Encapsulation of Huh-7 cells within alginate-poly(ethylene glycol) hybrid microspheres

Novel calcium alginate poly(ethylene glycol) hybrid microspheres (Ca-alg-PEG) were developed and evaluated as potentially suitable materials for cell microencapsulation. Grafting 5-13% of the backbone units of sodium alginate (Na-alg) with α-amine-ω-thiol PEG maintained the gelling capacity in presence of calcium ions, while thiol end groups allowed for preparing chemically crosslinked hydrogel via spontaneous disulfide bond formation. The combination of these two gelling mechanisms yielded Ca-alg-PEG. Human hepatocellular carcinoma cells (Huh-7) were encapsulated in Ca-alg-PEG and calcium alginate beads (Ca-alg), and cultured for 2weeks under agitation conditions. Immediately after completion of the microencapsulation, the cell viability was 60% and similar in Ca-alg-PEG and Ca-alg. The proliferation of Huh-7 encapsulated in Ca-alg-PEG was slightly higher than in Ca-alg. Accelerated proliferation after 2weeks was observed for the encapsulation in Ca-alg-PEG. The production of albumin confirmed the functionality of the encapsulated Huh-7 cells. The study confirms the suitability of Ca-alg-PEG and the one-step technology for cell microencapsulatio

Encapsulation of Huh-7 cells within alginate-poly(ethylene glycol) hybrid microspheres

Novel calcium alginate poly(ethylene glycol) hybrid microspheres (Ca-alg-PEG) were developed and evaluated as potentially suitable materials for cell microencapsulation. Grafting 5-13% of the backbone units of sodium alginate (Na-alg) with alpha-amine-omega-thiol PEG maintained the gelling capacity in presence of calcium ions, while thiol end groups allowed for preparing chemically crosslinked hydrogel via spontaneous disulfide bond formation. The combination of these two gelling mechanisms yielded Ca-alg-PEG. Human hepatocellular carcinoma cells (Huh-7) were encapsulated in Ca-alg-PEG and calcium alginate beads (Ca-alg), and cultured for 2 weeks under agitation conditions. Immediately after completion of the microencapsulation, the cell viability was 60% and similar in Ca-alg-PEG and Ca-alg. The proliferation of Huh-7 encapsulated in Ca-alg-PEG was slightly higher than in Ca-alg. Accelerated proliferation after 2 weeks was observed for the encapsulation in Ca-alg-PEG. The production of albumin confirmed the functionality of the encapsulated Huh-7 cells. The study confirms the suitability of Ca-alg-PEG and the one-step technology for cell microencapsulation

Multipotent mesenchymal stromal cells enhance insulin secretion from human islets via N-Cadherin interaction and prolong function of transplanted encapsulated islets in mice

Background: Multipotent mesenchymal stromal cells (MSC) enhance viability and function of islets of Langerhans. We aimed to examine the interactions between human MSC and human islets of Langerhans that influence the function of islets. Methods: Human MSC and human islets (or pseudoislets, obtained after digestion and reaggregation of islet cells) were cocultured with or without cellular contact and glucose-stimulated insulin secretion assays were performed to assess cell function. The expression of several adhesion molecules, notably ICAM-1 and N-cadherin on islets and MSC, was investigated by qPCR. The role of N-cadherin was analyzed by adding an anti-N-cadherin antibody in islets cultured with or without MSC for 24 h followed by insulin measurements in static incubation assays. Islets and MSC were coencapsulated in new hydrogel microspheres composed of calcium alginate and covalently crosslinked polyethylene glycol. Encapsulated cells were transplanted intraperitoneally in streptozotocin-induced diabetic mice and glycemia was monitored. Islet function was evaluated by the intraperitoneal glucose tolerance test. Results: In vitro, free islets and pseudoislets cocultured in contact with MSC showed a significantly increased insulin secretion when compared to islets or pseudoislets cultured alone or cocultured without cell-to-cell contact with MSC (p < 0.05). The expression of ICAM-1 and N-cadherin was present on islets and MSC. Blocking N-cadherin prevented the enhanced insulin secretion by islets cultured in contact with MSC whereas it did not affect insulin secretion by islets cultured alone. Upon transplantation in diabetic mice, islets microencapsulated together with MSC showed significantly prolonged normoglycemia when compared with islets alone (median 69 and 39 days,respectively, p < 0.01). The intraperitoneal glucose tolerance test revealed an improved glycemic response in mice treated with islets microencapsulated together with MSC compared to mice transplanted with islets alone (p < 0.001). Conclusions: MSC improve survival and function of islets of Lan gerhans by cell-to-cell contact mediated by the adhesion molecule N-cadherin

Hybrid Microspheres For Cell Encapsulation

Cell microencapsulation is an active interdisciplinary research field. It requires the input from materials sciences, organic and physical chemistry, biology and medicine. Hydrogels are particularly suitable materials to form microspheres for cell entrapment. The present work describes the development of two novel types of hybrid microspheres, their physical properties, in vitro and in vivo biocompatibility as well as the application for the microencapsulation of three different cell types. The first part describes an approach to produce hybrid microspheres by combining fast ionotropic gelation of sodium alginate (Na-alg) with calcium ions and slow covalent crosslinking of poly(ethylene glycol) (PEG) derivatives. A one-step extrusion process yields under physiological conditions alginate-poly(ethylene glycol) hybrid microspheres (alg-PEG-M) consisting of a PEG chemical hydrogel network interpenetrating the physical calcium alginate hydrogel network. The physical properties of alg-PEG-M such as mechanical resistance, elasticity, permeability, and water uptake are adjustable by the macromolecular characteristics of the components, their concentration as well as the process conditions. In vitro cytotoxicity tests did not show cytotoxic effects for EC219 rat endothelial cells, ECp23 mouse endothelial cells and RAW264.7 murine macrophages upon incubation with alg-PEG-M. The immune response to alg-PEG-M intraperitoneally implanted into mice was similar as the response to injected medium used as control. The feasibility of cell microencapsulation within alg-PEG-M was confirmed by encapsulating two cell models, human islets of Langerhans and human mesenchymal stem cells (MSC). Encapsulated human islets continued insulin secretion upon stimulation. Insulin release revealed technology dependency. The viability of encapsulated MSC, their proliferation, and their differentiation into adipocytes was confirmed. In the second part, eleven heterobifunctional PEG derivatives having variable functionalities were synthesized as potential candidates for biomaterials modification. The synthesis involves selective monotosylation of symmetrical PEG. For both end groups, a degree of functionalization > 95 % was achieved in all cases. Heterobifunctional α-amine-ω-thiol PEG was selected and grafted onto alginate chain units as an example of pegylation technology. The grafted alginate molecules maintained their gelling capacity in presence of divalent cations, while a novel chemically cross-linked hydrogel was obtained via simultaneous spontaneous disulfide bond formation. The combination of these two gelling mechanisms yields Ca-alg-PEG hybrid microspheres in one step under physiological conditions. Microencapsulation of human hepatocellular carcinoma cells (Huh-7) in Ca-alg-PEG demonstrated cell viability, metabolic activity, and proliferation post encapsulation in vitro, while applying microgravity culture conditions during storage for two weeks as model for bioartificial liver development. The two novel types of hybrid microspheres avoid the incorporation of polycations for mechanical reinforcement and tuning of the permeability and can be produced by a one-step microencapsulation technology under physiological conditions. They thus fulfill important requirements for biocompatible cell encapsulation materials

Alginate-Poly(ethylene glycol) Hybrid Microspheres with Adjustable Physical Properties

A one-step extrusion process under physiological conditions yielded calcium alginate-poly-(ethylene glycol) hybrid microspheres (Alg-PEG-M), for which the physical properties were adjustable by the macromolecular characteristics of the components, their concentration as well as the process conditions. A solution containing a mixture of sodium alginate (Naalg) and multiarm vinyl sulfone-terminated PEG(PEG-VS) was extruded into a receiving bath providing calcium ions and a thiol cross-linker. Covalent cross-linking of PEG-VS occurred in the rapidly gelled spherical calcium alginate (Caalg) matrix. After liquefaction of the Caalg, the cross-linked PEG remained spherical. The stoichiometric ratio thiol/VS was decisive for the PEG gel stability. The permeability of the hydrogels could be tuned by adequate choice of the arm length of PEG-VS, while the swelling behavior was influenced by its concentration, the quality of the storage solvent, and the presence or absence of the Caalg matrix. Only slight differences of the mechanical resistance were observed after the dissolution of Caalg

Alginate-Poly(ethylene glycol) Hybrid Microspheres for Primary Cell Microencapsulation

The progress of medical therapies, which rely on the transplantation of microencapsulated living cells, depends on the quality of the encapsulating material. Such material has to be biocompatible, and the microencapsulation process must be simple and not harm the cells. Alginate-poly(ethylene glycol) hybrid microspheres (alg-PEG-M) were produced by combining ionotropic gelation of sodium alginate (Na-alg) using calcium ions with covalent crosslinking of vinyl sulfone-terminated multi-arm poly(ethylene glycol) (PEG-VS). In a one-step microsphere formation process, fast ionotropic gelation yields spherical calcium alginate gel beads, which serve as a matrix for simultaneously but slowly occurring covalent cross-linking of the PEG-VS molecules. The feasibility of cell microencapsulation was studied using primary human foreskin fibroblasts (EDX cells) as a model. The use of cell culture media as polymer solvent, gelation bath, and storage medium did not negatively affect the alg-PEG-M properties. Microencapsulated EDX cells maintained their viability and proliferated. This study demonstrates the feasibility of primary cell microencapsulation within the novel microsphere type alg-PEG-M, serves as reference for future therapy development, and confirms the suitability of EDX cells as control model

Versatile Route to Synthesize Heterobifunctional Poly(ethylene glycol) of Variable Functionality for Subsequent Pegylation

Pegylation using heterotelechelic poly(ethylene glycol) (PEG) offers many possibilities to create high-performance molecules and materials. A versatile route is proposed to synthesize heterobifunctional PEG containing diverse combinations of azide, amine, thioacetate, thiol, pyridyl disulfide, as well as activated hydroxyl end groups. Asymmetric activation of one hydroxyl end group enables the heterobifunctionalization while applying selective monotosylation of linear, symmetrical PEG as a key step. The azide function is introduced by reacting monotosyl PEG with sodium azide. A thiol end group is obtained by reaction with sodium hydrosulfide. The activation of the hydroxyl end group and subsequent reaction with potassium carbonate/thioacetic acid yields a thioacetate end group. The hydrolysis of the thioester end group by ammonia in presence of 2,2′-dipyridyl disulfide provides PEG pyridyl disulfide. Amine terminated PEG is prepared either by reduction of the azide or by nucleophilic substitution of mesylate terminated PEG using ammonia. In all cases, &gt;95% functionalization of the PEG end groups is achieved. The PEG derivatives particularly support the development of materials for biomedical applications. For example, grafting up to 13% of the Na-alg monomer units with α-amine-ω-thiol PEG maintains the gelling capacity in presence of calcium ions but simultaneous, spontaneous disulfide bond formation reinforces the initial physical hydrogel

Interpenetrating Alginate-Collagen Polymer Network Microspheres for Modular Tissue Engineering

The lack of vascularization limits the creation of engineered tissue constructs with clinically relevant sizes. We pioneered a bottom-up process (modular tissue engineering) in which constructs with intrinsic vasculature were assembled from endothelialized building blocks. In this study, we prepared an interpenetrating polymer network (IPN) hydrogel from a collagen-alginate blend and evaluated its use as microspheres in modular tissue engineering. Ionotropic gelation of alginate was combined with collagen fibrillogenesis, and the resulting hydrogel was stiffer and had greater resistance to enzymatic degradation relative to that of collagen alone; the viability of embedded mesenchymal stromal cells (adMSC) was unaltered. IPN microspheres were fabricated by a coaxial air-flow technique, and an additional step of collagen coating was required to have human umbilical vein endothelial cells (HUVEC) attach and proliferate. When implanted subcutaneously in SCID/bg mice, adMSC-HUVEC microspheres promoted more blood vessels at day 7 relative to microspheres without adMSC but coated with HUVEC. Perfusion studies confirmed that these vessels were connected to the host vasculature. Fewer vessels were detected in both groups at day 21, but in adMSC-HUVEC explants, more smooth muscle cells had wrapped around vessels, and CLARITY processing of whole explants revealed a restricted leakage of blood. The capacity for rapid gelation and high throughput production are promising features for the use of these microspheres in modular tissue engineering

Coaxial micro-extrusion of a calcium phosphate ink with aqueous solvents improves printing stability, structure fidelity and mechanical properties.

Micro-extrusion-based 3D printing of complex geometrical and porous calcium phosphate (CaP) can improve treatment of bone defects through the production of personalized bone substitutes. However, achieving printing and post-printing shape stabilities for the efficient fabrication and application of rapid hardening protocol are still challenging. In this work, the coaxial printing of a self-setting CaP cement with water and ethanol mixtures aiming to increase the ink yield stress upon extrusion and the stability of fabricated structures was explored. Printing height of overhang structure was doubled when aqueous solvents were used and a 2 log increase of the stiffness was achieved post-printing. A standard and fast steam sterilization protocol applied as hardening step on the coaxial printed CaP cement (CPC) ink resulted in constructs with 4 to 5 times higher compressive moduli in comparison to extrusion process in the absence of solvent. This improved mechanical performance is likely due to rapid CPC setting, preventing cracks formation during hardening process. Thus, coaxial micro-extrusion-based 3D printing of a CPC ink with aqueous solvent enhances printability and allows the use of the widespread steam sterilization cycle as a standalone post-processing technique for production of 3D printed personalized CaP bone substitutes. STATEMENT OF SIGNIFICANCE: Coaxial micro-extrusion-based 3D printing of a self-setting CaP cement with water:ethanol mixtures increased the ink yield stress upon extrusion and the stability of fabricated structures. Printing height of overhang structure was doubled when aqueous solvents were used, and a 2 orders of magnitude log increase of the stiffness was achieved post-printing. A fast hardening step consisting of a standard steam sterilization was applied. Four to 5 times higher compressive moduli was obtained for hardened coaxially printed constructs. This improved mechanical performance is likely due to rapid CPC setting in the coaxial printing, preventing cracks formation during hardening process

Tuning the Properties of Hydrogel Microspheres by Adding Chemical Cross-linking Functionality to Sodium Alginate

The production of hydrogel microspheres (MS) for cell immobilization, maintaining the favorable properties of alginate gels but presenting enhanced performance in terms of in vivo durability and physical properties, is desirable to extend the therapeutic potential of cell transplantation. A novel type of hydrogel MS was produced by straightforward functionalization of sodium alginate (Na-alg) with heterotelechelic poly(ethylene glycol) (PEG) derivatives equipped with either end thiol or 1,2-dithiolane moieties. Activation of the hydroxyl moieties of the alginate backbone in the form of imidazolide intermediate allowed for fast conjugation to PEG oligomers through a covalent carbamate linkage. Evaluation of the modified alginates for the preparation of MS combining fast ionic gelation ability of the alginate carboxylate groups and slow covalent cross-linking provided by the PEG-end functionalities highlighted the influence of the chemical composition of the PEG-grafting units on the physical characteristics of the MS. The mechanical properties of the MS (resistance and shape recovery) and durability of PEG-grafted alginates in physiological environment can be adjusted by varying the nature of the end functionalities and the length of the PEG chains. In vitro cell microencapsulation studies and preliminary in vivo assessment suggested the potential of these hydrogels for cell transplantation applications