Yrr A. Mørch

Multiscale requirements for bioencapsulation in medicine and biotechnology

Bioencapsulation involves the envelopment of tissues or biological active substances in semipermeable membranes. Bioencapsulation has been shown to be efficacious in mimicking the cells natural environment and thereby improves the efficiency of production of different metabolites and therapeutic agents. The field of application is broad. It is being applied in bioindustry and biomedicine. It is clinically applied for the treatment of a wide variety of endocrine diseases. During the past decades many procedures to fabricate capsules have been described. Unfortunately, most of these procedures lack an adequate documentation of the characterization of the biocapsules. As a result many procedures show an extreme lab-to-lab variation and many results cannot be adequately reproduced. The characterization of capsules can no longer be neglected, especially since new clinical trials with bioencapsulated therapeutic cells have been initiated and the industrial application of bioencapsulation is growing. In the present review we discuss novel Approached to produce and characterize biocapsules in view of clinical and industrial application. A dominant factor in bioencapsulation is selection and characterization of suitable polymers. We present the adequacy of using high-resolution NMR for characterizing polymers. These polymers are applied for producing semipermeable membranes. We present the pitfalls of the currently applied methods and provide recommendations for standardization to avoid lab-to-lab variations. Also, we compare and present methodologies to produce biocompatible biocapsules for specific fields of applications and we demonstrate how physico-chemical technologies such as FT-IR, XPS, and TOF-SIMS contribute to reproducibility and standardization of the bioencapsulation process. During recent years it has become more and more clear that bioencapsulation requires a multidisciplinary approach in which biomedical, physical, and chemical technologies are combined. For adequate reproducibility and for understanding variations in outcome of biocapsules it is advisable if not mandatory to include the characterization processes presented in this review in future studies.

Encapsulation of Human Islets in Novel Inhomogeneous Alginate-Ca2+/Ba2+ Microbeads : In Vitro and In Vivo Function

Microencapsulation may allow for immunosuppression-free islet transplantation. Herein we investigated whether human islets can be shipped safely to a remote encapsulation core facility and maintain in vitro and in vivo functionality. In non-encapsulated islets before and encapsulated islets after shipment, viability was 88.3±2.5 and 87.5±2.7% (n=6, p=0.30). Stimulation index after static glucose incubation was 5.4±0.5 and 6.3±0.4 (n=6, p=0.18), respectively. After intraperitoneal transplantation, long-term normoglycemia was consistently achieved with 3,000, 5,000, and 10,000 IEQ encapsulated human islets. When transplanting 1,000 IEQ, mice returned to hyperglycemia after 30-55 (n=4/7) and 160 days (n=3/7). Transplanted mice showed human oral glucose tolerance with lower glucose levels than non-diabetic control mice. Capsules retrieved after transplantation were intact, with only minimal overgrowth. This study shows that human islets maintained the viability and in vitro function after encapsulation and the inhomogeneous alginate-Ca2+/Ba2+ microbeads allow for long-term in vivo human islet graft function, despite long-distance shipment.

Mechanical properties of C-5 epimerized alginates.

There is an increased need for alginate materials with both enhanced and controllable mechanical properties in the fields of food, pharmaceutical and specialty applications. In the present work, well-characterized algal polymers and mannuronan were enzymatically modified using C-5 epimerases converting mannuronic acid residues to guluronic acid in the polymer chain. Composition and sequential structure of controls and epimerized alginates were analyzed by (1)H NMR spectroscopy. Mechanical properties of Ca-alginate gels were further examined giving Youngs modulus, syneresis, rupture strength, and elasticity of the gels. Both mechanical strength and elasticity of hydrogels could be improved and manipulated by epimerization. In particular, alternating sequences were found to play an important role for the final mechanical properties of alginate gels, and interestingly, a pure polyalternating sample resulted in gels with extremely high syneresis and rupture strength. In conclusion, enzymatic modification was shown to be a valuable tool in modifying the mechanical properties of alginates in a highly specific manner.

Binding and Leakage of Barium in Alginate Microbeads

Microbeads of alginate crosslinked with Ca(2+) and/or Ba(2+) are popular matrices in cell-based therapy. The aim of this study was to quantify the binding of barium in alginate microbeads and its leakage under in vitro and accumulation under in vivo conditions. Low concentrations of barium (1 mM) in combination with calcium (50 mM) and high concentrations of barium (20 mM) in gelling solutions were used for preparation of microbeads made of high-G and high-M alginates. High-G microbeads accumulated barium from gelling solution and contained higher concentrations of divalent ions for both low- and high-Ba exposure compared with high-G microbeads exposed to calcium solely and to high-M microbeads for all gelling conditions. Although most of the unbound divalent ions were removed during the wash and culture steps, leakage of barium was still detected during storage. Barium accumulation in blood and femur bone of mice implanted with high-G beads was found to be dose-dependent. Estimated barium leakage relevant to transplantation to diabetic patients with islets in alginate microbeads showed that the leakage was 2.5 times lower than the tolerable intake value given by WHO for high-G microbeads made using low barium concentration. The similar estimate gave 1.5 times higher than is the tolerable intake value for the high-G microbeads made using high barium concentration. To reduce the risk of barium accumulation that may be of safety concern, the microbeads made of high-G alginate gelled with a combination of calcium and low concentration of barium ions is recommended for islet transplantation.

Effect of elongation of alternating sequences on swelling behavior and large deformation properties of natural alginate gels.

The physical properties of alginate gels correlate with alginate composition. Blocks of guluronic acid (G) strongly contribute to gel formation. Recently, the role of alternating sequences in calcium-alginate gels has been elucidated. The present contribution aimed at extending the analysis already reported (Donati, I.; Holtan, S.; Mørch, Y. A.; Borgogna, M.; Dentini, M.; Skjåk-Braek, G. Biomacromolecules 2005, 6, 1031) and at explaining some apparent mismatch of experimental data. In the present work, calcium hydrogels from different alginate samples have been analyzed by means of uniaxial compression and puncture tests to evaluate their Youngs modulus and work at break. The role of long MG blocks in mechanical deformations (small and large domains) as well as in swelling experiments was investigated with natural and MG-enriched (AlgE4 epimerized) alginate samples. Alginates with elongated alternating sequences displayed, upon treatment with saline solution, a notable increase in swelling behavior, which was not paralleled by increased mechanical properties (Youngs modulus). This behavior was traced back to the disentanglement of MG/MG junctions, which increased the local charge density, reducing the osmotic contribution to hydrogel swelling. The analyses of the large deformation curves for natural and epimerized alginates revealed an increase in the energy to breakage in the latter case caused by the dissipation effect of sliding MG/MG junctions.

Survival of human islets in microbeads containing high guluronic acid alginate crosslinked with Ca2+ and Ba2+

Qi M, Mørch Y, Lacík I, Formo K, Marchese E, Wang Y, Danielson KK, Kinzer K, Wang S, Barbaro B, Kolláriková G, Chorvát D Jr, Hunkeler D, Skjåk‐Bræk G, Oberholzer J, Strand BL. Survival of human islets in microbeads containing high guluronic acid alginate crosslinked with Ca2+ and Ba2+. Xenotransplantation 2012; 19: 355–364.