Reactor for making uniform capsules

The present invention provides a novel reactor for making capsules with uniform membrane. The reactor includes a source for providing a continuous flow of a first liquid through the reactor; a source for delivering a steady stream of drops of a second liquid to the entrance of the reactor; a main tube portion having at least one loop, and an exit opening, where the exit opening is at a height substantially equal to the entrance. In addition, a method for using the novel reactor is provided. This method involves providing a continuous stream of a first liquid; introducing uniformly-sized drops of the second liquid into the stream of the first liquid; allowing the drops to react in the stream for a pre-determined period of time; and collecting the capsules

Encapsulation system for the immunoisolation of living cells

The present invention is drawn to a composition of matter comprising high viscosity sodium alginate, cellulose sulfate and a multi-component polycation. Additionally, the present invention provides methods for making capsules, measuring capsule permeability to immunologically-relevant proteins and treating disease in an animal using encapsulated cells. Over one thousand combinations of polyanions and polycations were examined as polymer candidates suitable for encapsulation of living cells and thirty-three pairs were effective. The combination of sodium alginate, cellulose sulfate, poly(methylene-co-guanidine) hydrochloride, calcium chloride, and sodium chloride produced the most desirable results. Pancreatic islets encapsulated in this multicomponent capsule demonstrated glucose-stimulated insulin secretion in vitro and reversed diabetes without stimulating immune reaction in mice. The capsule formulation and system of the present invention allows independent adjustments of capsule size, wall thickness, mechanical strength and permeability, and offers distinct advantages for immunoisolating cells

Cell encapsulation:Promise and progress

In cell encapsulation, transplanted cells are protected from immune rejection by an artificial, semipermeable membrane, potentially allowing transplantation (allo-or xenotransplantation) without the need for immunosuppression. Yet, despite some promising results in animal studies, the field has not lived up to expectations, and clinical products based on encapsulated cell technology continue to elude the scientific community. This commentary discusses the reasons for this, summarizes recent progress in the field and outlines what is needed to bring this technology closer to clinical application

Advances in biocompatibility and physico-chemical characterization of microspheres for cell encapsulation

Cell encapsulation has already shown its high potential and holds the promise for future cell therapies to enter the clinics as a large scale treatment option for various types of diseases. The advancement in cell biology towards this goal has to be complemented with functional biomaterials suitable for cell encapsulation. This cannot be achieved without understanding the close correlation between cell performance and properties of microspheres. The ongoing challenges in the field of cell encapsulation require a critical view on techniques and approaches currently utilized to characterize microspheres. This review deals with both principal subjects of microspheres characterization in the cell encapsulation field: physico-chemical characterization and biocompatibility. The up-to-day knowledge is summarized and discussed with the focus to identify missing knowledge and uncertainties, and to propose the mandatory next steps in characterization of microspheres for cell encapsulation. The primary conclusion of this review is that further success in development of microspheres for cell therapies cannot be accomplished without careful selection of characterization techniques, which are employed in conjunction with biological tests. (C) 2013 Elsevier B.V. All rights reserved

A polysulfobetaine hydrogel for immobilization of a glucose-binding protein

© The Royal Society of Chemistry 2016. A hydrogel based on sulfobetaine methacrylate monomer N-(methacryloyloxyethyl)-N,N-dimethyl-N-(3-sulfopropyl)ammonium betaine and N,N-bis(methacryloyloxyethyl)-N-methyl-N-(3-sulfopropyl)ammonium betaine used as a crosslinker was investigated as a potential material for biosensor applications. The glucose diffusion coefficient of 1.2 × 10 -10 m 2 s -1 was determined from the glucose release experiment. Inverse size-exclusion chromatography was performed to determine the molecular weight cut-off of the hydrogel to be 8 kDa with respect to pullulans that corresponds to a viscosity radius of 2.1 nm. The narrow pore-size distribution suggests that using the sulfobetaine crosslinker suppresses the composition drift and results in a homogeneous hydrogel network. Furthermore, a glucose biosensor construct comprising the periplasmic glucose-binding protein of Escherichia coli fused to cyan and yellow fluorescent proteins was effectively entrapped in the hydrogel exhibiting no leakage for at least 7 days. The glucose-binding protein showed stability of its secondary structure and sensitivity to glucose as assessed by circular dichroism and Förster (fluorescence) resonance energy transfer measurements under physiological conditions and a physiological range of glucose concentration, respectively.crosscheck: This document is CrossCheck deposited copyright_licence: The Royal Society of Chemistry has an exclusive publication licence for this journal history: Received 3 June 2016; Accepted 31 August 2016; Accepted Manuscript published 31 August 2016; Version of Record published 5 September 2016status: publishe

SEC analysis of poly(acrylic acid) and poly(methacrylic acid)

The accurate characterization of molar-mass distributions of poly(acrylic acid) (PAA) and poly(methacrylic acid) (PMAA) by size-exclusion chromatography (SEC) is addressed. Two methods are employed: direct aqueous-phase SEC on P(M)AA and THF-based SEC after esterification of P(M)AA to the associated methyl esters, P(M)MA. P(M)AA calibration standards, P(M)AA samples prepared by pulsed-laser polymerization (PLP), and PAA samples prepared by reversible addition-fragmentation chain transfer (RAFT) are characterized in a joint initiative of seven laboratories, with satisfactory agreement achieved between the institutions. Both SEC methods provide reliable results for PMAA. In the case of PAA, close agreement between the two SEC methods is only observed for samples prepared by RAFT polymerization with weightaverage molar mass between 80 000 and 145 000 g mol-1 and for standards with peak molar masses below 20 000 g mol-1. For standards with higher molar masses and for PLP-prepared PAA, the values from THF-based SEC are as much as 40% below the molar masses determined by aqueous-phase SEC. This discrepancy may be due to branching or degradation of branched PAA during methylation. While both SEC methods can be recommended for PMAA, aqueous-phase SEC should be used for molar-mass analysis of PAA unless the sample is not branched. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Effect of prolonged gelling time on the intrinsic properties of barium alginate microcapsules and its biocompatibility

Pericapsular fibrotic overgrowth (PFO) may be attributed to an immune response against microcapsules themselves or to antigen shedding through microcapsule pores from encapsulated islet tissue. Modification of microcapsules aimed at reducing pore size should prevent PFO and improve graft survival. This study investigated the effect of increased gelling time (20 vs. 2min) in barium chloride on intrinsic properties of alginate microcapsules and tested their biocompatibility in-vivo. Prolonged gelling time affected neither permeability nor size of the microcapsules. However, prolonged gelling time for 20min produced brittle microcapsules compared to 2min during compression test. Encapsulation of human islets in both types of microcapsules affected neither islet viability nor function. The presence of PFO when transplanted into a large animal model such as baboon and its absence in small animal models such as rodents suggest that the host immune response towards alginate microcapsules is species rather than alginate specific.9 page(s

(Meth)acrylic monomers with heteroatom-containing ester side chains: a systematic PLP-SEC and polymerization study

The Arrhenius parameters of the propagation rate coefficient for two hetero-atom containing (meth)-acrylates (studied as 1 M solution in N,N-dimethylacetamide (DMAc)) are determined via the pulsed laser polymerization-size-exclusion chromatography (PLP-SEC) method. Absolute molar mass determination is achieved via SEC coupled to on-line multi-angle laser light scattering (MALLS). The data obtained for hydroxypropylcarbamate acrylate (HPCA, A = 3.97 (-1.44 to 1.63) × 106 L mol-1 s -1 and Ea = 14.3 (-1.38 to 5.13) kJ mol-1) are critically compared with the literature known data sets of two structural derivatives, i.e., 2-(phenylcarbamoyloxy)isopropyl acrylate (PhCPA) and 2-(hexylcarbamoyloxy)isopropyl acrylate (HCPA), indicating an increase in the propagation rate coefficient with increasing ester side chain length. Ureidoethyl methacrylate (UMA, A = 2.08 (-0.45 to 0.91) × 106 L mol-1 s-1 and Ea = 19.9 (-0.89 to 0.91) kJ mol-1) represents the first hetero-atom containing methacrylate to be studied via PLP-SEC, evidencing a significantly higher propagation rate coefficient compared to earlier investigated methacrylate-type monomers. Furthermore, the free-radical polymerization behavior of HPCA and UMA is studied via in situ1H-NMR experiments at elevated temperatures allowing for an estimation of average termination rate coefficients (at low conversion) in conjunction with the determined kp data. Furthermore, the polymerization of UMA was successfully controlled by reversible addition-fragmentation chain transfer (RAFT) polymerization as evidenced by the linear evolution of the number-average molar mass, Mn, with conversion (3000 g mol-1 ≤ Mn ≤ 23000 g mol -1, 1.15 ≤ D ≤ 1.3) as well as by nitroxide-mediated polymerization (NMP), as demonstrated by the linear evolution of Mn with conversion (4000 g mol-1 ≤ Mn ≤ 40000 g mol-1, 1.3 ≤ D ≤ 1.4). In addition, HPCA polymerization was successfully controlled by the RAFT process, as evidenced by the linear evolution of Mn with conversion (2000 g mol-1 ≤ M n ≤ 21000 g mol-1, 1.2 ≤ D ≤ 1.4) and successful chain extension experiments. Finally, the NMP of HPCA exhibited uniform shifts of the molar mass distributions in the range of 5000 g mol-1 ≤ Mn ≤ 70000 g mol-1 and successful chain extension experiments. © 2014 The Royal Society of Chemistry

Free radical exit in emulsion polymerization. II. Model discrimination via experiment

In emulsion polymerizations, desorption (exit) from latex particles of monomeric radical species that arise from transfer can be an important determinant of the overall kinetics. An examination of various methodologies for the testing of postulated free radical exit mechanisms is made. These utilize the model descriptions for the exit process presented in the accompanying article of Casey et al., employing data consisting of conversion as a function of time for the approach to steady state polymerization conditions. Experimental data are presented on the exit rate coefficients as a function of such experimental parameters as: particle size, monomer concentration, and aqueous-phase free-radical concentration for a series of styrene polymerizations at 50°C, where the average number of free radicals per particle (n̄) never exceeds 0.5. It is demonstrated for these systems that while the conversion/ time dependence from a single run, under conditions sensitive to exit, is insensitive to mechanistic assumptions as to the fate of desorbed free radicals, the variation of the exit rate coefficient with particle size so obtained suggests a second order dependence on n̄, implying the complete re-entry of desorbed free radicals under all conditions studied. Once the monomeric radicals have re-entered, they are more likely to remain inside the particle where they will either propagate or undergo termination rather than re-escape. The article also presents an estimate for the rate coefficient at 50°C of the first propagation step of the monomeric radical subsequent to transfer. The conclusions drawn here for seeded systems should prove useful for study of particle nucleation mechanisms, when exit is particularly likely in small, newly formed, particles

Beneficial effects of coating alginate microcapsules with macromolecular heparin conjugates-in vitro and in vivo study

Pericapsular fibrotic overgrowth (PFO) is associated with poor survival of encapsulated pancreatic islets. Modification of the microcapsule membrane aimed at preventing PFO should improve graft survival. This study investigated the effect of macromolecular Corline Heparin Conjugate (CHC) binding on intrinsic properties of alginate microcapsules and assessed the anti-fibrotic potential of this strategy both in vitro and in vivo. CHC was bound to alginate microcapsules using a layer-by-layer approach incorporating avidin. CHC binding to alginate microcapsule was visualized by confocal microscopy. Effects of CHC binding on microcapsule size, strength, and permeability were assessed, and the anti-clotting activity of bound CHC was determined by coagulation assay. Effect of CHC binding on the viability of encapsulated human islets was assessed in vitro, and their ability to function was assessed both in vitro and in vivo in diabetic immunodeficient mice. The potential of bound CHC to reduce PFO was assessed in vivo in different rat transplantation models. Confocal microscopy demonstrated a uniform coating of CHC onto the surface of microcapsules. CHC binding affected neither size nor permeability but significantly increased the tensile strength of alginate microcapsules by ~1.3-fold. The bound CHC molecules were stable and retained their anti-clotting activity for 3 weeks in culture. CHC binding affected neither viability nor function of the encapsulated human islets in vitro. In vivo CHC binding did not compromise islet function, and diabetes was reversed in all recipients with mice exhibiting lower blood glucose levels similar to controls in oral glucose tolerance tests. CHC binding was beneficial and significantly reduced PFO in both syngeneic and allogeneic rat transplantation models by ~65% and ~43%, respectively. In conclusion, our results show a new method to successfully coat CHC on alginate microcapsules and demonstrate its beneficial effect in increasing capsule strength and reduce PFO. This strategy has the potential to improve graft survival of encapsulated human islets.11 page(s