[Cell transplantation: current treatments and future prospects]. / Transplantation cellulaire: traitements actuels et perspectives d'avenir.

Regenerative medicine aims to replace a body function or specific cell loss. It includes therapies at the forefront of modem medicine, issuing from translational biomedical research. Transplantation of organs and cells has revolutionized the management of patients for whom medical treatment is a failure. Unfortunately, organ shortage is limiting treatment possibility. As an example, among the 15,000 patients with type I diabetes in Switzerland, only approximately 30 can receive a pancreas or an islet transplant per year. Second example, 500 patients die each year in Switzerland from alcoholic cirrhosis because no treatment is available. Transplantation of islet cells, hepatocytes, mesenchymal stem cells or dopaminergic neurons represents hope fora therapy available for large populations of patients.

Use of the mitochondria toxicity assay for quantifying the viable cell density of microencapsulated jurkat cells.

The mitochondria toxicity assay (MTT assay) is an established method for monitoring cell viability based on mitochondrial activity. Here the MTT assay is proposed for the in situ quantification of the living cell density of microencapsulated Jurkat cells. Three systems were used to encapsulate the cells, namely a membrane consisting of an interpenetrating polyelectrolyte network of sodium cellulose sulphate/poly(diallyldimethylammonium chloride) (NaCS/PDADMAC), a calcium alginate hydrogel covered with poly(L-lysine) (Ca-alg-PLL), and a novel calcium alginate-poly(ethylene glycol) hybrid material (Ca-alg-PEG). MTT results were correlated to data obtained by the trypan blue exclusion assay after release of the cells from the NaCS/PDADMAC and Ca-alg-PLL capsules, while a resazurin-based assay was used for comparison in case of the Ca-alg-PEG material. Analysis by MTT assay allows quick and reliable determination of viable cell densities of encapsulated cells independent of the capsule material. The assay is highly reproducible with inter-assay relative standard deviations below 10%.

Novel nano-particles as fillers for an experimental resin-based restorative material.

The purpose of this study is to compare the properties of two experimental materials, nano-material (Nano) and Microhybrid, and two trade products, Clearfil AP-X and Filtek Supreme XT. The flexural strength and modulus after 24h water storage and 5000 thermocycles, water sorption, solubility and X-ray opacity were determined according to ISO 4049. The volumetric behavior (DeltaV) after curing and after water storage was investigated with the Archimedes principle. ANOVA was calculated with p<0.05. Clearfil AP-X showed the highest flexural strength (154+/-14 MPa) and flexural modulus (11,600+/-550 MPa) prior to and after thermocycling (117+/-14 MPa and 13,000+/-300 MPa). The flexural strength of all materials decreased after thermocycling, but the flexural modulus decreased only for Filtek Supreme XT. After thermocycling, there were no significant differences in flexural strength and modulus between Filtek Supreme XT, Microhybrid and Nano. Clearfil AP-X had the lowest water sorption (22+/-1.1 microg mm(-3)) and Nano had the highest water sorption (82+/-2.6 microg mm(-3)) and solubility (27+/-2.9 microg mm(-3)) of all the materials. No significant differences occurred between the solubility of Clearfil AP-X, Filtek Supreme XT and Microhybrid. Microhybrid and Nano provided the highest X-ray opacity. Owing to the lower filler content, Nano showed higher shrinkage than the commercial materials. Nano had the highest expansion after water storage. After thermocycling, Nano performed as well as Filtek Supreme XT for flexural strength, even better for X-ray opacity but significantly worse for flexural modulus, water sorption and solubility. The performances of microhybrids were superior to those of the nano-materials.

Synthesis of enantiopure (5R)-hydroxyhexane-2-one with immobilised whole cells of Lactobacillus kefiri.

Whole-cell reduction of (2,5)-hexanedione to yield highly enantiopure (5R)-hydroxyhexane-2-one (enantiomeric excess >99%) with Lactobacillus kefiri DSM 20587 was investigated. Cell immobilisation with sodium cellulose sulphate was chosen as the most suitable encapsulation matrix, giving an immobilisation yield of 40%. Despite the lowered biocatalytic activity from cell immobilisation, the bioreduction process was vastly improved with the help of reaction engineering techniques (batch to a plug flow reactor set-up). High selectivity (95%) and space-time yield (87 g L(-1) day(-1)) were achieved in the plug flow reactor. The biocatalyst remained active (68% residual activity) after 6 days of operation.

Indirect electrochemical reduction of nicotinamide coenzymes.

Nicotinamide coenzymes nicotinamide adenine dinucleotide (NAD(+)) and nicotinamide adenine dinucleotide phosphate (NADP(+)) were electrochemically reduced to NADH and NADPH, respectively. As direct reduction of nicotinamide coenzymes leads to inactive by-products, an indirect method using (pentamethylcyclopentadienyl-2,2'-bipyridine aqua) rhodium (III) as the mediator, was applied. A phosphate buffer solution, pH 8, with 1-10 mM NAD(P)(+) and 2.5-200 microM mediator, was pumped through a glassy carbon packed bed cathode. Virtually all the NAD(P)(+) was reduced to NAD(P)H in the cell. No sign of mediator loss due to side-reactions was detected though the mediator molecules shuttled hundreds of times between the oxidised and the reduced form. Adsorption of mediator molecules on the surface of the carbon cathode was found to be important for the reduction process. Due to strong adsorption, only minute amounts of mediator were consumed.

Short-term immunosuppression reduces fibrotic cellular infiltration around barium-M-alginate microbeads injected intraportally.

INTRODUCTION: One of the major barriers affecting the viability of encapsulated islets is pericapsular fibrotic infiltration (PFI). This study aimed to design strategies to reduce PFI around intraportally injected alginate microbeads. METHODS: Empty, highly purified, barium-M-alginate microbeads (400 microm) were injected intraportally into Lewis rats (3000 beads/rat). Rats (n = 9/group) were treated daily with either rapamycine (RAPA; 1 mg/kg/d p.o.), tacrolimus (TAC; 2 mg/kg/d p.o.), a combination of both, or gadolinium-chloride (GdC13, 20 mg/kg/d i.v., at day -1 and day +4). Treatment was discontinued at 10 days. Three rats/group were sacrificed at 3, 7, and 42 days after transplantation. Cellular composition of PFI was evaluated by immunohistochemistry. Severity of the reaction to the beads was determined by measuring the thickness of PFI on histology. RESULTS: The main cellular components of PFI in the liver were macrophages and myofibroblasts. There was a significant (P <.05) reduction in the thickness of PFI in all treated groups, even 6 weeks after transplantation. Encapsulated rat islets showed excellent insulin response to glucose in vitro, with a stimulation index of 3.6 +/- 2.0. CONCLUSION: Combination of highly purified alginate with short-term immunosuppression reduces fibrotic overgrowth around microbeads injected intraportally.

Modeling of the pyruvate production with Escherichia coli in a fed-batch bioreactor.

A family of 10 competing, unstructured models has been developed to model cell growth, substrate consumption, and product formation of the pyruvate producing strain Escherichia coli YYC202 ldhA::Kan strain used in fed-batch processes. The strain is completely blocked in its ability to convert pyruvate into acetyl-CoA or acetate (using glucose as the carbon source) resulting in an acetate auxotrophy during growth in glucose minimal medium. Parameter estimation was carried out using data from fed-batch fermentation performed at constant glucose feed rates of q(VG)=10 mL h(-1). Acetate was fed according to the previously developed feeding strategy. While the model identification was realized by least-square fit, the model discrimination was based on the model selection criterion (MSC). The validation of model parameters was performed applying data from two different fed-batch experiments with glucose feed rate q(VG)=20 and 30 mL h(-1), respectively. Consequently, the most suitable model was identified that reflected the pyruvate and biomass curves adequately by considering a pyruvate inhibited growth (Jerusalimsky approach) and pyruvate inhibited product formation (described by modified Luedeking-Piret/Levenspiel term).

Influence of alginate characteristics on the properties of multi-component microcapsules.

A variety of sodium alginates, differing in molar mass and structural composition, have been evaluated in the preparation of multi-component microbeads and microcapsules. Bead formation occurred by gelation with calcium chloride. Capsules were produced by reacting the pre-formed beads with the oligocation poly(methylene-co-guanidine). Despite the equiponderous (1:1) mixing with a second polyanion, sodium cellulose sulphate, the influence of the alginate properties remains evident. Specifically, the effect of the chemical composition was found to be more significant than that of the molar mass for both the mechanical and transport properties. Furthermore, for alginates of 73% alpha-l-guluronic acid content less shrinking was observed compared to the 38% guluronic materials. This results in the case of the same encapsulator settings in larger microsphere diameters and thicker membranes accompanied by enhanced mechanical resistance though, also, in a higher permeability for the high-G capsules. However, subsequent coating with lower molar mass alginate allows one to adjust the permeability over a broad range, suitable for cell encapsulation and immunoprotection, without compromising the durability.

Continuous gluconic acid production by isolated yeast-like mould strains of Aureobasidium pullulans.

By extensive microbial screening, about 50 strains with the ability to secrete gluconic acid were isolated from wild flowers. The strains belong to the yeast-like mould Aureobasidium pullulans (de Bary) Arnaud. In shake flask experiments, gluconic acid concentrations between 23 and 140 g/l were produced within 2 days using a mineral medium. In batch experiments, various important fermentation parameters influencing gluconic acid production by A. pullulans isolate 70 (DSM 7085) were identified. Continuous production of gluconic acid with free-growing cells of the isolated yeast-like microorganisms was studied. About 260 g/l gluconic acid at total glucose conversion could be achieved using continuous stirred tank reactors in defined media with residence times (RT) of about 26 h. The highest space-time-yield of 19.3 g l(-1) x h(-1)) with a gluconic acid concentration of 207.5 g/l was achieved with a RT of 10.8 h. The possibility of gluconic acid production with biomass retention by immobilised cells on porous sinter glass is discussed. The new continuous gluconate fermentation process provides significant advantages over traditional discontinuous operation employing Aspergillus niger. The aim of this work was the development of a continuous fermentation process for the production of gluconic acid. Process control becomes easier, offering constant product quality and quantity.

Citric acid production by Candida strains under intracellular nitrogen limitation.

A suitable strain and important factors influencing citric acid formation in yeasts were identified. Candida oleophila ATCC 20177 was chosen as the best citric acid producer from several Candida strains. Yields of 50 g/l citric acid were produced in shake flask and 80 g/l in fed-batch fermentations with 1.5 and 3 g/l NH(4)Cl under non-optimized conditions. Ammonium nitrogen was identified as the limiting substrate for citrate formation. Citric acid excretion begins a few hours after exhaustion of nitrogen in the medium. The importance of intracellular nitrogen limitation was clarified by elemental analysis of C. oleophila biomass. The nitrogen content of C. oleophila biomass decreased from 7.45% during the growth phase to 3.96% in the production phase. The biomass contained less carbon and more trace elements in the growth phase compared with the production phase. Relatively high intracellular NH(4)(+) concentration of about 1.2 mg/g biomass (~37.4 mM) was found during the production phase. The low intracellular nitrogen content and increase of intracellular NH(4)(+) concentration, possibly caused by proteolysis following extracellular nitrogen exhaustion, trigger citric acid production. Intracellular nitrogen limitation and the increase in intracellular NH(4)(+) concentration are the most important factors influencing citric acid formation in yeasts.

Chemo-enzymatic synthesis of the Galili epitope Gal(alpha)(1-->3)Galbeta(1-->4)GlcNAc on a homogeneously soluble PEG polymer by a multi-enzyme system.

The alpha-Gal trisaccharide Gal(alpha)(1-->3)Galbeta(1-->4)GlcNAc 11 was synthesized on a homogeneously soluble polymeric support (polyethylene glycol, PEG) by use of a multi-enzyme system consisting of beta-1,4-galactosyltransferase (EC 2.4.1.38), alpha-1,3-galactosyltransferase (EC 2.4.1.151), sucrose synthase (EC 2.4.1.13) and UDP-glucose-4-epimerase (EC 5.1.3.2). In addition workup was simplified by use of dia-ultrafiltration. Thus the advantages of classic chemistry/enzymology and solid-phase synthesis could be united in one. Subsequent hydrogenolytic cleavage afforded the free alpha-Gal trisaccharide.

Quantification of intracellular metabolites in Escherichia coli K12 using liquid chromatographic-electrospray ionization tandem mass spectrometric techniques.

The quantitative comprehension of microbial metabolic networks is a prerequisite for an efficient rational strain improvement ("metabolic engineering"). It is therefore necessary to accurately determine the concentration of a large number of reactants (i.e., metabolites, nucleotides, cofactors) in order to understand "in vivo" reaction kinetics. Quantification of intracellular concentrations of glycolytic intermediates and nucleotides in Escherichia coli K12 using a perchloric acid extraction and an LC-ESI-MS method was achieved. Intracellular metabolites (e.g., glucose 6-phosphate, fructose 1,6-bisphosphate, 6-phospho gluconate, acetyl-CoA, adenine nucleotides) were quantified under defined (glucose-limited steady-state) growth conditions. The method was verified by comparing the intracellular metabolite concentrations measured via LC-ESI-MS with enzymatic determinations. It is thus possible to identify and quantify more than 15 intracellular metabolites in parallel with a minimal amount of sample volume.

Human chymotrypsinogen B production with Pichia pastoris by integrated development of fermentation and downstream processing. Part 1. Fermentation.

Based on an integrated approach of genetic engineering, fermentation process development, and downstream processing, a fermentative chymotrypsinogen B production process using recombinant Pichia pastoris is presented. Making use of the P. pastoris AOX1-promotor, the demand for methanol as the single carbon source as well as an inducer of protein secretion enforced the use of an optimized feeding strategy by help of on-line analysis and an advanced controller algorithm. By using an experimental system of six parallel sparged column bioreactors, proteolytic product degradation could be minimized while also optimizing starting conditions for the following downstream processing. This optimization of process conditions resulted in the production of authentic chymotrypsinogen at a final concentration level of 480 mg.L(-)(1) in the whole broth and a biomass concentration of 150 g.L(-)(1) cell dry weight, thus comprising a space-time yield of 5.2 mg.L(-)(1).h(-)(1). Alternatively to the high cell density fermentation approach, a continuous fermentation process was developed to study the effects of reduced cell density toward oxygen demand, cooling energy, and biomass separation. This development led to a process with a highly increased space-time yield of 25 mg.L(-)(1).h(-)(1) while reducing the cell dry weight concentration from 150 g.L(-)(1) in fed-batch to 65 g.L(-)(1) in continuous cultivation.

Evaluation of parallel operated small-scale bubble columns for microbial process development using Staphylococcus carnosus.

Shake flasks and pH-controlled small-scale bubble columns were compared with respect to their usefulness as a basic tool for process development for human calcitonin precursor fusion-protein production with Staphylococcus carnosus. Parallel control of the pH (and making use of the base addition data) is necessary to study the effects of medium composition, to identify pH-optima and to develop a medium, which minimizes the acid excretion of S. carnosus. This medium with glycerol as energy source and yeast extract as carbon and nitrogen source resulted in cell dry weight concentration in shake flasks of 5 g l(-1), which were thus improved by a factor of 10. Cell dry weight concentrations of up to 12.5 g l(-1) were measured in the batch process with pH-controlled small-scale bubble columns due to their higher oxygen transfer capability. In contrast to shake flasks it was demonstrated, that the batch process performance of recombinant S. carnosus secreting the human calcitonin precursor fusion-protein was identical within the estimation error in pH-controlled small-scale bubble columns compared to the stirred-tank reactor.

Kinetic resolution of chiral amines with omega-transaminase using an enzyme-membrane reactor.

A kinetic resolution process for the production of chiral amines was developed using an enzyme-membrane reactor (EMR) and a hollow-fiber membrane contactor with (S)-specific omega-transaminases (omega-TA) from Vibrio fluvialis JS17 and Bacillus thuringiensis JS64. The substrate solution containing racemic amine and pyruvate was recirculated through the EMR and inhibitory ketone product was selectively extracted by the membrane contactor until enantiomeric excess of (R)-amine exceeded 95%. Using the reactor set-up with flat membrane reactor (10-mL working volume), kinetic resolutions of alpha-methylbenzylamine (alpha-MBA) and 1-aminotetralin (200 mM, 50 mL) were carried out. During the operation, concentration of ketone product, i.e., acetophenone or alpha-tetralone, in a substrate reservoir was maintained below 0.1 mM, suggesting efficient removal of the inhibitory ketone by the membrane contactor. After 47 and 32.5 h of operation using 5 U/mL of enzyme, 98.0 and 95.5% ee of (R)-alpha-MBA and (R)-1-aminotetralin were obtained at 49.5 and 48.8% of conversion, respectively. A hollow-fiber membrane reactor (39-mL working volume) was used for a preparative-scale kinetic resolution of 1-aminotetralin (200 mM, 1 L). After 133 h of operation, enantiomeric excess reached 95.6% and 14.3 g of (R)-1-aminotetralin was recovered (97.4% of yield). Mathematical modeling of the EMR process including the membrane contactor was performed to evaluate the effect of residence time. The simulation results suggest that residence time should be short to maintain the concentration of the ketone product in EMR sufficiently low so as to decrease conversion per cycle and, in turn, reduce the inhibition of the omega-TA activity.

Purification of polymeric biomaterials.

Employing a combined filtration and precipitation method, the endotoxin concentration in sodium alginate (SA) and sodium cellulose sulfate (SCS) was reduced to a value of 200 EU/g polymer. This is one tenth of the regulatory threshold calculated, for example, for an appropriate bioartificial pancreas that consists of approximately 420,000 encapsulated islets of Langerhans. The low endotoxin (ET) levels were maintained below this threshold during a six-month storage period. The purification procedure of the polymers did not negatively influence the final microcapsule properties. The mechanical stability of microcapsules from purified material is even slightly higher than that of microcapsules from the original polymers. A second approach to avoid endotoxin release from the device is its direct complexation during the bead or capsule formation process. The durability of endotoxin binding in binary, ternary, and quaternary complexes could be demonstrated for storage in culture medium and saline. Very low total endotoxin release from the complexes was detected after three months in culture medium and five months in saline. This complexation is primarily based on electrostatic interactions with the participating cationic components and provides additional security for the final bioartificial organ or delivery device.

Fed-batch production of recombinant human calcitonin precursor fusion protein using Staphylococcus carnosus as an expression-secretion system.

A pH-auxostatic fed-batch process was developed for the secretory production of a fusion protein consisting of the pro-part of Staphylococcus hyicus lipase and two synthetic human calcitonin (hCT) precursor repeats under the control of a xylose-inducible promotor from Staphylococcus xylosus. Using glycerol as the energy source and pH-controlled addition of yeast extract resulted in the production of 2000 mg 1(-1) of the fusion protein (420 mg 1(-1) of the recombinant hCT precursor) within 14 h, reaching 45 g 1(-1) cell dry mass with Staphylococcus carnosus in a stirred-tank reactor. Product titer and space-time yield (30 mg calcitonin precursor 1(-1) h(-1)) were thus improved by a factor of 2, and 4.5, respectively, compared to Escherichia coli expression-secretion systems for the production of calcitonin precursors. Two hundred grams of the fusion protein was secreted by the recombinant S. carnosus on a 150-1 scale (scale-up factor of 50) with a minimum use of technical-grade yeast extract (40 mg fusion protein g(-1) yeast extract).

Continuous enzymatic transformation in an enzyme membrane reactor with simultaneous NAD(H) regeneration. Reprinted from Biotechnology and Bioengineering, Vol. XXIII, No. 12, Pages 2789-2802 (1981).

Multienzyme reaction systems with simultaneous coenzyme regeneration have been investigated in a continuously operated membrane reactor at bench scale. NAD(H) covalently bound to polyethylene glycol with a molecular weight of 10(4) [PEG-10,000-NAD(H)] was used as coenzyme. It could be retained in the membrane reactor together with the enzymes. L-leucine dehydrogenase (LEUDH) was used as catalyst for the reductive amination of alpha-ketoisocaproate (2-oxo-4-methylpentanoic acid) to L-leucine. Formate dehydrogenase (FDH) was used for the regeneration of NADH. Kinetic experiments were carried out to obtain data which could be used in a kinetic model in order to predict the performance of an enzyme membrane reactor for the continuous production of L-leucine. The kinetic constants V(max) and k(m) of the enzymes are all in the same range regardless of whether native NAD(H) or PEG-10,000-NAD(H) is used as coenzyme. L-leucine was produced continuously out of alpha-ketoisocaproate for 48 days; a maximal conversion of 99.7% was reached. The space-time yield was 324 mmol/L day (or 42.5 g/L day).

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.

Polycations as displacer in high-performance bioseparation.

Displacement chromatography is an interesting but up to now rarely used type of preparative biochromatography. The lack of well-engineered and accessible displacer contributes to this phenomenon. In this paper a novel type of displacer is introduced for cation-exchange displacement chromatography, which will soon become commercially available. The molecule is a well-defined PolyDADMAC [poly(diallyldimethylammonium chloride)] with a molar mass of less than 35000 g/mol, an exclusively linear structure and a molar mass polydispersity of less than 1.5. A method for synthesizing such a polymer at high yields is described. The PolyDADMAC is shown to be an efficient displacer of basic proteins from strong cation-exchange columns.