京都大学 / 0048 / 新制・課程博士 / 博士(人間健康科学) / 甲第18197号 / 人健博第14号 / 新制||人健||2(附属図書館) / 31055 / 京都大学大学院医学研究科人間健康科学系専攻 / (主査)教授 藤井 康友, 教授 岡 昌吾, 教授 横出 正之 / 学位規則第4条第1項該当 / Doctor of Human Health Sciences / Kyoto University / DFAM

Diabetes Mellitus is a group of complex and heterogeneous metabolic disorders characterized by hyperglycemia. Even though the condition has been extensively studied, its causes and complex pathologies are still not fully understood. The occurring damage to the pancreatic islets is strikingly heterogeneous. In type 1 diabetes, the insulin producing beta cells are all destroyed within some islets, and similarly in type 2 diabetes, some islets may be severely affected by amyloid. At the same time other islets, in the near vicinity of the ones that are affected by disease, may appear fully normal in both diseases. Little is known about this heterogeneity in susceptibility to disease between pancreatic islets. This thesis examines the physiological and pathophysiological characteristics of islet subpopulations. Two subpopulations of islets were studied; one constituting highly vascularized islets with superior beta cell functionality, and one of low-oxygenated islets with low metabolic activity. The highly functional islets were found to be more susceptible to cellular stress both in vitro and in vivo, and developed more islet amyloid when metabolically challenged. Highly functional islets preferentially had a direct venous drainage, facilitating the distribution of islet hormones to the peripheral tissues. Further, these islets had an increased capacity for insulin secretion at low glucose levels, a response that was observed abolished in patients with recent onset type 1 diabetes.  The second investigated islet subpopulation, low-oxygenated islets, was found to be an over time stable subpopulation of islets with low vascular density and beta cell proliferation. In summary, two subpopulations of islets can be identified in the pancreas based on dissimilarities in vascular support and blood flow. These subpopulations appear to have different physiological functions of importance for the maintenance of glucose homeostasis. However, they also seem to differ in vulnerability, and a preferential death of the highly functional islets may accelerate the progression of both type 1 and type 2 diabetes.

Thesis (PhD)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Insights into the role of oxidative stress and pancreatic β-cell dysfunction in the pathogenesis of type 2 diabetes (T2D) reveals an opportunity for the development of novel therapeutics that directly protect and preserve β-cells. The protective role of dietary antioxidants, such as plant polyphenols, against oxidative stress induced diseases, including T2D, is increasingly under scrutiny. Polyphenol-rich extracts of Cyclopia spp, containing mangiferin, may provide novel therapeutics. An aqueous extract of unfermented Cyclopia maculata, containing more than 6 % mangiferin, was assessed for its protective effect in pancreatic β-cells in vitro, ex vivo and in vivo under conditions characteristic of T2D. The effect of mangiferin was also evaluated in vitro and ex vivo, with N-acetyl cysteine (NAC) as an antioxidant control. In this study, we established in vitro toxicity models in RIN-5F insulinoma cells based on conditions β-cells are exposed to in T2D; i.e. lipotoxicity, inflammation and oxidative stress conditions. To achieve this, cells were exposed to the following stressors: palmitic acid (PA), a pro-inflammatory cytokine combination and streptozotocin (STZ), respectively. Thereafter, the ability of the C. maculata extract, mangiferin and NAC to protect RIN-5F cells from the effects of these stressors was assessed by measuring β-cell viability, function and oxidative stress. Cell viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, adenosine triphosphate and annexin-V and propidium iodide assays. Cell function was evaluated by measuring glucose stimulated insulin secretion, cell proliferation and cellular calcium. To assess oxidative stress in the RIN-5F cells, diaminofluorescein-FM and dihydroethidium fluorescence, and superoxide dismutase enzyme activity were measured. The in vitro findings were then verified in isolated pancreatic rat islets using methods and models established in the RIN-5F experiments. The protective effect of the extract, NAC and metformin was assessed in STZ induced diabetic Wistar rats, using two treatment regimes, i.e. by treating rats with established diabetes and by pretreating rats prior to induction of diabetes by STZ. Glucose metabolism, oxidative stress and pancreatic morphology were assessed by performing an oral glucose tolerance test, measuring serum insulin, triglycerides, nitrites, catalase and glutathione. Hepatic thiobarbituric acid reactive substances and nitrotyrosine were also assessed. Immunohistochemical labelling of pancreata with insulin, glucagon and MIB-5 was used for morphological assessment. The extract improved β-cell viability, function and attenuated oxidative stress, most apparently in STZ and PA induced toxicity models comparable with NAC both in vitro and in isolated islets. Mangiferin was not as effective, showing only marginal improvement in RIN-5F cell and islet function, and oxidative stress. Pretreatment of STZ induced diabetic Wistar rats with extract was as effective as, if not better than, metformin in improving glucose tolerance, hypertriglyceridaemia and pancreatic islet morphology related to improved β-cell function. This study demonstrated that the aqueous extract of unfermented C. maculata was able to protect pancreatic β-cells from STZ and PA induced toxicity in vitro and ex vivo. In vivo, pretreatment with the extract improved glucose metabolism and pancreatic islet morphology in STZ induced diabetic Wistar rats. / AFRIKAANSE OPSOMMING: Insigte oor die rol wat oksidatiewe stres en pankreas β-sel disfunksie in die patogenese van tipe 2-diabetes (T2D) speel, bied 'n geleentheid vir die ontwikkeling van nuwe terapeutiese middels wat β-selle direk daarteen beskerm. Die beskermende rol van antioksidante in die dieët soos plantaardige polifenole teen oksidatiewe stres geinduseerde siektes soos T2D, is toenemend onder die soeklig. Polifenolryk ekstrakte van Cyclopia spp wat mangiferin bevat mag nuwe terapeutiese middels lewer. ‘n Waterekstrak van ongefermenteerde Cyclopia maculata wat meer as 6% mangiferin bevat, is ondersoek vir sy beskermende effek op pankreas ß-selle in vitro, ex vivo en in vivo teen kondisies kenmerkend aan T2D. Die effek van mangiferin is ook in vitro en ex vivo geëvalueer, met N-asetielsistien (NAC) as 'n antioksidant kontrole. In hierdie studie is in vitro toksisiteitsmodelle in RIN-5F insulinoomselle gevestig. Die modelle is gebaseer op toestande waaraan β-selle blootgestel word tydens T2D; d.w.s. lipotoksisiteit, inflammasie en oksidatiewe stres. Hiervoor is die selle aan die volgende stressors blootgestel: palmitiensuur (PA), ‘n pro-inflammatoriese sitokien mengsel en streptozotosien (STZ). Vervolgens is die vermoë van die C. maculata ekstrak, mangiferin en NAC om die RIN-5Fselle teen hierdie stressors te beskerm, beoordeel deur die meting van β-sellewensvatbaarheid, funksie en oksidatiewe stres. Sellewensvatbaarheid is bepaal met 3-(4,5-dimetielthiazol-2-yl)-2,5-difenieltetrazolium bromied, adenosientrifosfaat en anneksien-V and propidium jodied toetse. Selfunksie is geëvalueer d.m.v. glukose gestimuleerde insuliensekresie, selproliferasie en sellulêre kalsium bepaling. Oksidatiewe stres in die RIN-5Fselle is geëvalueer d.m.v. diaminofluorescein-FM en dihidroethidium fluoressensie bepalings, asook meting van superoksied dismutase ensiemaktiwiteit. Die in vitro bevindings is daarna in geїsoleerde rot pankreaseilande bevestig deur die metodes en modelle wat in die RIN-5F eksperimente gebruik is. Die antidiabetiese effekte van die ekstrak, NAC en metformien in STZ-geїnduseerde diabetiese Wistar rotte is bepaal d.m.v. twee behandlingsregimes, d.w.s. die behandeling van rotte met gevestigde diabetes of deur die behandeling voor die induksie van diabetes te begin. Glukose metabolisme, oksidatiewe stres en veranderinge in die pankreasmorfologie is ondersoek d.m.v. orale glukose toleransie toetse en die bepaling van serum insulien, trigliseriedes, nitriete, katalase en glutationien. Hepatiese tiobarbituursuur reaktiewe stowwe en nitrotirosien is ook geëvalueer. Immunohistochemiese kleuring van pankreas snitte is gebruik vir morfologiese assessering van insulien, glukagon en MIB-5. Die ekstrak het mees opvallend β-sel lewensvatbaarheid en funksie verbeter, terwyl oksidatiewe stres verminder is in die STZ- en PA-geїnduseerde toksisiteitmodelle. Bogenoemde effekte van die ekstrak in vitro en in die geїsoleerde eilande was vergelykbaar met die van NAC. Mangiferin was minder effektief, met slegs ‘n marginale verbetering in die funksie van RIN-5Fselle en eilande, asook t.o.v. oksidatiewe stres. Behandeling van die Wistar rotte met die ekstrak voor induksie van diabetes met STZ was net so effektief, of selfs beter as metformien in terme van verbeterde glukosetoleransie, trigliseriedvlakke en die morfologie van pankreas eilande wat verband gehou het met β-sel funksie. Hierdie studie het getoon dat die waterekstrak van ongefermenteerde C. maculata pankreas β-selle teen veral STZ- en PA-geїnduseerde toksisiteit in vitro en ex vivo beskerm het. In vivo het behandeling met die ekstrak voor en na induksie van diabetes, glukosemetabolisme en die morfologie van pankreas eilande in STZ-geїnduseerde diabetiese Wistar rotte verbeter.

O Diabetes mellitus (DM) do tipo 1 é uma doença causada pela destruição, por mecanismo auto-imune, das células beta das ilhotas pancreáticas, produtoras de insulina. O tratamento convencional da doença é realizado por meio de injeções diárias de insulina exógena. O transplante de ilhotas pancreáticas inclui-se, atualmente, como uma das alternativas terapêuticas à insulinoterapia. Entretanto, para atingir a insulino-independência, é necessário transplantar um grande número de ilhotas por paciente. O conhecimento do mecanismo de proliferação das células beta pode possibilitar a realização do transplante a partir da expansão celular ex vivo. A glicose é um dos principais indutores da proliferação de células beta. Neste trabalho, foi estabelecida e executada a tecnologia de isolamento e purificação de ilhotas pancreáticas humanas, visando sua estimulação com glicose. Para identificar genes regulados por glicose nestas ilhotas, foi utilizada a técnica de hibridização subtrativa SSH, associada ao rastreamento da biblioteca através de macroarranjos de DNA. Num primeiro rastreamento, foram identificados dois fragmentos gênicos induzidos pela glicose. Um destes apresentou homologia com uma proteína hipotética humana de função desconhecida e o segundo com o receptor de polipetídeo pancreático. Este trabalho permitiu a identificação de novos genes regulados pela glicose em ilhotas pancreáticas humanas, os quais podem estar relacionados à proliferação celular deste tecido. / Type 1 Diabetes mellitus (T1DM) is caused by autoimmune destruction of the insulin-producing pancreatic islet b-cells. Treatment is generally approached by daily subcutaneous injections of exogenous insulin. Nowadays, pancreatic islet transplantation is considered as an effective alternative treatment to insulin therapy. However, in order to reach insulin-independence, a large number of islets is required for each patient. Knowledge of the mechanisms regulating islet b-cell proliferation may allow ex-vivo b-cell expansion prior to transplant. Glucose is considered one of the main inducers of islet b-cells proliferation. We established and executed the technology of human islet isolation and purification. The islets were then stimulated in culture with glucose. In order to identify glucose-regulated genes in cultured human islets, we utilized the suppression subtractive hybridization (SSH) method, followed by cDNA library screening by DNA macroarrays. Preliminary screening allowed us to isolate two cDNAs displaying glucose regulation, one of which is similar to a human hypothetical protein of unknown function and the other shows similarity to the pancreatic polypeptide receptor. This work allowed identification of glucose-regulated genes in human pancreatic islets, which may be related to cell proliferation in this tissue.

O Diabetes mellitus (DM) do tipo 1 é uma doença causada pela destruição, por mecanismo auto-imune, das células beta das ilhotas pancreáticas, produtoras de insulina. O tratamento convencional da doença é realizado por meio de injeções diárias de insulina exógena. O transplante de ilhotas pancreáticas inclui-se, atualmente, como uma das alternativas terapêuticas à insulinoterapia. Entretanto, para atingir a insulino-independência, é necessário transplantar um grande número de ilhotas por paciente. O conhecimento do mecanismo de proliferação das células beta pode possibilitar a realização do transplante a partir da expansão celular ex vivo. A glicose é um dos principais indutores da proliferação de células beta. Neste trabalho, foi estabelecida e executada a tecnologia de isolamento e purificação de ilhotas pancreáticas humanas, visando sua estimulação com glicose. Para identificar genes regulados por glicose nestas ilhotas, foi utilizada a técnica de hibridização subtrativa SSH, associada ao rastreamento da biblioteca através de macroarranjos de DNA. Num primeiro rastreamento, foram identificados dois fragmentos gênicos induzidos pela glicose. Um destes apresentou homologia com uma proteína hipotética humana de função desconhecida e o segundo com o receptor de polipetídeo pancreático. Este trabalho permitiu a identificação de novos genes regulados pela glicose em ilhotas pancreáticas humanas, os quais podem estar relacionados à proliferação celular deste tecido. / Type 1 Diabetes mellitus (T1DM) is caused by autoimmune destruction of the insulin-producing pancreatic islet b-cells. Treatment is generally approached by daily subcutaneous injections of exogenous insulin. Nowadays, pancreatic islet transplantation is considered as an effective alternative treatment to insulin therapy. However, in order to reach insulin-independence, a large number of islets is required for each patient. Knowledge of the mechanisms regulating islet b-cell proliferation may allow ex-vivo b-cell expansion prior to transplant. Glucose is considered one of the main inducers of islet b-cells proliferation. We established and executed the technology of human islet isolation and purification. The islets were then stimulated in culture with glucose. In order to identify glucose-regulated genes in cultured human islets, we utilized the suppression subtractive hybridization (SSH) method, followed by cDNA library screening by DNA macroarrays. Preliminary screening allowed us to isolate two cDNAs displaying glucose regulation, one of which is similar to a human hypothetical protein of unknown function and the other shows similarity to the pancreatic polypeptide receptor. This work allowed identification of glucose-regulated genes in human pancreatic islets, which may be related to cell proliferation in this tissue.

O transplante de ilhotas pancreáticas humanas representa uma estratégia promissora para a cura do diabetes mellitus tipo 1 (DM1), mas a aplicação a todos os pacientes diabéticos ainda é impraticável devido à limitada disponibilidade de ilhotas ou células β e à necessidade de utilização de drogas imunossupressoras pelo paciente transplantado. O tratamento com imunossupressores após o transplante de ilhotas pode ser abolido quando se realiza o microencapsulamento das ilhotas pancreáticas. Neste trabalho investigou-se um novo biomaterial, Biodritina® (alginato/sulfato de condroitina) adequado ao microencapsulamento que gelifica na presença de íons de cálcio ou bário. A biocompatibilidade das microcápsulas tem sido avaliada segundo o grau de pureza do alginato utilizado na sua confecção. Amostras de alginato comercial purificado foram analisadas, comprovando-se a presença de impurezas (polifenóis, endotoxinas, proteínas) em níveis elevados, que impedem sua aplicação clínica. Optou-se, portanto pela utilização do alginato comercial ultrapurificado nos experimentos descritos neste trabalho. Das formulações de biomateriais avaliadas, as microcápsulas de bário-Biodritina apresentaram o melhor desempenho em testes de estabilidade físico-química. Estas microcápsulas mantiveram sua morfologia e estabilidade estrutural após permanecerem 30 dias na cavidade peritoneal de camundongos, conforme demonstrado por microscopia eletrônica de varredura (MEV). Análises histológicas mostraram que microcápsulas de bário-Biodritina explantadas, não possuíam adesão celular em sua superfície. Estudos de permeabilidade demonstraram que o tamanho médio dos poros das microcápsulas de bário-Biodritina permite passagem de proteínas de até 70 kDa, enquanto os poros daquelas de cálcio-Biodritina comportam proteínas de até 100 kDa. Experimentos de coResumo | x cultivo de macrófagos peritoneais com ilhotas de rato microencapsuladas demonstraram uma capacidade imunoprotetora maior das microcápsulas de bário-Biodritina em relação às de cálcio- Biodritina, sendo que as primeiras não ativaram os macrófagos. A manutenção da viabilidade e função de ilhotas humanas microencapsuladas com bário-Biodritina foi confirmada através de ensaio funcional in vitro, no qual ilhotas microencapsuladas apresentaram níveis de secreção de insulina idênticos aos de ilhotas nuas. A prova de conceito do biomaterial foi realizada através do implante de ilhotas humanas microencapsuladas em bário-Biodritina em camundongos com DM1 induzido por estreptozotocina. A hiperglicemia desses animais foi corrigida pelo implante por um período superior a 60 dias, durante os quais o teste oral de tolerância à glicose mostrou-se normal, demonstrando completa funcionalidade e eficiência das ilhotas microencapsuladas com bário-Biodritina. Partindo de observações de que animais inoculados com a peçonha do escorpião Tityus serrulatus apresentam nesidioblastose, foi realizado o fracionamento do veneno por HPLC de fase reversa e 24 frações obtidas foram submetidas a ensaios de proliferação celular através da incorporação de 3H-timidina em células de insulinoma de rato RINm5F. Uma dessas frações foi capaz de induzir a proliferação das células RINm5F e quando aplicada a ilhotas humanas isoladas, elevou o índice de secreção de insulina e induziu um aumento da expressão dos mRNAs de insulina e PCNA. Portanto, demonstrou-se que o biomaterial bário-Biodritina possui as características necessárias para microencapsular células/ilhotas com eficiência e que a \"fração ativa\" do veneno do escorpião T. serrulatus induz proliferação de células RINm5F e melhora a secreção de insulina de ilhotas humanas. / Islet transplantation has been proposed as a promising therapeutic strategy for the cure of type 1 diabetes mellitus (DM), however, its application to all diabetic patients is still not possible due to the limited source of islets or β cells and to the need of an immunosuppressive treatment of the recipient to avoid graft rejection. The use of immunosupressors may be abolished when pancreatic islets are microencapsulated prior to transplantation. Here, we investigated the use of a new biomaterial suitable for cell microencapsulation, namely, Biodritin®, composed of alginate and chondroitin sulphate, which is capable of gelation in the presence of barium or calcium ions. Microcapsules biocompatibility has been evaluated according to the purity of the alginate used in its production. Samples of purified commercial alginate were analyzed, but the high levels of contaminants (proteins, endotoxins and polyphenols) detected prevented its use in clinical applications. On the other hand, also commercially available ultrapure alginate fulfills the requirements for this application, therefore, this biomaterial was chosen for our experiments. Among the different biomaterial formulations evaluated, barium-Biodritin microcapsules displayed the best performance in the physico-chemical tests. Scanning electronic microscopy revealed that barium-Biodritin microcapsules maintained their morphology and structural stability after being implanted for 30 days in the peritoneal cavity of mice. No cellular adhesion was detected on the surface of explanted barium-Biodritin microcapsules by histological analysis. Permeability studies determined the medium pore size of barium-Biodritin microcapsules, which allows proteins of up to 70 kDa to pass through the biomaterial, while calcium-Biodritin pores accomodate proteins of up to 100 kDa. Co-culture of peritoneal macrophages with microencapsulated rat islets, revealed a superior immunoprotective capacity of barium-Biodritin microcapsules, which were capable of protecting the islets with no macrophage activation. Microencapsulated and naked human islets presented identical insulin secretion levels upon stimulation with glucose in vitro, confirming that barium-Biodritin microencapsulation maintains the function and viability of human islets. Proof-of-concept experiments in which barium-Biodritin microencapsulated human islets were implanted into chemically-induced diabetic mice, showed that these animals maintained normal blood glucose levels for more than 60 days, during which oral glucose tolerance tests were normal, demonstrating the complete functionality and efficiency of barium-Biodritin microencapsulated human islets. From the observation that animals inoculated with the venom of the scorpion Tityus serrulatus presented nesidioblastosis, we decided to fractionate the venom to isolate the active principle. The venom was fractionated by reversed phase HPLC and 24 fractions were obtained and submitted to cellular proliferation assays, in which rat insulinoma RINm5F cells evaluated for 3H-timidina incorporation. One of these fractions was capable of inducing cell proliferation and was also applied to isolated human islets. Treated islets presented a higher insulin secretion index and an increase in insulin and PCNA mRNA expression. In conclusion, we demonstrated that the barium-Biodritin biomaterial possesses all characteristics required for efficient cell/islet microencapsulation and that the active fraction of Tityus serrulatus venom induces the proliferation of RINm5F cells and improves insulin secretion in human islets.

O transplante de ilhotas pancreáticas humanas representa uma estratégia promissora para a cura do diabetes mellitus tipo 1 (DM1), mas a aplicação a todos os pacientes diabéticos ainda é impraticável devido à limitada disponibilidade de ilhotas ou células β e à necessidade de utilização de drogas imunossupressoras pelo paciente transplantado. O tratamento com imunossupressores após o transplante de ilhotas pode ser abolido quando se realiza o microencapsulamento das ilhotas pancreáticas. Neste trabalho investigou-se um novo biomaterial, Biodritina® (alginato/sulfato de condroitina) adequado ao microencapsulamento que gelifica na presença de íons de cálcio ou bário. A biocompatibilidade das microcápsulas tem sido avaliada segundo o grau de pureza do alginato utilizado na sua confecção. Amostras de alginato comercial purificado foram analisadas, comprovando-se a presença de impurezas (polifenóis, endotoxinas, proteínas) em níveis elevados, que impedem sua aplicação clínica. Optou-se, portanto pela utilização do alginato comercial ultrapurificado nos experimentos descritos neste trabalho. Das formulações de biomateriais avaliadas, as microcápsulas de bário-Biodritina apresentaram o melhor desempenho em testes de estabilidade físico-química. Estas microcápsulas mantiveram sua morfologia e estabilidade estrutural após permanecerem 30 dias na cavidade peritoneal de camundongos, conforme demonstrado por microscopia eletrônica de varredura (MEV). Análises histológicas mostraram que microcápsulas de bário-Biodritina explantadas, não possuíam adesão celular em sua superfície. Estudos de permeabilidade demonstraram que o tamanho médio dos poros das microcápsulas de bário-Biodritina permite passagem de proteínas de até 70 kDa, enquanto os poros daquelas de cálcio-Biodritina comportam proteínas de até 100 kDa. Experimentos de coResumo | x cultivo de macrófagos peritoneais com ilhotas de rato microencapsuladas demonstraram uma capacidade imunoprotetora maior das microcápsulas de bário-Biodritina em relação às de cálcio- Biodritina, sendo que as primeiras não ativaram os macrófagos. A manutenção da viabilidade e função de ilhotas humanas microencapsuladas com bário-Biodritina foi confirmada através de ensaio funcional in vitro, no qual ilhotas microencapsuladas apresentaram níveis de secreção de insulina idênticos aos de ilhotas nuas. A prova de conceito do biomaterial foi realizada através do implante de ilhotas humanas microencapsuladas em bário-Biodritina em camundongos com DM1 induzido por estreptozotocina. A hiperglicemia desses animais foi corrigida pelo implante por um período superior a 60 dias, durante os quais o teste oral de tolerância à glicose mostrou-se normal, demonstrando completa funcionalidade e eficiência das ilhotas microencapsuladas com bário-Biodritina. Partindo de observações de que animais inoculados com a peçonha do escorpião Tityus serrulatus apresentam nesidioblastose, foi realizado o fracionamento do veneno por HPLC de fase reversa e 24 frações obtidas foram submetidas a ensaios de proliferação celular através da incorporação de 3H-timidina em células de insulinoma de rato RINm5F. Uma dessas frações foi capaz de induzir a proliferação das células RINm5F e quando aplicada a ilhotas humanas isoladas, elevou o índice de secreção de insulina e induziu um aumento da expressão dos mRNAs de insulina e PCNA. Portanto, demonstrou-se que o biomaterial bário-Biodritina possui as características necessárias para microencapsular células/ilhotas com eficiência e que a \"fração ativa\" do veneno do escorpião T. serrulatus induz proliferação de células RINm5F e melhora a secreção de insulina de ilhotas humanas. / Islet transplantation has been proposed as a promising therapeutic strategy for the cure of type 1 diabetes mellitus (DM), however, its application to all diabetic patients is still not possible due to the limited source of islets or β cells and to the need of an immunosuppressive treatment of the recipient to avoid graft rejection. The use of immunosupressors may be abolished when pancreatic islets are microencapsulated prior to transplantation. Here, we investigated the use of a new biomaterial suitable for cell microencapsulation, namely, Biodritin®, composed of alginate and chondroitin sulphate, which is capable of gelation in the presence of barium or calcium ions. Microcapsules biocompatibility has been evaluated according to the purity of the alginate used in its production. Samples of purified commercial alginate were analyzed, but the high levels of contaminants (proteins, endotoxins and polyphenols) detected prevented its use in clinical applications. On the other hand, also commercially available ultrapure alginate fulfills the requirements for this application, therefore, this biomaterial was chosen for our experiments. Among the different biomaterial formulations evaluated, barium-Biodritin microcapsules displayed the best performance in the physico-chemical tests. Scanning electronic microscopy revealed that barium-Biodritin microcapsules maintained their morphology and structural stability after being implanted for 30 days in the peritoneal cavity of mice. No cellular adhesion was detected on the surface of explanted barium-Biodritin microcapsules by histological analysis. Permeability studies determined the medium pore size of barium-Biodritin microcapsules, which allows proteins of up to 70 kDa to pass through the biomaterial, while calcium-Biodritin pores accomodate proteins of up to 100 kDa. Co-culture of peritoneal macrophages with microencapsulated rat islets, revealed a superior immunoprotective capacity of barium-Biodritin microcapsules, which were capable of protecting the islets with no macrophage activation. Microencapsulated and naked human islets presented identical insulin secretion levels upon stimulation with glucose in vitro, confirming that barium-Biodritin microencapsulation maintains the function and viability of human islets. Proof-of-concept experiments in which barium-Biodritin microencapsulated human islets were implanted into chemically-induced diabetic mice, showed that these animals maintained normal blood glucose levels for more than 60 days, during which oral glucose tolerance tests were normal, demonstrating the complete functionality and efficiency of barium-Biodritin microencapsulated human islets. From the observation that animals inoculated with the venom of the scorpion Tityus serrulatus presented nesidioblastosis, we decided to fractionate the venom to isolate the active principle. The venom was fractionated by reversed phase HPLC and 24 fractions were obtained and submitted to cellular proliferation assays, in which rat insulinoma RINm5F cells evaluated for 3H-timidina incorporation. One of these fractions was capable of inducing cell proliferation and was also applied to isolated human islets. Treated islets presented a higher insulin secretion index and an increase in insulin and PCNA mRNA expression. In conclusion, we demonstrated that the barium-Biodritin biomaterial possesses all characteristics required for efficient cell/islet microencapsulation and that the active fraction of Tityus serrulatus venom induces the proliferation of RINm5F cells and improves insulin secretion in human islets.