In an attempt to improve the outcome of pancreatic islet transplantation, we performed in-vitro and in-vivo experiments with isolated human islets coated by multi-layer nano-encapsulation. Human islets were isolated from 32 non-diabetic donors (age: 63±17 yrs, BMI: 26.8 ± 3.5 kg/m2, M/F: 17/15) by enzymatic digestion and gradient purifi cation. Multi-layer nano-encapsulation was performed by electrostatic binding of differently charged polymers [chitosan and poly(sodium styrene sulfonate)], up to 9 layers. Morphological, ultrastructural, functional and transplantation studies where then accomplished with the nano-coated human islets. The procedure provided full coating of the human islets (thickness: 104.2±4.2 nm), as assessed by fl uorescence, confocal and electron microscopy (EM). Vital staining and EM showed ≥90% cell survival and well maintained beta and alpha cell ultrastructure, with unchanged morphology and morphometry of intracellular organelles. Insulin secretion from nanoencapsulated islets was 44±33 μU/ml (mean±SD) at 3.3 mM glucose and increased to 176±120 μU/ml at 16.7 mM glucose (p<0.01), with a stimulation index of 4.6±2.7. Perifusion studies showed maintained dynamics of insulin secretion. Toxicity by palmitate or cytokine exposure was signifi cantly reduced in nano-coated islets. Transplantation of nano-encapsulated islets under the kidney capsule of C57Bl/6J mice with streptozotocin-induced diabetes allowed long term normal or near normal glycemia, with intra-peritoneal glucose tolerance test results similar to those of non-diabetic mice. Light and electron microscopy of nano-coated islet grafts was performed at one month post-transplantation, showing minimal mononuclear cell infi ltration, preservation of islet cell ultrastructure and signs of revascularization. Isolated human islets were effi ciently encapsulated by this multi-layer nano-coating approach, with preserved in-vitro and in-vivo function. Supported By: CariPisa Foundation
The use of multilayer nano-encapsulation for the immunoprotection of isolated human islets: in vitro and in vivo studies.
SYED, FAROOQ;BUGLIANI, MARCO;NOVELLI, MICHELA;MASINI, MATILDE;DE TATA, VINCENZO;SULEIMAN, MARA;MARSELLI, LORELLA;BOGGI, UGO;FILIPPONI, FRANCO;RAFFA, VITTORIA;MASIELLO, PELLEGRINO;MARCHETTI, PIERO
2014-01-01
Abstract
In an attempt to improve the outcome of pancreatic islet transplantation, we performed in-vitro and in-vivo experiments with isolated human islets coated by multi-layer nano-encapsulation. Human islets were isolated from 32 non-diabetic donors (age: 63±17 yrs, BMI: 26.8 ± 3.5 kg/m2, M/F: 17/15) by enzymatic digestion and gradient purifi cation. Multi-layer nano-encapsulation was performed by electrostatic binding of differently charged polymers [chitosan and poly(sodium styrene sulfonate)], up to 9 layers. Morphological, ultrastructural, functional and transplantation studies where then accomplished with the nano-coated human islets. The procedure provided full coating of the human islets (thickness: 104.2±4.2 nm), as assessed by fl uorescence, confocal and electron microscopy (EM). Vital staining and EM showed ≥90% cell survival and well maintained beta and alpha cell ultrastructure, with unchanged morphology and morphometry of intracellular organelles. Insulin secretion from nanoencapsulated islets was 44±33 μU/ml (mean±SD) at 3.3 mM glucose and increased to 176±120 μU/ml at 16.7 mM glucose (p<0.01), with a stimulation index of 4.6±2.7. Perifusion studies showed maintained dynamics of insulin secretion. Toxicity by palmitate or cytokine exposure was signifi cantly reduced in nano-coated islets. Transplantation of nano-encapsulated islets under the kidney capsule of C57Bl/6J mice with streptozotocin-induced diabetes allowed long term normal or near normal glycemia, with intra-peritoneal glucose tolerance test results similar to those of non-diabetic mice. Light and electron microscopy of nano-coated islet grafts was performed at one month post-transplantation, showing minimal mononuclear cell infi ltration, preservation of islet cell ultrastructure and signs of revascularization. Isolated human islets were effi ciently encapsulated by this multi-layer nano-coating approach, with preserved in-vitro and in-vivo function. Supported By: CariPisa FoundationI documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
