Real-time 3-D dark-field microscopy for the validation of the cross-linking process of alginate microcapsules.

Alginate-based microencapsulation is a promising method for long-term maintenance of cellular and membrane function of the cells and tissue fragments required for in vitro and in vivo biosensors, for tissue engineering and particularly for immunoisolation of non-autologous transplants. Microcapsules of high mechanical strength and optimum permeability can be produced by injection of BaCl2 crystals into alginate droplets before they come into contact with external Ba2+. A key requirement is that the system parameters (number of crystals, speed of the crystal stream etc.) are properly adjusted according to the mannuronic and guluronic acid ratio and the average molecular mass of the alginate as well as to the diameter of the microcapsules. Robust, reliable, rapid and low-cost validation tools are, therefore, needed for assurance of the microcapsule quality. Here, we describe a novel three-dimensional (3-D) dark-field microscopy that allows the real-time measurement of the number and spatial distribution of the injected Ba2+ ions throughout the microcapsules after treatment with sulphate. This novel method requires only a conventional microscope equipped with three polarising filters and a double aperture stop. In contrast to confocal laser scanning microscopy images, peripherally attached BaSO4 precipitates can clearly be distinguished from internal ones. The data also demonstrate that several steps of the alginate gelling process must be improved before such immunoisolation can be used in patients.

Hepatocytes cultured in alginate microspheres: an optimized technique to study enzyme induction.

An important application of hepatocyte cultures is identification of drugs acting as inducers of biotransformation enzymes that alter metabolic clearance of other therapeutic agents. In the present study we optimized an in vitro system with hepatocytes cultured in alginate microspheres that allow studies of enzyme induction with excellent sensitivity. Induction factors obtained with standard inducers, such as 3-methylcholanthrene or phenobarbital, were higher compared to those with conventional hepatocyte co-cultures on collagen coated dishes. This is illustrated by activities of 7-ethoxyresorufin-O-deethylase (EROD) after incubation with 5 microM 3-methylcholanthrene (3-MC), a standard inducer for cytochrome P4501A1 and 1A2. Mean activities for solvent controls and 3-MC exposed cells were 2.99 and 449 pmol/min/mg protein (induction factor: 150) for hepatocytes cultured in microspheres compared to 2.72 and 80.6 pmol/min/mg (induction factor: 29.6) for hepatocytes on collagen coated dishes. To compare these in vitro data to the in vivo situation male Sprague Dawley rats, the same strain that was used also for the in vitro studies, were exposed to 3-MC in vivo using a protocol that guarantees maximal induction. Activities were 29.2 and 1656 pmol/min/mg in liver homogenate of solvent and 3-MC treated animals (induction factor: 56.7). Thus, the absolute activities of 3-MC exposed hepatocytes in microspheres are lower compared to the in vivo situation. However, the induction factor in vitro was even higher compared to the in vivo situation (150-fold versus 56.7-fold). A similar scenario was observed using phenobarbital (0.75 mM) for induction of CYP2B and 3A isoenzymes: induction factors for testosterone hydroxylation in position 16beta were 127.5- and 50.4-fold for hepatocytes in microspheres and conventionally cultured hepatocytes, respectively. The new in vitro system with hepatocytes embedded in solid alginate microspheres offers several technical advantages: (i) the solid alginate microspheres can be liquefied within 60s, allowing a fast and complete harvest of hepatocytes; (ii) alginate capsules are stable allowing transport and mechanical stress; (iii) high numbers of hepatocytes can be encapsulated in short periods; (iv) defined cell numbers between 600 hepatocytes, the approximate number of cells in one capsule, and 18 x 10(6) hepatocytes, the number of hepatocytes in 6 ml alginate, can be transferred to a culture dish or flask. Thus, encapsulated hepatocytes allow a flexible organization of experiments with respect to cell number. In conclusion, we optimized a technique for encapsulation of hepatocytes in alginate microspheres that allows identification of enzyme induction with an improved sensitivity compared to existing systems.

In vitro and in vivo evaluation of novel glibenclamide derivatives as imaging agents for the non-invasive assessment of the pancreatic islet cell mass in animals and humans.

Pancreatic islet cell mass (PICM) is a major determinant of the insulin secretory capacity in humans. Currently, the only method for accurate assessment of the PICM is an autopsy study. Thus, development of a technique allowing the non-invasive quantification of PICM is of great interest. The aim of this study was to develop such a non-invasive technique featuring novel fluorine- and (99m)Tc-labelled glibenclamide derivatives. Despite the structural modifications necessary to introduce fluorine into the glibenclamide molecule, all derivatives retained insulin stimulating capacity as well as high affinity binding to human SUR1 when compared to the original glibenclamide. Contrastingly, the lipophilicity of the fluorine-labelled derivatives was altered depending on the particular modification. In the human PET-study a constant but weak radioactive signal could be detected in the pancreas using a fluorine-labelled glibenclamide derivative. However, a reliable assessment and visualisation of the PICM could not be obtained. It can be assumed that the high uptake of the fluorine-labelled tracer e.g. into the the liver and the high plasma protein binding leads to a relatively low signal-to-noise ratio. In case of the presented fluorine-labelled glibenclamide based compounds this could be the result of their invariably high lipophilicity. The development of a (99 m)Tc-labelled glibenclamide derivative with a lower lipophilicity and differing in vivo behaviour, glibenclamide based compounds for non-invasive imaging of the pancreatic islet cell mass may be possible.

Fabrication of homogeneously cross-linked, functional alginate microcapsules validated by NMR-, CLSM- and AFM-imaging.

Cross-linked alginate microcapsules of sufficient mechanical strength can immunoisolate cells for the long-term treatment of hormone and other deficiency diseases in human beings. However, gelation of alginate by external Ba(2+) (or other divalent cations) produces non-homogeneous cross-linking of the polymeric mannuronic (M) and guluronic (G) acid chains. The stability of such microcapsules is rather limited. Here, we show that homogeneous cross-linking can be achieved by injecting BaCl(2) crystals into alginate droplets before they come into contact with external BaCl(2). The high effectiveness of this crystal gun method is demonstrated by confocal laser scanning microscopy and by advanced nuclear magnetic resonance imaging. Both techniques gave clear-cut evidence that homogeneous cross-linkage throughout the microcapsule is only obtained with simultaneous internal and external gelation. Atomic force microscopy showed a very smooth surface topography for microcapsules made by the crystal gun method, provided that excess Ba(2+) ions were removed immediately after gelation. In vitro experiments showed greatly suppressed swelling for crystal gun microcapsules. Even alginate extracted from Lessonia nigrescens (highly biocompatible) yielded microcapsules with long-term mechanical stability not hitherto possible. Encapsulation of rat islets, human monoclonal antibodies secreting hybridoma cells and murine mesenchymal stem cells transfected with cDNA encoding for bone morphogenetic protein (BMP-4) revealed that injection of BaCl(2) crystals has no adverse side effects on cell viability and function. However, the release of low-molecular weight factors (such as insulin) may be delayed when using alginate concentrations in the usual range.

Multilayer capsules: a promising microencapsulation system for transplantation of pancreatic islets.

In 1980, Lim and Sun introduced a microcapsule coated with an alginate/polylysine complex for encapsulation of pancreatic islets. Characteristic to this type of capsule is, that it consists of a plain membrane which is formed during a single procedural step. With such a simple process it is difficult to obtain instantly a membrane optimized with respect to all the properties requested for islet transplantation. To overcome these difficulties, it is recommended to build up the membrane in several consecutive steps, each optimized for a certain property. In this study, we have analysed such a multilayer microcapsule for the encapsulation of pancreatic islets. Therefore, empty and islet containing alginate beads were coated with alternating layers of polyethyleneimine, polyacrylacid or carboxymethylcellulose and alginate. By scanning electron microscopy the thickness of the covering multilayer-membrane was estimated to be less than 800 nm by comparison with an apparatus scale. Ellipsometric measurements showed that the membrane thickness is in the range of 145 nm. Neither the encapsulation procedure, nor the membrane-forming step did impede the stimulatory response of the islets. The encapsulation even lead to a significantly better stimulatory response of the encapsulated islets during week three and five of cell culture. Furthermore, the multilayer-membrane did not deteriorate the biocompatibility of the transplanted microcapsules, allowing an easy tuning of the molecular cut-off and the mechanical stability depending on the polycation-polyanion combination used. The multilayer membrane capsule has obvious advantages compared to a one-step encapsulation procedure. These beads guarantee a high biocompatibility, a precisely adjusted cut-off, an optimal insulin-response and high mechanical stability although the membrane is only 145 nm thick.

Synthesis and evaluation of (S)-2-(2-[18F]fluoroethoxy)-4-([3-methyl-1-(2-piperidin-1-yl-phenyl)-butyl-carbamoyl]-methyl)-benzoic acid ([18F]repaglinide): a promising radioligand for quantification of pancreatic beta-cell mass with positron emission tomography (PET).

18F-labeled non-sulfonylurea hypoglycemic agent (S)-2-(2-[(18)F]fluoroethoxy)-4-((3-methyl-1-(2-piperidin-1-yl-phenyl)-butylcarbamoyl)-methyl)-benzoic acid ([(18)F]repaglinide), a derivative of the sulfonylurea-receptor (SUR) ligand repaglinide, was synthesized as a potential tracer for the non-invasive investigation of the sulfonylurea 1 receptor status of pancreatic beta-cells by positron emission tomography (PET) in the context of type 1 and type 2 diabetes. [(18)F]Repaglinide could be obtained in an overall radiochemical yield (RCY) of 20% after 135 min with a radiochemical purity higher than 98% applying the secondary labeling precursor 2-[(18)F]fluoroethyltosylate. Specific activity was in the range of 50-60 GBq/micromol. Labeling was conducted by exchanging the ethoxy-moiety into a 2-[(18)F]fluoroethoxy group. To characterize the properties of fluorinated repaglinide, the affinity of the analogous non-radioactive (19)F-compound for binding to the human SUR1 isoform was assessed. [(19)F]Repaglinide induced a complete monophasic inhibition curve with a Hill coefficient close to 1 (1.03) yielding a dissociation constant (K(D)) of 134 nM. Biological activity was proven via insulin secretion experiments on isolated rat islets and was comparable to that of repaglinide. Finally, biodistribution of [(18)F]repaglinide was investigated in rats by measuring the concentration of the compound in different organs after i.v. injection. Pancreatic tissue displayed a stable accumulation of approximately 0.12% of the injected dose from 10 min to 30 min p.i. 50% of the radioactive tracer could be displaced by additional injection of unlabeled repaglinide, indicating that [(18)F]repaglinide might be suitable for in vivo investigation with PET.

Size of pancreatic islets of Langerhans: a key parameter for viability after cryopreservation.

Large amounts and excellent viabilities of pancreatic islets are prerequisites for recent advances in islet transplantation. Cryopreservation has been shown to enlarge transplanted cell mass, but has been accompanied by reduced viability. In this study rat pancreatic islets were differentiated into small (<200 micro m), medium (200-400 micrometers) and large (>400 micrometers) categories and their susceptibilities to different freezing conditions were evaluated: concentration of cryoprotectant (0.7-3.1 M), equilibration (15 vs. 45 min, 22 degrees C vs. on ice) and post-thaw removal of cryoprotectant (15 vs. 30 min, stepwise vs. one-step). The most prominent finding was a negative correlation between islet size and viability observed in non-frozen islets to a minor degree (r=-0.44) and significantly enhanced after cryopreservation (r<-0.8). The concentration of cryoprotectant showed the most significant influence on viability affecting small, medium and large islets. Different techniques of equilibration with the cryoprotectant resulted in significant changes of islet viability of medium islets, whereas small and large islets were unaffected. For different techniques of removal of the cryoprotectant, no significant influence on viabilities was found. We conclude that large islets represented a highly susceptible population concerning damage due to cryopreservation.

Towards a medically approved technology for alginate-based microcapsules allowing long-term immunoisolated transplantation.

The concept of encapsulated-cell therapy is very appealing, but in practice a great deal of technology and know-how is needed for the production of long-term functional transplants. Alginate is one of the most promising biomaterials for immunoisolation of allogeneic and xenogeneic cells and tissues (such as Langerhans islets). Although great advances in alginate-based cell encapsulation have been reported, several improvements need to be made before routine clinical applications can be considered. Among these is the production of purified alginates with consistently high transplantation-grade quality. This depends to a great extent on the purity of the input algal source as well as on the development of alginate extraction and purification processes that can be validated. A key engineering challenge in designing immunoisolating alginate-based microcapsules is that of maintaining unimpeded exchange of nutrients, oxygen and therapeutic factors (released by the encapsulated cells), while simultaneously avoiding swelling and subsequent rupture of the microcapsules. This requires the development of efficient, validated and well-documented technology for cross-linking alginates with divalent cations. Clinical applications also require validated technology for long-term cryopreservation of encapsulated cells to maintaining a product inventory in order to meet end-user demands. As shown here these demands could be met by the development of novel, validated technologies for production of transplantation-grade alginate and microcapsule engineering and storage. The advances in alginate-based therapy are demonstrated by transplantation of encapsulated rat and human islet grafts that functioned properly for about 1 year in diabetic mice.

Beneficial effects of human serum albumin on stability and functionality of alginate microcapsules fabricated in different ways.

A key engineering challenge in designing microcapsules made from biocompatible alginate is maintaining adequate exchange of nutrients and oxygen between the entrapped cells and the environment, while simultaneously avoiding swelling and subsequent failure of the microcapsule. Approval for the use of alginate in pharmaceutical and/or biomedical applications also strictly requires that the components of the microcapsule material must meet the safety criteria of the ASTM and FDA. Incorporation of foetal calf serum (FCS) into the microcapsules for stabilization is not in accordance with the guidelines affirmed by these organizations. FCS should be substituted by microcapsule-stabilizing additives that are medically approved. In this communication, it is shown that 10% FCS can be replaced by 1% human serum albumin (i.e. by an agent for which medical approval is granted) without compromising effects on long-term in vitro stability. Furthermore, it is demonstrated that human serum albumin (HSA) significantly enhances cell survival and, particularly, insulin secretion of encapsulated rat islets over a time period of 3 weeks when incubated in culture medium. Thus, HSA-stabilized microcapsules made from UHV(Lam) alginate are apparently a promising system for immunoisolation of cells, particularly when alginate is cross-linked by injection of BaCl(2) crystals into the alginate droplets. Slight adjustments of the alginate concentration can tailor the microcapsule permeability to the released therapeutic factor.