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.

Pancreatic islets are composed of endocrine cells that secrete hormones essential for blood-glucose homeostasis. Prior research has revealed that the gene expression and functionality of human islet cells is heterogenous. However, it is not currently understood how the heterogeneity correlates to normal islet cell function and dysfunction in diabetes pathogenesis. Subsequently, an international collaborative project has been initiated to elucidate what constitutes islet cell heterogeneity from a transcriptional, proteomic, and functional perspective in both health and disease. In this study, a highly multiplex tissue image assay was developed to allow for the study of the localization and distribution of proteins previously identified to correlate with functional activity and heterogeneity in islet cells using the CO-Detection by indEXing (CODEX) platform. In total, 22 proteins were studied simultaneously of which 10 were specifically expressed in islet cells. These included generic pancreatic markers such as C-peptide (C-pep) marking insulin-secreting β-cells, glucagon (GCG) and somatostatin (SST), but also less well-characterized proteins such as Shisa like 2B (FAM159B) and Neural proliferation, differentiation and control 1 (NPDC1). The multiplex tissue imaging allowed for single-cell analysis of protein expression in human islet cells showing that most islet specific proteins were heterogeneously expressed. The observations made in this study serves as a validation to that the human islet microenvironment is highly complex due to islet cell heterogeneity. Additionally, the study demonstrated that multiplex tissue imaging has the potential to reveal novel cell types and interactions. / Langerhanska öar består av endokrina celler som utsöndrar hormoner nödvändiga för reglering av blodsockernivåerna. Tidigare forskning har visat att genuttrycket och funktionaliteten är heterogen i de celler som utgör mänskliga Langerhanska öar. Dock är förståelsen för hur heterogeniteten korrelerar till normal cellfunktion och dysfunktion i diabetespatogenes fortfarande ofullständig. Följaktligen har ett internationellt samarbete inletts i syfte att  undersöka  vad som utgör heterogenitet  i  Langerhanska öar ur ett transkriptionellt, proteomiskt och funktionellt perspektiv i såväl friska som sjuka individer. I denna studie utvecklades en metod för multiplex mikroskopisk avbildning av vävnad för att möjliggöra undersökningen av hur proteiner som tidigare korrelerats med endokrin cellspecifik aktivitet och heterogenitet i Langerhanska öar var lokaliserade med hjälp av plattformen för Co-Detection by indEXing (CODEX). Totalt undersöktes 22 proteiner samtidigt varav 10 var specifikt uttryckta i celler som utgör Langerhanska öar. Bland dessa proteiner fanns generella markörer för vanligt förekommande celltyper i Langerhanska öar såsom C-peptid (C-pep) som markör för insulinsekreterande β-celler, glukagon (GCG) och somatostatin (SST) såväl som proteiner med färre kända funktioner såsom Shisa like B (FAM159B) och Neural proliferation, differentiation and control 1 (NPDC1). Med hjälp av multiplex mikroskopisk avbildning av vävnad kunde uttrycket av proteiner specifikt uttryckta i celler som utgör Langerhanska öar analyseras för enskilda celler i vävnaden. Denna analys visade att de flesta proteiner specifikt uttryckta i celler som Langerhanska öar består av var heterogent uttrycka. Resultaten från denna studie validerar att mikromiljön i Langerhanska öar är mycket komplex på grund av cellernas heterogenitet. Vidare visade denna studie att multiplex mikroskopisk avbildning av vävnad har potentialen att identifiera nya celltyper och interaktioner.