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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.
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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.
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Hepatocitos , Hidrogeles , Animales , Proliferación Celular , Células Cultivadas , Medios de Cultivo , RatasRESUMEN
BACKGROUND: Mobile 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. OBJECTIVE: This 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). METHODS: This 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. RESULTS: Of 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<.001). Statistically significant differences were also found for the change of body weight, BMI, and waist circumference. These findings did not change after adjusting for VFA at the baseline. The intervention had no significant effect on any of the metabolic parameters. An exploratory analysis showed significant associations between the change in VFA and steps per day and between the change in VFA and calorie intake per day within the intervention group. CONCLUSIONS: Our findings indicate that an mHealth intervention facilitating the daily monitoring of several physical parameters and lifestyle behavior can be highly effective in inducing visceral fat loss and weight loss among adults eligible for SHG. TRIAL REGISTRATION: UMIN Clinical Trials Registry UMIN000042045; https://tinyurl.com/4vat3v53.
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Microneedle (MN) is a key technology of the biomedical engineering field due to its capability of accessing the biological information in a minimally invasive manner. One of the huge demands for next-generation healthcare monitoring is continuous monitoring, especially of blood glucose concentration. For this, MN should be kept inserted into the human skin for a certain period of time, enduring stresses induced by daily human motion and at the same time measuring biomarkers in ISF. However, conventional MNs for biosensing are not suitable for a long term insertion due to the rigid structure and biological risks of MN breakage. In this study, a novel MN structure is proposed and investigated by combining flexible "sponge-like" porous PDMS matrix and coating by biodissolving hyaluronic acid (HA). The fabricated porous MNs coated with HA show ideal mechanical characteristics, by which the MNs are rigid enough to penetrate the skin and become flexible after insertion into the skin. It is also shown that the MN array successfully extracts ISF in vitro and in vivo not by capillary action but by repeated compressions. The results show the applicability of the flexible MNs to continuous blood glucose monitoring.
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Automonitorización de la Glucosa Sanguínea/instrumentación , Fenómenos Mecánicos , Agujas , Microinyecciones , PorosidadRESUMEN
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.
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Transplantation technologies of pancreatic islets as well as stem cell-derived pancreatic beta cells encapsulated in hydrogel for the induction of immunoprotection could advance to treat type 1 diabetes mellitus, if the hydrogel transplants acquire retrievability through mitigating foreign body reactions after transplantation. Here, we demonstrate that the diameter of the fiber-shaped hydrogel transplants determines both in vivo cellular deposition onto themselves and their retrievability. Specifically, we found that the in vivo cellular deposition is significantly mitigated when the diameter is 1.0 mm and larger, and that 1.0 mm-thick xenoislet-laden fiber-shaped hydrogel transplants can be retrieved after being placed in the intraperitoneal cavities of immunocompetent diabetic mice for more than 100 days, during which period the hydrogel transplants can normalize the blood glucose concentrations of the mice. These findings could provide an innovative concept of a transplant that would promote the clinical application of stem cell-derived functional cells through improving their in vivo efficacy and safety.
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Diabetes Mellitus Experimental , Trasplante de Islotes Pancreáticos , Islotes Pancreáticos , Animales , Glucemia , Diabetes Mellitus Experimental/terapia , Reacción a Cuerpo Extraño/prevención & control , Control Glucémico , RatonesRESUMEN
Organ-on-chip technology is a promising tool for investigating physiological in vitro responses in drug screening development, and in advanced disease models. Within this framework, we investigated the behavior of rat islets of Langerhans in an organ-on-chip model. The islets were trapped by sedimentation in a biochip with a microstructure based on microwells, and perfused for 5 days of culture. The live/dead assay confirmed the high viability of the islets in the biochip cultures. The microfluidic culture leads to upregulation of mRNA levels of important pancreatic islet genes: Ins1, App, Insr, Gcgr, Reg3a and Neurod. Furthermore, insulin and glucagon secretion were higher in the biochips compared to the Petri conditions after 5 days of culture. We also confirmed glucose-induced insulin secretion in biochips via high and low glucose stimulations leading to high/low insulin secretion. The high responsiveness of the pancreatic islets to glucagon-like peptide 1 (GLP-1) stimulation in the biochips was reflected by the upregulation of mRNA levels of Gcgr, Reg3a, Neurog3, Ins1, Ins2, Stt and Glp-1r and by increased insulin secretion. The results obtained highlighted the functionality of the islets in the biochips and illustrated the potential of our pancreas-on-chip model for future pancreatic disease modeling and anti-diabetic drugs screening.
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Islotes Pancreáticos/fisiología , Páncreas/citología , Técnicas de Cultivo de Tejidos/métodos , Animales , Supervivencia Celular , Células Cultivadas , Expresión Génica , Glucagón/metabolismo , Glucosa/metabolismo , Insulina/metabolismo , Secreción de Insulina/genética , Islotes Pancreáticos/citología , Islotes Pancreáticos/metabolismo , Masculino , Procedimientos Analíticos en Microchip/métodos , Modelos Biológicos , Páncreas/metabolismo , Ratas , Ratas Wistar , Técnicas de Cultivo de Tejidos/instrumentación , Andamios del Tejido/químicaRESUMEN
Collagenase products are crucial to isolate primary cells in basic research and clinical therapies, where their stability in collagenolytic activity is required. However, currently standard collagenase products from Clostridium histolyticum lack such stability. Previously, we produced a recombinant 74-kDa collagenase from Grimontia hollisae, which spontaneously became truncated to ~60 kDa and possessed no stability. In this study, to generate G. hollisae collagenase useful as a collagenase product, we designed recombinant 62-kDa collagenase consisting only of the catalytic domain, which exhibits high production efficiency. We demonstrated that this recombinant collagenase is stable and active under physiological conditions. Moreover, it possesses higher specific activity against collagen and cleaves a wider variety of collagens than a standard collagenase product from C. histolyticum. Furthermore, it dissociated murine pancreata by digesting the collagens within the pancreata in a dose-dependent manner, and this dissociation facilitated isolation of pancreatic islets with masses and numbers comparable to those isolated using the standard collagenase from C. histolyticum. Implantation of these isolated islets into five diabetic mice led to normalisation of the blood glucose concentrations of all the recipients. These findings suggest that recombinant 62-kDa collagenase from G. hollisae can be used as a collagenase product to isolate primary cells.
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Separación Celular/métodos , Colagenasas/metabolismo , Proteínas Recombinantes/metabolismo , Vibrionaceae/enzimología , Animales , Islotes Pancreáticos/citología , Islotes Pancreáticos/metabolismo , Ratones , Vibrionaceae/genéticaRESUMEN
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.
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BACKGROUND: Reperitonealization has attracted increasing attention for its potential to prevent postoperative abdominal adhesions and subsequent related complications. We studied the effect of an autologous peritoneal graft on reperitonealization and prevention of adhesions in a rat model. METHODS: A standardized peritoneal lesion was induced on the parietal peritoneum by electrocoagulation and sutures. Twenty adult rats sustaining these lesions were randomized to 1 of 4 groups: (1) autologuous peritoneal graft with the side of mesothelial cells exposed to the abdominal cavity; (2) autologuous peritoneal graft with the side of subserosa containing fibroblasts exposed to the abdominal cavity; (3) cell sheet consisting of autologuous mesothelial cells and fibroblasts; or (4) nontreated group (Control). Fourteen days after the operation, abdominal adhesions were evaluated by macroscopic observation and histologic assessment. RESULTS: Macroscopic observation revealed that in mesothelial cells/fibroblasts grafts, there was no adhesion on the surface of the peritoneal graft covering the lesion. In contrast, in the other 3 groups, all rats obviously revealed extended and severe adhesions. Histology showed that mesothelial cells exist on the surface of the graft in mesothelial cells/fibroblasts graft, but no mesothelial cells were observed in the samples from the other groups. CONCLUSION: Autologous peritoneal grafts prevented postoperative abdominal adhesions in this rat model. As the mechanism of this prevention, the mesothelial cells survived and contributed to reperitonealization, only when they were transplanted as a part of the autologous peritoneal grafts and were located on the surface exposed to the abdomen.
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Técnicas de Cierre de Herida Abdominal , Peritoneo/trasplante , Complicaciones Posoperatorias/prevención & control , Adherencias Tisulares/prevención & control , Animales , Modelos Animales de Enfermedad , Masculino , Complicaciones Posoperatorias/patología , Distribución Aleatoria , Ratas , Ratas Sprague-Dawley , Adherencias Tisulares/patología , Trasplante Autólogo , Cicatrización de HeridasRESUMEN
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.
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Técnicas de Cultivo de Célula , Células Madre Pluripotentes Inducidas/citología , Animales , Recuento de Células , Técnicas de Cultivo de Célula/métodos , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Matriz Extracelular , Humanos , RatonesRESUMEN
Microencapsulation of cells is a promising technique in biomedical applications such as cell therapy. Recently, cell-laden hydrogel microfibers have been proposed as another shape microcapsule instead of microbeads; however, these are brittle with little stretching capability. This paper describes a cell-laden hydrogel microfiber that showed enhanced mechanical properties and handleability by using a double-network (DN) hydrogel consisting of alginate and polyacrylamide. The DN hydrogel microfiber supported approximately 6-fold higher strain and exhibited 10-fold higher tensile strength than the conventional alginate form. The DN hydrogel microfiber could also encapsulate pancreatic ß cells while maintaining cell viability and function. The in vivo functionality of the DN hydrogel microfiber was demonstrated by transplanting 3D assemblies of the microfibers into the intraperitoneal or subcutaneous space of diabetic mice, which successfully decreased their blood glucose levels. Thus, cell-laden DN hydrogel microfibers may represent a promising material for various biomedical applications.
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OBJECTIVES: Because therapeutic options for severe diabetes are currently limited, there is a continuing need for new therapeutic strategies, especially in the field of regenerative medicine. Collaborative efforts across the fields of tissue engineering technology and islet biology may be able to create functionally engineered islets capable of restoring endocrine function in patients with insulin-dependent diabetes. METHODS: This engineered scaffold was seeded with isolated primary porcine islets via the pancreatic duct using a multi-step infusion technique. Endocrine function of perfusion-cultured islets in the native scaffold was analyzed by immunohistochemical staining of insulin and glucagon as well as by the insulin stimulation test. RESULTS: The pancreas in this large animal could be uniformly decellularized by perfusion with trypsin and TritonX-100 via the pancreatic duct, as shown by positive staining of extracellular matrix (ECM) components. These scaffolds derived from porcine pancreas were able to maintain the cellular integrity of islets that had repopulated the parenchymal space, which is fundamental for the restoration of endocrine function. Insulin release up to four days after islet infusion was maintained. CONCLUSIONS: This scaffold from a large animal maintained islet survival and function in the short-term, retaining the cells as a solid organ in the parenchymal space after infusion through the pancreatic duct. These results suggest that this scaffold is suitable for further fabrication of fully functional bioengineered endocrine pancreases when implanted in vivo. Therefore, it may represent a key improvement in the field of beta-cell replacement therapy. Nonetheless, the facilitation of longer-term islet survival and studies of implantation in vivo is required for successful clinical translation.
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Sistemas de Infusión de Insulina , Islotes Pancreáticos/crecimiento & desarrollo , Andamios del Tejido , Animales , Separación Celular , Matriz Extracelular , Femenino , Insulina/metabolismo , Células Secretoras de Insulina , Islotes Pancreáticos/metabolismo , Conductos Pancreáticos/crecimiento & desarrollo , PorcinosRESUMEN
Piceatannol is a phytochemical in the seeds of passion fruit that has a hypoglycemic effect when orally administered. To elucidate the contribution of intact and metabolites of piceatannol after gastro-intestinal absorption to hypoglycemic effect, we examined the influence of piceatannol and isorhapontigenin on blood glucose concentrations during fasting and glucose tolerance tests by administering them intravascularly to freely moving healthy rats. We found that intravascularly administered piceatannol reduced the blood glucose concentrations during both fasting and glucose tolerance tests, but isorhapontigenin did not during either of them. Furthermore, we found that piceatannol increased the insulinogenic index during glucose tolerance tests and that piceatannol had no influence on insulin sensitivity by performing hyperinsulinemic euglycemic clamping tests. These results suggest that piceatannol orally intaken may enhance glucose tolerance by the effect of intact piceatannol through enhanced early-phase secretion of insulin. Therefore, oral intake of piceatannol might contribute to proper control of postprandial glycemic excursions in healthy subjects.
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Glucemia/efectos de los fármacos , Glucemia/metabolismo , Ayuno/sangre , Prueba de Tolerancia a la Glucosa , Resistencia a la Insulina/fisiología , Estilbenos/administración & dosificación , Administración Oral , Animales , Relación Dosis-Respuesta a Droga , Hipoglucemiantes/administración & dosificación , Inyecciones Intraarteriales , Inyecciones Intravenosas , Masculino , Ratas , Ratas Sprague-Dawley , Resultado del TratamientoRESUMEN
Adipose tissue, an active metabolic and endocrine organ mainly composed of unilocular adipocytes, is implicated in various obesity related diseases. Developing morphologically and functionally accurate in vitro models of the adipose tissue is therefore critically important for basic biological studies, drug screening/testing, and clinical implants to advance the understanding and treatment of these diseases. However, current adipose tissue engineering technologies either cannot replicate the unilocular morphologies of mature adipocytes, or lack the ease of monitoring, handling, and scaling up required in the above mentioned applications. This paper presents the differentiation of adipose derived stem cells (ADSCs) to mature adipocytes in highly observable and highly handleable 3D fiber shaped constructs exhibiting morphologies and functions of native adipose tissues. Using the cell fiber technology, ADSCs were encapsulated in hydrogel microfibers, allowed to form into fiber shaped constructs, and differentiated to mature unilocular adipocytes. These adipocyte fibers are observed and maintained for up to 91 d, and secretion of adipose tissue-specific factor, adiponectin, is further confirmed. The handleability of the adipocyte fibers is demonstrated by assembling the adipocyte fibers into doll shaped constructs. Such highly observable, highly handleable, and scalable characteristics of the adipocyte fibers make them suitable for biological studies, high-throughput drug screening/testing, and clinical applications.
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Adipocitos/citología , Hidrogel de Polietilenoglicol-Dimetacrilato/farmacología , Ingeniería de Tejidos/métodos , Adipocitos/efectos de los fármacos , Adipocitos/metabolismo , Adipocitos/ultraestructura , Adipogénesis/efectos de los fármacos , Adiponectina/metabolismo , Diferenciación Celular/efectos de los fármacos , Forma de la Célula/efectos de los fármacos , Células Cultivadas , Humanos , Imagenología Tridimensional , Factores de TiempoRESUMEN
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.
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Adipocitos/citología , Técnicas de Cultivo de Célula/instrumentación , Miocitos del Músculo Liso/citología , Ingeniería de Tejidos/métodos , Adipocitos/metabolismo , Alginatos/química , Animales , Autoanticuerpos/química , Biomarcadores/metabolismo , Proteínas de Unión al Calcio/metabolismo , Diferenciación Celular , Células Cultivadas , Diseño de Equipo , Ácido Glucurónico/química , Ácidos Hexurónicos/química , Hidrogeles/química , Dispositivos Laboratorio en un Chip , Proteínas de Microfilamentos/metabolismo , Músculo Liso/citología , Músculo Liso/metabolismo , Miocitos del Músculo Liso/metabolismo , Conejos , Ingeniería de Tejidos/instrumentación , Andamios del Tejido , CalponinasRESUMEN
A CMOS image sensor-based implantable glucose sensor based on an optical-sensing scheme is proposed and experimentally verified. A glucose-responsive fluorescent hydrogel is used as the mediator in the measurement scheme. The wired implantable glucose sensor was realized by integrating a CMOS image sensor, hydrogel, UV light emitting diodes, and an optical filter on a flexible polyimide substrate. Feasibility of the glucose sensor was verified by both in vitro and in vivo experiments.
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In living tissues, a cell is exposed to chemical substances delivered partially to its surface. Such a heterogeneous chemical environment potentially induces cell polarity. To evaluate this effect, we developed a microfluidic device that realizes spatially confined delivery of chemical substances at subcellular resolution. Our microfluidic device allows simple setup and stable operation for over 4 h to deliver chemicals partially to a single cell. Using the device, we showed that subcellular glucose exposure triggers an intracellular [Ca(2+)] change in the ß-cells. In addition, the imaging of a cell expressing GFP-tagged insulin showed that continuous subcellular exposure to glucose biased the spatial distribution of insulin granules toward the site where the glucose was delivered. Our approach illustrates an experimental technique that will be applicable to many biological experiments for imaging the response to subcellular chemical exposure and will also provide new insights about the development of polarity of ß-cells.
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Gránulos Citoplasmáticos/metabolismo , Glucosa/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Animales , Línea Celular , RatonesRESUMEN
After producing α1-3-galactosyltransferase knockout (GKO) pigs, most of the organs of these pigs showed less antigenicity to the human body. However, wild-type adult pig islets (API) that originally contained negligible levels of α-galactosidase now showed a clear antigenicity to human serum. In this study, N-glycans were isolated from both APIs and human islets. Their structures were then analyzed by a mapping technique based on their high-performance liquid chromatography elution positions and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometric data. Both preparations contained substantial amounts of high-mannose structures. The N-glycans from human islets were separated into 17 neutral, 8 mono-sialyl and 4 di-sialyl glycans, and the API glycans were comprised of 11 neutral, 8 mono-sialyl, 3 di-sialyl, 2 mono-sulfated, 3 mono-sialyl-mono-sulfated and 1 di-sulfated glycans. Among them, the API preparation contained one neutral, five mono-sialyl glycans and six sulfated glycans that were not detected in human islets. The structures of 9 of these 12 could be clearly determined. In addition, a study of the sulfate-depleted API suggests that sulfate residues could be antigenic to humans. The data herein will be helpful for future studies of the antigenicity associated with API.
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Islotes Pancreáticos/metabolismo , Polisacáridos/química , Porcinos , Animales , Animales Modificados Genéticamente , Secuencia de Carbohidratos , Galactosiltransferasas/genética , Galactosiltransferasas/metabolismo , Técnicas de Inactivación de Genes , Humanos , Datos de Secuencia Molecular , Polisacáridos/metabolismo , Porcinos/genética , Porcinos/metabolismoRESUMEN
BACKGROUND: Besides α-Gal expression, the differences of glycosylation and antigenicity between adult pig islets (APIs) and neonatal porcine islet-like cell clusters (NPCCs) are altogether unclear. In this study, lectin microarray analyses of NPCCs were performed and the results compared with the corresponding values for wild-type APIs and NPCCs from α-Gal transferase knockout (GalT-KO) pig. METHODS: NPCCs were isolated from 1-3-d-old neonatal wild-type pigs and cultured for 1 d, 5 d, and 9 d, using a previously described technique. Alternatively, the isoration of APIs were isolated based on the method for human islets. RESULTS: In a comparison between NPCCs and APIs, all of the NPCCs showed higher signals for Sambucus nigra, Sambucus sieboldiana, and Trichosanthes japonica I and the binding of α2,6 sialc acid, whereas the APIs showed stronger signals for Lotus tetragonolobus, Aleuria aurantia, Narcissus pseudonarcissus, and Galanthus nivalis, suggesting that APIs contain high levels of high-mannose forms. Among the NPCCs, NPCC (day1) appeared to be richer than the others in Lotus tetragonolobus, Narcissus pseudonarcissus, Galanthus nivalis, and Urtica dioica, implying the presence of high-mannose forms. However, as a whole, the signals for many lectins for NPCCs were very similar. The NPCCs from a GalT-KO pig indicated not only the downregulation of α-Gal expression but α-GalNAc as well, and α2-6 sialic acid was upregulated. CONCLUSIONS: The results reported herein contain useful information for the future production of immunomodified pigs with less antigenicity than GalT-KO pigs toward clinical applications of NPCCs.