Objective The purity and activity of islets will greatly affect the outcome of xenotransplantation therapy of type 1 diabetes mell itus. To set up an improved method of the isolation and purification of rat islets, which can obtain highpurity,high-yield, and high-viabil ity islets. Methods Ten healthy and adult male SD rats, weighing 250-300 g were used asorgan donors. Collagenase V was perfused into pancreas via pancreatic duct. Pancreas was digested with collagenase in water bath at 38℃ about 15 minutes, islet purification was performed using two techniques: with Ficoll 400 density gradient (group A), and Ficoll-Paque™ PLUS (group B). Dithizone (DTZ) was util ized for identifying islets, counting islets equivalent quantity (IEQ) and islets’ purity. Trypan blue staining was used to detect the viabil ity of islets. Islets of group B was encapsulated with alginate/poly-L-lysine/alginate (APA). Islets function of microencapsulated and nonmicroencapsulated was evaluated by the insul in release test. Results DTZ staining showed that islets shape were round, ell ipse and irregular with a clear edge and a diameter range of 50-300 μm. The IEQ values were 338.04 ± 76.61 and 834.80 ± 54.00 in groups A and B, respectively, showing significant difference (P lt; 0.05). The purities were 88.31% ± 2.67% and 95.63% ± 1.96% in groups A and B, respectively, showing no significant difference (P gt; 0.05). The activities of islets were 67.40% ± 5.15% and 86.05% ± 2.52% in groups A and B, showing significant difference (P lt; 0.05). Islet APA microcapsules had round shape, unified size, and its diameter was between 1.5 and 2.0 mm. Each microcapsule was encapsulated of 1 to 3 islets. The result of insul in release assay was that the concentrations of insul in secretion with islets of microencapsulated and nonmicroencapsulated were (5.53 ± 1.64) ng/ mL and (4.76 ± 0.26) ng/mL in low glucose, and its concentrations of insul in secretion in high glucose were (11.95 ± 2.07) ng/ mL and (14.34 ± 3.18) ng/mL. Stimulated insul in secretion in high glucose was 2 times more than that in low glucose (P lt; 0.05), but there was no significant difference (P gt; 0.05) in the stimulation index between group A (2.16 ± 0.30) and group B (3.01 ± 0.59). Conclusion The method of islets isolation and purification using Ficoll-Paque™ PLUS own the virtues of more convenient, high islet yield, and high islet purity. Both microencapsulated and nonmicroencapsulated islets show high-viabil ity while culture in vitro.
Objective To review the general approaches in isolation and purification of pancreatic islets and progress in several aspects. Methods The latest l iterature concerning acquisition of pancreatic islets was reviewed and analyzed interms of the choice of pancreatic islet donors, the digestion and isolation of pancreas, the purification of islet and the assay of outcome. Results The profile of the isolation and purification depends on the selection of reagents and methods of operation in every step and l inkup between every step. Conclusion Pancreatic islet transplantation is the most effective method to treat type 1 diabetes, the problem of inadequate sources of pancreatic islets could be resolved by the optimal process and the establ ishment of standardized operation.
To set up an economic and effective method for islet isolation from rat, and thereby prove a laboratory protocol of animal model for cl inical islet transplantation. Methods Twenty-five adult male SD rats weighing 230-380 g were used as organ donor. In each of 5 repeated experiments, pancreatic islets of 5 animals were isolated by intraductal infusion of compound sodium chloride injection (CSCI), and subsequently, digested with low concentration (0.5 mg/mL)of collagenase V solution. Islet purification was performed by using a discontinuous density gradient centrifugation thatwas prepared with 27.0%, 23.0%, 20.5% and 11.0% of Ficoll 400. Islet yield and purity were determined by dithizon (DTZ)stain, and propidium iodide (PI)/fluorescein diacetate (FDA) double stain was used to check viabil ity of islets. The endocrine secretory function was assessed by insul in secretion in either low (2.8 mmol/L) or high (25.0 mmol/L) glucose incubation after 3 days of culture in RPMI1640 media. Results Average islet digestion time of 5 experiments was (13.8 ± 1.6) min. Before purification, average isolated number was (5 626 ± 422) islets, and the number was significantly reduced to (2 914 ± 485) islets after purification (P lt; 0.01). The average recovery rate was 51.6% ± 6.0%, and the average yield was (583 ± 97) islets/pancreas. The average purity and viabil ity of islets were 90.2% ± 3.4% and 81.6% ± 7.0%, respectively. After 3 days of culture, insul in secretion of the islets was (116.1 ± 17.4) EU/L in high glucose incubation, which was significantly higher than that of low glucose environment [(39.7 ± 7.5) EU/L, P lt; 0.01)]. The average insul in stimulation index was 3.0 ± 0.4. Conclusion The islet isolation with the CSCI solution and digestion with low concentration of collagenase V decrease experimental cost and also have a beneficial effect on islet recovery and their function.
ObjectiveTo review the current progresses in purification strategies, biological characters, and functions of endothelial progenitor cells (EPCs) derived extracellular vesicles (EVs) (EPC-EVs). MethodsRecent relevant publications on the EPC-EVs were extensively reviewed, analyzed, and summarized. ResultsEPC-EVs are usually isolated by differential centrifugation and exhibit a homogenous pattern of spheroid particles with a diameter ranging from 60 to 160 nm under transmission electron microscopy. EPC-EVs are positive for cell-surface markers of EPCs (CD31, CD34, and CD133), and negative for markers of platelets (P-selectin and CD42b) and monocytes (CD14). Recent studies have shown the effectiveness of EPC-EVs in ischemic injuries, anti-Thy1 glomerulonephritis, and cardiomyocyte hypertrophy, and also shown their predictive role in cardio-cerebral-vascular diseases. ConclusionAn alluring prospect exists on the EPC-EVs-related research. Further studies are required to decipher the composition of EPC-EVs and their precise role in pathophysiological processes, and to investigate the molecular mechanisms for their targeting and function.