difficult to control the thickness of cell aggregations in these systems, leading to increased cell death likely caused by limited diffusion of gases and nutrients into the aggregations. Here, we describe a scalable culture system using the cell fiber technology for the expansion of human induced pluripotent stem (iPS) cells. The cells were encapsulated and cultured within the core region of core-shell hydrogel microfibers, resulting in the formation of rod-shaped or fiber-shaped cell aggregations with sustained thickness and high viability. By encapsulating the cells with type I collagen, we demonstrated a long-term culture of the cells by serial passaging at a high expansion rate (14-fold in four days) while retaining its pluripotency. Therefore, our culture system could be used for large-scale expansion of human pluripotent stem cells for use in regenerative medicine.

The proper functioning of many organs and tissues containing smooth muscles greatly depends on the intricate organization of the smooth muscle cells oriented in appropriate directions. Consequently controlling the cellular orientation in three-dimensional (3D) cellular constructs is an important issue in engineering tissues of smooth muscles. However, the ability to precisely control the cellular orientation at the microscale cannot be achieved by various commonly used 3D tissue engineering building blocks such as spheroids. This paper presents the formation of coiled spring-shaped 3D cellular constructs containing circumferentially oriented smooth muscle-like cells differentiated from dedifferentiated fat (DFAT) cells. By using the cell fiber technology, DFAT cells suspended in a mixture of extracellular proteins possessing an optimized stiffness were encapsulated in the core region of alginate shell microfibers and uniformly aligned to the longitudinal direction. Upon differentiation induction to the smooth muscle lineage, DFAT cell fibers self-assembled to coiled spring structures where the cells became circumferentially oriented. By changing the initial core-shell microfiber diameter, we demonstrated that the spring pitch and diameter could be controlled. 21 days after differentiation induction, the cell fibers contained high percentages of ASMA-positive and calponin-positive cells. Our technology to create these smooth muscle-like spring constructs enabled precise control of cellular alignment and orientation in 3D. These constructs can further serve as tissue engineering building blocks for larger organs and cellular implants used in clinical treatments.

For islet transplantation, maintaining organ viability after pancreas procurement is critically important for optimal graft function and survival. We recently reported that islet yield was significantly higher in the modified ET-Kyoto (MK) solution, which includes a trypsin inhibitor (ulinastatin), compared with the UW solution, and that the advantages of MK solution are trypsin inhibition and less collagenase inhibition. In this study, we compared ulinastatin with other trypsin inhibitors, gabexate mesilate, and nafamostat mesilate, in preservation solution for islet isolation. Ulinastatin was easily dissolved in ET-Kyoto solution, while ET-Kyoto with gabexate mesilate and nafamostat mesilate became cloudy immediately after addition. Although there were no significant differences in islet yield among the three groups, viability was significantly higher for the MK group than for the GK group or the NK group. The stimulation index was significantly higher for the MK group than for the GK group. In summary, there are no other trypsin inhibitors that are more effective than ulinastatin. Based on these data, we now use ET-Kyoto solution with ulinastatin for clinical islet transplantation.

In diabetes, a loss of pancreatic β-cells causes insulin dependency. When insulin dependency is caused by type 1 diabetes or pancreatic diabetes, for example, pancreatic β-cells need to be regenerated for definitive treatment. The methods for generating pancreatic β-cells include a method of creating pancreatic β-cells in vitro and implanting them into the body and a method of regenerating pancreatic β-cells in the body via gene introduction or the administration of differential proliferation factors to the body. Moreover, the number of pancreatic β-cells is also low in type 2 diabetes, caused by the compounding factors of insulin secretory failure and insulin resistance; therefore, if pancreatic β-cells can be regenerated in a living body, then a further amelioration of the pathology can be expected. The development of pancreatic β-cell-targeting regenerative medicine can lead to the next generation of diabetes treatment.

The development of a bioartificial pancreas (BAP) with immunoisolating fashion has been gaining attention as a new method for treating diabetes. We have been proceeding with the development of a bag-type BAP that can be easily implanted and that allows for the optional injection or rejection of cells at any time. If fibrosis develops around a BAP device, then the permeability of substances transmitted through a semipermeable membrane will decrease, thereby reducing the reactivity with glucose, so it is necessary for the material of the device to have an excellent histocompatibility. Furthermore, in order to improve the efficacy of BAP treatment, it is important to maintain an environment of ample blood flow around the device. We have created a bag-type device for BAP that is 20 × 20 mm in size and comprises two layers of membranes. We have used an EVAL membrane for the outer membrane of the two layers. The EVAL membrane is a semipermeable membrane with good insulin permeability, which functions as an immunoisolation membrane. The inner membrane consists of PAU-coated HD-PE (nonwoven material processed with polyaminourethan) and it is designed to function as a scaffold for cells. We used Lewis rats to determine whether the effectiveness of fibroblast growth factor 2 (bFGF) can be improved by concomitantly using bFGF with a capacity for blood vessel regeneration as well as bFGF immersed in a sheet of gelatin. We placed the BAP in the abdominal cavity and covered it with the greater omentum. We were able to significantly increase the blood flow and the number of new blood vessels in the tissue surrounding the BAP device by using gelatinized bFGF. There were only a few instances of fibrosis as a biological reaction to the EVAL membrane, and the infiltration of inflammatory cells was mild. There were no adverse effects related to implantation of the device. We confirmed in this study that the use of an implantable BAP device and bFGF allowed for a better blood flow around the BAP device. There were only minor instances of fibrosis and inflammation reaction around the BAP, thus indicating the BAP that we are currently developing to have an excellent histocompatibility.

Islet transplantation has recently emerged as an effective therapy and potential cure for type 1 diabetes mellitus. Recent reports show that the two-layer method (TLM), which employs oxygenated perfluorochemical (PFC) and University of Wisconsin (UW) solution, is superior to simple cold storage in UW for pancreas preservation in islet transplantation. Moreover, we recently reported that islet yield was significantly higher in the ET-Kyoto solution with ulinastatin (MK)/PFC preservation solution compared with the UW/PFC preservation solution in the porcine model and that the advantages of MK solution are trypsin inhibition and less collagenase inhibition. In this study, we compared ulinastatin with another trypsin inhibitor, Pefabloc, in preservation solution for islet isolation. Islet yield before purification was higher in the MK/PFC group compared with the ET-Kyoto with Pefabloc (PK)/PFC group. The stimulation index was higher for the MK/PFC group than for the PK/PFC group. These data suggest that ET-Kyoto with ulinastatin was the better combination for pancreas preservation than ET-Kyoto with Pefabloc. Based on these data, we now use ET-Kyoto solution with ulinastatin for clinical islet transplantation.

Recently, we demonstrated that islet transplantation from non-heart-beating donors (NHBDs) using the Kyoto islet isolation method (KIIM) successfully reversed patients' diabetes state. In this study, we evaluated the effects of donor- and isolation-related variables on islet isolation results from NHBDs by KIIM. Twenty-one islet preparations from the pancreata of NHBDs were isolated by KIIM. Islet preparations that met transplantation criteria and achieved improved patient diabetes control after transplantation were defined as successful isolations. Potential risk factors deemed to affect islet isolation results, such as age, gender, body mass index, hospital stay, donors' blood biochemical tests, a modified pancreata procurement method, and isolation and purification procedure-related variables, were analyzed. Seventeen out of 21 islet isolations (81%) were successful isolations. Postpurification islet yield was 447,639 ± 39,902 islet equivalents (IE) in the successful isolation group and 108,007 ± 31,532 IE in the failure group. Donor age was significantly younger in the success group (41.9 ± 4.0 years old in the success group vs. 57.5 ± 2.2 years old in the failure group, p = 0.003). Chronic pancreatitis significantly decreased islet yields (p = 0.006). Phase I time was significantly shorter (p = 0.010) and undigested tissue volume was significantly smaller (p = 0.020) in the success group. Purity was in positive correlation to postpurification islet yield, while donor age was in reverse correlation to postpurification islet yield. KIIM enables us to perform islet transplantation from NHBDs; however, the decision to use pancreata from older donors or those with chronic pancreatitis requires careful consideration.

Islet transplantation is associated with an elevated rate of early graft failure. The isolation process leads to structural and functional abnormalities. The reestablishment of the cell–matrix relationship is important to modulate the survival and function of islets. Thus, we evaluated the effect of human fibronectin (hFN) and self-assembling peptide nanofiber (SAPNF) in the ability to support islet function in vitro and after transplantation into streptozotocin (STZ)-induced diabetic severe combined immunodeficiency (SCID) mice. Human isolated islets were cultured with hFN or SAPNF for 7 days. Their ability to maintain insulin production/glucose responsiveness over time was evaluated. Islets embedded in hFN, SAPNF, or alone were transplanted into STZ-induced diabetic SCID mice. Islet grafts were removed after 14 days to evaluate insulin content, insulin expression, and apoptosis. SAPNF-entrapped islets maintained satisfactory morphology/viability and capability of glucose-dependent insulin secretion for over 7 days, whereas islets cultured in hFN underwent widespread deterioration. In vivo grafts containing human islets in SAPNF showed remarkably higher insulin content and expression when compared with human islets in hFn or alone. RT-PCR revealed lower caspase-3 expression in SAPNF islets grafts. These studies indicate that the reestablishment of the cell–matrix interactions by a synthetic matrix in the immediate postisolation period is a useful tool to maintain islet functions in vitro and in vivo.

For islet transplantation, it is important to obtain an available islet mass adequate for diabetes reversal from a single donor pancreas. A recent report demonstrated that the use of M-Kyoto solution instead of UW solution improved islet yields in the two-layer method for pancreas preservation. The present study investigated whether the ductal injection of a large volume of preservation solution (UW and M-Kyoto solution) before pancreas storage improves islet yields. Islet yield both before and after purification was significantly higher in the ductal injection (+) group compared with the ductal injection (–) group. TUNEL-positive cells in the ductal injection (+) group were significantly decreased in comparison to the ductal injection (–) group. The ductal injection of preservation solution increased the ATP level in the pancreas tissue and reduced trypsin activity during the digestion step. Annexin V and PI assays showed that the ductal injection prevents islet apoptosis. In a transplant model, the ductal injection improved islet graft function. These findings suggest that the ductal injection of preservation solution, especially the M-Kyoto solution, leads to improved outcomes for pancreatic islet transplantation. Based on these data, this technique is now used for clinical islet transplantation from non-heart-beating donor pancreata or living donor pancreas.

The evaluation of engraftment is important to assess the success of islet transplantation, but it is complex because islet transplantation usually requires two or more donors to achieve euglycemia. Islet transplantation from NHBDs was evaluated using new assessment forms for the secretory unit of islet in transplantation (SUIT) and engrafted islet rate (EIR) indexes. Insulin independence was obtained when the SUIT index was more than 28, which might indicate that 28% of the β-cell mass of a normal subject is required for insulin independence. Because the average EIR for a single transplantation is about 30, the percentage of engrafted islets following one transplantation is about 30%, assuming that a normal subject has 1 million islet equivalents. Although few cultured islet transplants have been performed, the increase of the SUIT and EIR indexes in patients who received cultured islets was significantly lower than in patients who received fresh islets, suggesting that fresh islets may be more effective than cultured islets. The SUIT and EIR indexes are thus considered to be useful values for evaluating islet transplantation, especially for single islet transplantation.

Development of a subcutaneously implantable bioartificial pancreas (BAP) with immunoisolatory function could have a great impact on the treatment of diabetes mellitus. We have developed an implantable BAP device with an ethylene vinyl alcohol (EVAL) membrane. In the present study, we used basic fibroblast growth factors (bFGF), which was incorporated in a carrier for sustained release, in order to induce neovascularization when the device was implanted subcutaneously. To maintain the vasculature thus formed, a cell infusion port was attached to the BAP device, through which the device was filled with human liver vascular endothelial cell line TMNK-1, and the vasculature could be adequately maintained. Mice were divided into the following three groups. In group 1, a bFGF-free BAP device was implanted subcutaneously. In group 2, a sustained-release bFGF-impregnated BAP device was implanted. In group 3, a sustained-release bFGF-impregnated BAP device was implanted, and 3 × 106 TMNK-1 cells were infused into the implanted device every week. Neovascularization induced in the subcutaneous tissue around the implanted BAP device was macroscopically examined and histologically evaluated. In addition, the tissue blood flow was measured using a laser blood flow meter. In mice in group 3, neovascularization was significantly induced and maintained until week 8 postimplantation. It was confirmed by scanning electron microscopy that infused TMNK-1 cells adhered to the inner polyethylene surface of the device. It was demonstrated that the use of bFGF and vascular endothelial TMNK-1 cells induced and maintained adequate vasculature and tissue blood flow surrounding the implantable bag-type BAP device. We believe that the present study will contribute to BAP development for the treatment of diabetes.

Fibroblast growth factor (FGF)-2 has been recognized to be a key element involved in angiogenesis and a putative factor involved in stem cell-mediated islet regeneration. However, the usefulness of FGF-2 in an islet transplantation setting has not yet been explored. We therefore evaluated the effect of FGF-2 on both islet culture and islet transplantation. Isolated islets were cultured in the presence of 100 ng/ml FGF-2 for a week and then the glucose-responding insulin secretion and insulin contents were measured. Gelatinized FGF-2 (100 ng), which allowed the controlled release of FGF-2, was used for islet transplantation of streptozotocin-induced diabetic mice. Islets (150 IEQ), obtained from a single donor, mixed with gelatinized FGF-2, were transplanted into the subrenal capsule of the mice and the animals were observed for 30 days. Revascularization around the islet grafts was examined. The blood glucose levels were measured and the intraperitoneal glucose tolerance test (IPGTT) was performed. The supplementation of FGF-2 maintained proper insulin secretion and insulin contents in an in vitro culture. The use of gelatinized FGF-2 facilitated revascularization and favorable islet engraftment, thus resulting in an amelioration of the blood glucose levels in diabetic mice. The utilization of FGF-2 showed increased contents of insulin in the islet grafts and revealed a similar pattern as that of normal healthy mice in IPGTT. In contrast, the transplantation of islets without FGF-2 supplementation showed poor revascularization and failed to control the blood glucose levels in the diabetic mice.

Development of an efficient preculture system of islets is ideal. Toward that goal, we constructed a human pancreatic islet-derived fibroblast cell line MNNK-1 for a source as a coculture system for freshly isolated islets to maintain islet functions. Human pancreatic islet cells were nucleofected with a plasmid vector pYK-1 expressing simian virus 40 large T antigen gene (SV40T) and hygromycin resistance gene (HygroR). One of the transduced cell lines, MNNK-1, was established and served as a feeder cell in the coculture for freshly isolated mouse, rat, and pig islets. Morphology, viability, and glucose-responding insulin secretion were analyzed in the coculture system. MNNK-1 cells were morphologically spindle shaped and were negative for pancreatic endocrine markers. MNNK-1 cells were positive for α-smooth muscle actin and collagen type I and produced fibroblast growth factor. Coculture of the mouse, rat, and pig islets with MNNK-1 cells maintained their viability and insulin secretion with glucose responsiveness. A human pancreatic islet-derived fibroblast cell line MNNK-1 was established. MNNK-1 cells were a useful means for maintaining morphology and insulin secretion of islets in the coculture system.

Achieving good islet isolation is one of the most important factors for successful islet transplantation. Porcine pancreas is suitable for islet isolation research due to its anatomical and physiological similarities to human pancreas. In this study, we evaluated a new porcine islet isolation method designed to maximize islet yield and compared it with our previous open pan method and the standard method using a Ricordi chamber (Ricordi method). We performed 15 porcine islet isolations, five each with the new method, the open pan method, and the Ricordi method. The new method features several important improvements. Pancreata remain uncut and are kept intact during collagenase intraductal injection, a large filtration chamber to handle whole pancreata, low concentration of collagenase (Liberase™ HI) for digestion, and large plastic containers for large-scale islet purification. All isolated islets were assessed for yield, purity, viability and in vitro function. Islets isolated with this new method were transplanted under the kidney capsules of SCID mice with chemically induced diabetes for in vivo functional assessment (n = 8). With the new method, we obtained on average more than 1,000,000 islet equivalents (IE) (1,236,266 ± 213,486 IE) (mean ± SE) before purification and 800,000 IE (879,815 ± 222,729 IE) after purification from one adult pig. Islet yield per pancreas was significantly higher compared with our previous open pan method (30,666 ± 11,532 IE, p < 0.01) and the Ricordi method (317,073 ± 86,093 IE, p < 0.05). All mice, transplanted with 1000 islets from the new method, returned to normoglycemia within 4 days after transplantation. Our new method makes it possible to obtain extremely high porcine islet yield with good function. It should produce useful information for human islet isolation and transplantation, and might be applied to single donor clinical xenogeneic transplantation.

PDX-1 plays a central role in regulating insulin gene transcription and differentiation of insulin-producing cells. It was previously reported that, due to its own Antennapedia-like protein transduction domain (PTD), exogenous PDX-1 protein can permeate cells and induces insulin gene expression in pancreatic ducts, thought to be islet progenitor cells. These data suggest that PDX-1 protein transduction could be a safe and valuable strategy for facilitating differentiation of progenitor cells into insulin-producing cells without requiring gene transfer technology. Here it is shown that after an initial ionic cell–surface interaction, PDX-1 proteins are rapidly internalized by lipid raft-dependent macropinocytosis. HeLa cells were treated with both FITC-conjugated PDX-1 PTD and FM 4–64, a general fluorescent marker of endocytosis. A punctate cytoplasmic distribution of PDX-1 PTD, which colocalized with FM 4–64, was observed in treated cells. Because expression of dominant-negative dynamin-1 did not block PDX-1 PTD uptake, PDX-1 protein transduction is independent on phagocytosis and clathrin- or caveolar-mediated endocytosis. Cells were pretreated with amiloride, a specific inhibitor of the Na+/H+ exchange required for macropinocytosis, or cytochalasin D, an F-actin elongation inhibitor. Treatment of cells with both macropinosome inhibitors resulted in the reduction in PDX-1 PTD transduction into vesicles, suggesting that PDX-1 PTD-mediated cellular entry occurs by lipid raft-mediated macropinocytosis. Taken together, these observations provide the mechanism of PDX-1 protein transduction and suggest that the protein transduction system could work for experimental and therapeutic strategies.

Development of liver-targeted cell therapies, such as hepatocyte transplantation and bioartificial livers, requires a large amount of functional hepatocytes as needed. To achieve this development, establishing an excellent cryopreservation method of hepatocytes is an extremely important issue. Therefore, we performed a comparative review of cryoprotective effects of various cryopreservation solutions using primarily isolated porcine hepatocytes. Porcine hepatocytes were isolated with a four-step dispase and collagenase perfusion method. The obtained hepatocytes with the initial viabilities of 76%, 84%, and 96% were assigned to the following four groups for cryopreservation at −80°C: Dulbecco's modified Eagle's medium (DMEM) + 10% fetal bovine serum (FBS) + 12% dimethyl sulfoxide (DMSO) (group A), University of Wisconsin (UW) solution + 12% DMSO (group B), Cell Banker 1 (group C), and Cell Banker 2 (group D). The hepatocytes in each group were thawed at 3 days, 10 days, and 5 months of cryopreservation and subjected to comparative analyses, including viability, plating efficiency, LDH release, ammonia removal test, and lentiviral gene transfer. These parameters were the most favorable in the hepatocytes cryopreserved with UW solution. Approximately 5% of thawed cryopreserved porcine hepatocytes expressed LacZ activity after lentiviral transduction. Intrasplenic transplantation of UW solution-cryopreserved hepatocytes improved the survival of rats treated with D-galactosamine. UW solution maintained the functions of cryopreserved porcine hepatocytes.

Normal human hepatocytes are an ideal source of liver-targeted cell therapies, such as hepatocyte transplantation and bioartificial livers, but availability of human donor livers for liver cell isolation is severely limited. To effectively utilize scarce donor organs for cell therapies, it is of extreme importance to establish an efficient isolation technique and an effective cold preservation solution for transportation of isolated cells. A lateral segment of the liver was surgically resected from pigs weighing 10 kg and a four-step collagenase and dispase digestion was conducted. Isolated hepatocytes were subjected to 8-h cold storage on ice. The following preservation solutions were tested: 1) University of Wisconsin (UW) solution, 2) UW with 100 μg/ml of ascorbic acid-2 glucoside (AA2G), 3) 100% fetal bovine serum (FBS), and 4) Dulbecco's modified Eagle's medium (DMEM) supplemented with 100% FBS. The mean viability of porcine hepatocytes was 95.5 ± 2.5% when isolated in three independent experiments. Viability, plating efficiency, membrane stability, and ammonia metabolic capacity of cold-preserved hepatocytes were significantly better maintained by the use of UW solution. When AA2G (100 μg/ml) was combined with UW solution, such parameters were further improved. It was explained by inhibition of caspase-3 activation and retention of ATP at high levels of hepatocytes preserved with UW solution containing AA2G. The present work demonstrates that a combination of UW solution with AA2G (100 μg/ml) would be a useful cold preservation means for the development of cell therapies.

Because human hepatic stellate cells (HSCs) perform a crucial role in the progress of hepatic fibrosis, it is of great value to establish an immortalized human cell line that exhibits HSC characteristics and grows well in tissue cultures for the development of antifibrotic therapies. Thus, we engineered an immortalized human hepatic stellate cell (HSC) line TWNT-4 by retrovirally inducing human telomerase reverse transcriptase (hTERT) into LI 90 cells established from a human liver mesenchymal tumor. Parental LI 90 entered replicative senescence, whereas TWNT-4 showed telomerase activity and proliferated for more than population doubling level (PDL) 200 without any crisis. TWNT-4 expressed platelet-derived growth factor-β receptor (PDGF-βR), α-smooth muscle actin (α-SMA), and type I collagen (α1) and was considered to be an activated form of HSCs. Treatment of TWNT-4 cells with either 100 U/ml of IFN-γ or 1 ng/ml of rapamycin (Rapa) for 14 days led to lower expression of type I collagen (α1) at RNA and protein levels. Exposure of TWNT-4 cells to both of IFN-γ (10 U/ml) and Rapa (0.1 ng/ml) for 14 days effectively decreased the expression of type I collagen (α1), PDGF-βR, and α-SMA expression and suppressed TGF-β1 secretion of TWNT-4 cells. We successfully induced apoptosis by transducing TNF-related apoptosis-inducing ligand (TRAIL) into TWNT-4 cells using adenovirus vectors Ad/GT-TRAIL and Ad/PGK-GV-17. These findings suggested that immortalized activated HSC line TWNT-4 would be a useful means to develop antifibrotic therapies.

We previously constructed an immortal human hepatocyte line NKNT-3 with a simian virus 40 T antigen (SV40T) to develop cell-based biological therapies. p21 is a molecule that regulates the transition from the G 1 phase to the S phase of the cell cycle. Investigators have demonstrated that overexpression of p21 induces differentiation in various cell lines. In the current study we examined the effect of p21 on differentiated phenotypes of SV40T-immortalized NKNT-3 cells. A replication-deficient adenovirus vector expressing a human wild-type p21 cDNA under the control of the CMV promoter (Ad5CMVp21) and a human wild-type p21 protein fused to the protein transduction domain from the human immunodeficiency virus (HIV) TAT protein (TAT/p21) were utilized to achieve efficient delivery of p21 into NKNT-3 cells. Morphological alterations, cell cycle progression, and expression of albumin and p-450 associated enzymes (CYPs) 3A4 and 2C9 were evaluated in NKNT-3 cells treated with Ad5CMVp21 and TAT/p21. Efficient adenovirus-based p21 transfer and TAT-mediated p21 protein delivery were confirmed in NKNT-3 cells in an immuno-fluorescence study and Western blotting analysis. Transduction of NKNT-3 cells with p21 predominantly arrested the cell cycle at the G 1 checkpoint, resulting in differentiated hepatic phenotypes in morphology and improvement in protein expression of albumin, CYP 3A4, and CYP C29. We here show that exogenous expression of p21 augments cellular differentiation in immortalized human NKNT-3 cells.

Because one of the attractive characteristics of human immunodeficiency virus type 1 (HIV-1)-based lentiviral vectors is that it can infect even nondividing cells, a lentivirus-mediated gene delivery system is currently being paid a great deal of attention as an innovative tool for gene transfer into target cells. The purpose of the work was to investigate the efficacy of lentiviral transfer of the LacZ gene into human umbilical vein endothelial cells (HUVECs) and human bone marrow mesenchymal stem cells (HMSCs) in vitro. For the present study, a vesicular stomatitis virus G-protein (VSV-G)-pseudotyped lentiviral vector encoding the E. coli LacZ gene tagged with nuclear localization signal (NLS) was generated in 293T cells by means of the three-plasmid system. The resulting lentiviral vector, LtV-NLS/LacZ, was allowed to infect HUVECs and HMSCs. Approximately 70% of HUVECs were positive for LacZ expression and 50% of HMSCs showed LacZ activity. There was no significant difference in transduction efficacy between early and late-passage phases in both cells. LtV-NLS/LacZ-transduced HUVECs showed gene expression of endothelial markers including CD34 and flt-1 and KDR/flk-1 of vascular endothelial growth factor (VEGF) receptors and had angiogenic potential as efficiently as primarily cultured HUVECs in a Matrigel assay. These findings provide evidence that lentiviral vectors are efficient tools for gene transfer and expression in human endothelial cells and stem cells that could be useful for tissue engineering.

With the development of biotechnology, hepatic support by a hybrid artificial liver (HAL) using hepatocytes has been given much attention. Because the availability of human livers is limited, we have established a tightly regulated immortal human hepatocyte cell line, NKNT-3, for developing HAL. Because high-density cell culture allows the compactness of the HAL device and its easy use under emergency circumstances, we have developed cell adhesive GRGDS peptide-containing cellulose microspheres (GRGDS/CMS). The GRGDS/CMS efficiently immobilized NKNT-3 cells within 24 h in a stirred suspension culture. Electron microscopic examinations demonstrated glycogen granules and well-developed endoplasmic reticulum and mitochondria in NKNT-3 cells attached to the GRGDS/CMS. The cells showed ammonia clearance activity, whereas HepG2-transformed human liver cells did not remove the loaded ammonia. An efficient adenoviral delivery of the lacZ reporter gene was performed in GRGDS/CMS-immobilized NKNT-3 cells. In this study we present rapid immobilization of NKNT-3 immortal human hepatocytes using cellulose microspheres carrying GRGDS peptides. These microspheres satisfied immediate preparation of NKNT-3 cells in sufficient quantity and of adequate quality.