RESUMO
BACKGROUND@#Islet transplantation is currently considered the most promising method for treating insulin-dependent diabetes. The two most-studied artificial islets are alginate-encapsulated b cells or b cell spheroids. As three-dimensional (3D) models, both artificial islets have better insulin secretory functions and transplantation efficiencies than cells in twodimensional (2D) monolayer culture. However, the effects of these two methods have not been compared yet. Therefore, in this study, cells from the mouse islet b cell line Min6 were constructed as scaffold-free spheroids or alginate-encapsulated dispersed cells. @*METHODS@#MIN6 cell spheroids were prepared by using Agarose-base microwell arrays. The insulin secretion level was determined by mouse insulin ELISA kit, and the gene and protein expression status of the MIN6 were performed by Quantitative polymerase chain reaction and immunoblot, respectively. @*RESULTS@#Both 3D cultures effectively promoted the proliferation and glucose-stimulated insulin release (GSIS) of MIN6 cells compared to 2D adherent cells. Furthermore, 1% alginate-encapsulated MIN6 cells demonstrated more significant effects than the spheroids. In general, three pancreatic genes were expressed at higher levels in response to the 3D culture than to the 2D culture, and pancreatic/duodenal homeobox-1 (PDX1) expression was higher in the cells encapsulated in 1% alginate than that in the spheroids. A western blot analysis showed that 1% alginate-encapsulated MIN6 cells activated the phosphoinositide 3-kinase (PI3K)/serine/threonine protein kinase (AKT)/forkhead transcription factor FKHR (FoxO1) pathway more than the spheroids, 0.5% alginate-, or 2% alginate-encapsulated cells did. The 3D MIN6 culture, therefore, showed improved effects compared to the 2D culture, and the 1% alginate-encapsulated MIN6 cells exhibited better effects than the spheroids. The upregulation of PDX1 expression through the activation of the PI3K/AKT/FoxO1 pathway may mediate the improved cell proliferation and GSIS in 1% alginate-encapsulated MIN6 cells. @*CONCLUSION@#This study may contribute to the construction of in vitro culture systems for pancreatic islets to meet clinical requirements.
RESUMO
OBJECTIVE: The aim of this study is to evaluate the performance of different platelet counting methods (optical, impedance, fluorescence and hand counting) applied in different analysers by comparing with the international flow cytometric reference method (IRM). METHODS: A total of 333 blood samples from different subgroups (168 cases with thrombocytopenia, 136 cases with normal platelet counts and 29 cases with thrombocytosis) were tested. Regarding IRM as the gold standard, we compared the accuracy and precision of different platelet count methods; i.e. LH780 (impedance), BC-6000 Plus (optical (O) and impedance (I)), Sysmex XN-9000 (optical (O), impedance (I), fluorescence (F)), and hand counting. RESULTS: Sysmex XN-9000-F (r = 0.988) had the best correlation with IRM for thrombocytopenic samples; BC-6000 Plus-I (r = 0.966) was more relevant to IRM than any other method for samples with normal platelet counts. Correlation between Sysmex XN-9000-I (r = 0.960) and IRM was the highest among these methods for samples with thrombocytosis. For bias evaluation, the average bias of Sysmex XN-9000-F was -1.5 × 109/L (95% LA = -9.4 to +6.4) for samples with thrombocytopenia, compared with IRM. BC-6000 Plus-I had a small mean difference with IRM for samples with normal platelet counts or thrombocytosis. Moreover, all evaluated methods had acceptable sensitivity, specificity, and concordance rates as compared with IRM in the diagnosis of thrombocytopenia and thrombocytosis. CONCLUSIONS: Platelet counting by Sysmex XN-9000-F is more accurate than other methods for thrombocytopenic samples. BC-6000 Plus-I has superior association and consistency for normal platelet counts. As for thrombocytosis patients, Sysmex XN-9000-I has the highest correlation with IRM while Sysmex XN-9000-O has the highest diagnosis efficacy.
