The compressive deformation of multicomponent microcapsules: influence of size, membrane thickness, and compression speed.

The clinical application of microcapsules for the immunoisolation of living tissue requires knowledge about the mechanical stability of polymer membranes. Microcapsules of 400-1000 microm in diameter were formed through the gelation of sodium alginate/sodium cellulose sulfate droplets through calcium chloride, with the membrane produced via complex coacervation between polyanions and poly(methylene-co-guanidine) hydrochloride. The deformation behavior of these multicomponent microcapsules was investigated by uniaxial compression experiments. Specifically, the influence of the deformation speed, capsule diameter, and membrane thickness on the mechanical properties was evaluated. The bursting force was found to be dependent on the deformation speed. Therefore, the measurement of the bursting work, a speed-independent value of the resistance to high stresses and deformations, was recommended as the most valid for capsule mechanical resistance. Furthermore, the bursting force was positively correlated with membrane thickness only for membrane-radius ratios up to 20%. For thicker membranes, the bursting event occurred because the opposite membranes touched each other, and not, because of insufficient strength. Indeed, the resistance to smaller deformations was positively correlated to the membrane thickness over the whole range of membrane-radius ratios. Moreover, the forces for constant deformation were linearly correlated to the total membrane volume, independently of capsule size and membrane thickness.

Maintenance of primary murine hepatocyte functions in multicomponent polymer capsules--in vitro cryopreservation studies.

BACKGROUND/AIMS: The potential of a new encapsulation system has been evaluated as an artificial housing for liver cells. METHODS: Murine hepatocytes were encapsulated in specially designed multicomponent capsules formed by polyelectrolyte complexation of sodium alginate, cellulose sulphate and poly(methylene-co-guanidine) hydrochloride, the permeability of which has previously been characterised. RESULTS: We demonstrate here the absence of cytotoxicity and the excellent biocompatibility of these capsules towards primary culture of murine hepatocytes. Experimental results demonstrated that the encapsulated hepatocytes retained their specific functions--transaminase activity, urea synthesis and protein secretion--over the first 4 days of culture in minimum medium. The cryopreservation of encapsulated hepatocytes, for periods of up to 4 months, did not alter their functional capacities, as no major differences were observed between unfrozen and frozen encapsulated cells for the functions tested. CONCLUSIONS: Because of the absence of cytotoxicity, and the ease of handling and cryopreservation, while maintaining liver specific functions, the described system appears to be valuable for murine liver cell encapsulation. It is also a promising tool for fundamental research into drug metabolism, intercellular regulation, metabolic pathways, and the establishment of banks for the supply and storage of murine hepatocytes.

Development of a coculture model of encapsulated cells.

In the whole animal, metabolic regulations are set by reciprocal interactions between various organs, via the blood circulation. At present, analyses of such interactions require numerous and uneasily controlled in vivo experiments. In a search for an alternative to in vivo experiments, our work aims at developing a coculture system in which different cell types are isolated in polymer capsules and grown in a common environment. The signals exchanged between cells from various origins are, thus, reproducing the in vivo intertissular communications. With this perspective, we evaluated a new encapsulation system as an artificial housing for liver cells on the one hand and adipocytes on the other hand. Murine hepatocytes were encapsulated with specially designed multicomponent capsules formed by polyelectrolyte complexation between sodium alginate, cellulose sulphate and poly(methylene-coguanidine) hydrochloride, of which the permeability has been characterized. We demonstrated the absence of cytotoxicity and the excellent biocompatibility of these capsules towards primary culture of murine hepatocytes. Encapsulated hepatocytes retain their specific functions--transaminase activity, urea synthesis, and protein secretion--during the first four days of culture in minimum medium. Mature adipocytes, isolated from mouse epidydimal fat, were embedded in alginate beads. Measurement of protein secretion shows an identical profile between free and embedded adipocytes. We finally assessed the properties of encapsulated hepatocytes, cryopreserved over a periods of up to four months. The perspective of using encapsulated cells in coculture are discussed, since this system may represent a promising tool for fundamental research, such as analyses of drug metabolism, intercellular regulations, and metabolic pathways, as well as for the establishment of a tissue bank for storage and supply of murine hepatocytes.

Objectively assessing bioartificial organs.

The metrics used, thus far, to assess bioartificial organ function are shown to be subjective and requiring validation. Therefore, four categories of correlations are proposed based on, respectively, device, in vitro and in vivo evaluations, and clinical function. Examples are presented whereby the correlations among individual indicators are used as a means to expedite the development of immunoisolated cells. Specifically, a case study illustrating the validation of in vitro indicators of in vivo graft function for the bioartificial pancreas (microencapsulated islets) is summarized. This has revealed thresholds with respect to given metrics relating to in vivo device function, the necessity to couple bioartificial organ design with transplant site selection, as well as the lack of objectivity involved in the evaluation and establishment of hypotheses. Specific quantitative indicators illustrate the need for quality-controlled measures, for example, relating to the tolerance of microcapsule diameter and membrane thickness distributions. Qualitative indices representing fibrosis and device properties (e.g., sphericity) are also used to describe the need for in vitro experiments in the development of bioartificial organs.

Activation of peroxisome proliferator-activated receptors (PPARs) by their ligands and protein kinase A activators.

The nuclear peroxisome proliferator-activated receptors (PPARs) alpha, beta, and gamma activate the transcription of multiple genes involved in lipid metabolism. Several natural and synthetic ligands have been identified for each PPAR isotype but little is known about the phosphorylation state of these receptors. We show here that activators of protein kinase A (PKA) can enhance mouse PPAR activity in the absence and the presence of exogenous ligands in transient transfection experiments. Activation function 1 (AF-1) of PPARs was dispensable for transcriptional enhancement, whereas activation function 2 (AF-2) was required for this effect. We also show that several domains of PPAR can be phosphorylated by PKA in vitro. Moreover, gel retardation experiments suggest that PKA stabilizes binding of the liganded PPAR to DNA. PKA inhibitors decreased not only the kinase-dependent induction of PPARs but also their ligand-dependent induction, suggesting an interaction between both pathways that leads to maximal transcriptional induction by PPARs. Moreover, comparing PPAR alpha knockout (KO) with PPAR alpha WT mice, we show that the expression of the acyl CoA oxidase (ACO) gene can be regulated by PKA-activated PPAR alpha in liver. These data demonstrate that the PKA pathway is an important modulator of PPAR activity, and we propose a model associating this pathway in the control of fatty acid beta-oxidation under conditions of fasting, stress, and exercise.

New multicomponent capsules for immunoisolation.

A new generation of microcapsules based on the use of oligomers which participate in polyelectrolyte complexation reactions has been developed. These freeze-thaw stable capsules have been applied as a bioartificial pancreas and have resulted in normoglycemia for periods of six months in concordant xenotransplantations. The new chemistry permits the control of permeability and mechanical properties over a wide range and can be adapted both to microcapsule and hollow fiber geometries rendering it a robust tool for encapsulation in general. Methods, and metrics, for the characterization of the mechanical properties and permeability of microcapsules are presented.

DSP1 gene of Drosophila melanogaster encodes an HMG-domain protein that plays multiple roles in development.

DSP1 is an HMG-box containing protein of Drosophila melanogaster which was first identified as a co-repressor of the Dorsal protein. Recently, the analysis of the structure of the gene has led us to propose that DSP1 is the Drosophila equivalent of the ubiquitous vertebrate HMG 1/2 proteins. In the present paper, the patterns of expression of DSP1 protein and RNA in adult flies and during development are reported. In the adults DSP1 protein is located in nurse cells of ovaries and in brain. During eggs development uniform expression of DSP1 protein persists until the end of germband retraction. At later stages, expression is restricted to the ventral nerve chord and brain. Using P-element mutagenesis, we have isolated a mutant deficient in DSP1 functions. Genetic studies of this mutant show that DSP1 protein is essential for the growth and the development of Drosophila. In addition to be a co-repressor of the transcriptional activator Dorsal our results provide compelling evidence that DSP1 is a regulator involved in several pathways necessary for the development of the fly.

The Drosophila DSP1 gene encoding an HMG 1-like protein: genomic organization, evolutionary conservation and expression.

The gene that encodes the dorsal switch protein (DSP1) has been isolated from a Drosophila melanogaster cosmid library. It is organized into seven exons and six introns. The relative position of the introns within the region coding for the high mobility group (HMG) domains are identical to those of vertebrate HMG 1/2 genes. The close similarity between DSP1 and HMG 1/2 genes strongly suggests that these genes derived from a common ancestral gene. DSP1 encodes, at least, two distinct mRNAs that differ in the length of their 5'-untranslated region and coding sequence. Detailed sequence analysis shows that alternative splicing of precursor mRNA gives rise to the two isoform mRNAs found in Drosophila cells.