Dynamic clamp with StdpC software

Dynamic clamp is a powerful method that allows the introduction of artificial electrical components into target cells to simulate ionic conductances and synaptic inputs. This method is based on a fast cycle of measuring the membrane potential of a cell, calculating the current of a desired simulated component using an appropriate model and injecting this current into the cell. Here we present a dynamic clamp protocol using free, fully integrated, open-source software (StdpC, for spike timing-dependent plasticity clamp). Use of this protocol does not require specialist hardware, costly commercial software, experience in real-time operating systems or a strong programming background. The software enables the configuration and operation of a wide range of complex and fully automated dynamic clamp experiments through an intuitive and powerful interface with a minimal initial lead time of a few hours. After initial configuration, experimental results can be generated within minutes of establishing cell recording

Investigating the Role of Islet Cytoarchitecture in Its Oscillation Using a New β-Cell Cluster Model

The oscillatory insulin release is fundamental to normal glycemic control. The basis of the oscillation is the intercellular coupling and bursting synchronization of β cells in each islet. The functional role of islet β cell mass organization with respect to its oscillatory bursting is not well understood. This is of special interest in view of the recent finding of islet cytoarchitectural differences between human and animal models. In this study we developed a new hexagonal closest packing (HCP) cell cluster model. The model captures more accurately the real islet cell organization than the simple cubic packing (SCP) cluster that is conventionally used. Using our new model we investigated the functional characteristics of β-cell clusters, including the fraction of cells able to burst fb, the synchronization index λ of the bursting β cells, the bursting period Tb, the plateau fraction pf, and the amplitude of intracellular calcium oscillation [Ca]. We determined their dependence on cluster architectural parameters including number of cells nβ, number of inter-β cell couplings of each β cell nc, and the coupling strength gc. We found that at low values of nβ, nc and gc, the oscillation regularity improves with their increasing values. This functional gain plateaus around their physiological values in real islets, at nβ∼100, nc∼6 and gc∼200 pS. In addition, normal β-cell clusters are robust against significant perturbation to their architecture, including the presence of non-β cells or dead β cells. In clusters with nβ>∼100, coordinated β-cell bursting can be maintained at up to 70% of β-cell loss, which is consistent with laboratory and clinical findings of islets. Our results suggest that the bursting characteristics of a β-cell cluster depend quantitatively on its architecture in a non-linear fashion. These findings are important to understand the islet bursting phenomenon and the regulation of insulin secretion, under both physiological and pathological conditions

Synthesis of oligo(poly(ethylene glycol) fumarate)

This protocol describes the synthesis of oligo(poly(ethylene glycol) fumarate) (OPF) (1-35 kDa)(a polymer useful for tissue engineering applications) by a one-pot reaction of poly(ethylene glycol) (PEG) and fumaryl chloride. The procedure involves three parts: dichloromethane and PEG are first dried; the reaction step follows in which fumaryl chloride and triethylamine are added dropwise to a solution of PEG in dichloromethane; and finally the product solution is filtered to remove byproduct salt, and the OPF product is twice crystallized, washed, and dried under vacuum. The reaction is affected by PEG molecular weight and reactant molar ratio. The OPF product is cross-linked by radical polymerization by either a thermally induced or UV-induced radical initiator, and the physical properties of the OPF oligomer and resulting cross-linked hydrogel are easily tailored by varying PEG molecular weight. OPF hydrogels are injectable, polymerize in situ, and undergo biodegradation by hydrolysis of ester bonds. The expected time required to complete this protocol is 6 d