Intra-Abdominal Cooling System Limits Ischemia-Reperfusion Injury During Robot-Assisted Renal Transplantation.

Robot-assisted kidney transplantation is feasible; however, concerns have been raised about possible increases in warm ischemia times. We describe a novel intra-abdominal cooling system to continuously cool the kidney during the procedure. Porcine kidneys were procured by standard open technique. Groups were as follows: Robotic renal transplantation with (n = 11) and without (n = 6) continuous intra-abdominal cooling and conventional open technique with intermittent 4°C saline cooling (n = 6). Renal cortex temperature, magnetic resonance imaging, and histology were analyzed. Robotic renal transplantation required a longer anastomosis time, either with or without the cooling system, compared to the open approach (70.4 ± 17.7 min and 74.0 ± 21.5 min vs. 48.7 ± 11.2 min, p-values < 0.05). The temperature was lower in the robotic group with cooling system compared to the open approach group (6.5 ± 3.1°C vs. 22.5 ± 6.5°C; p = 0.001) or compared to the robotic group without the cooling system (28.7 ± 3.3°C; p < 0.001). Magnetic resonance imaging parenchymal heterogeneities and histologic ischemia-reperfusion lesions were more severe in the robotic group without cooling than in the cooled (open and robotic) groups. Robot-assisted kidney transplantation prolongs the warm ischemia time of the donor kidney. We developed a novel intra-abdominal cooling system that suppresses the noncontrolled rewarming of donor kidneys during the transplant procedure and prevents ischemia-reperfusion injuries.

Parallel substrate supply and pH stabilization for optimal screening of E. coli with the membrane-based fed-batch shake flask.

BACKGROUND: Screening in the fed-batch operation mode is essential for biological cultivations facing challenges as oxygen limitation, osmotic inhibition, catabolite repression, substrate inhibition or overflow metabolism. As a screening tool on shake flask level, the membrane-based fed-batch shake flask was developed. While a controlled supply of a substrate was realized with the in-built membrane tip, the possibilities for replenishing nutrients and stabilizing pH values was not yet exploited. High buffer concentrations were initially used, shifting the medium osmolality out of the biological optimum. As the growth rate is predefined by the glucose release kinetics from the reservoir, the resulting medium acidification can be compensated with a controlled continuous supply of an alkaline compound. The focus of this research is to establish a simultaneous multi-component release of glucose and an alkaline compound from the reservoir to enable cultivations within the optimal physiological range of Escherichia coli. RESULTS: In combination with the Respiratory Activity MOnitoring System, the membrane-based fed-batch shake flask enabled the detection of an ammonium limitation. The multi-component release of ammonium carbonate along with glucose from the reservoir resulted not only in the replenishment of the nitrogen source but also in the stabilization of the pH value in the culture medium. A biomass concentration up to 25 g/L was achieved, which is one of the highest values obtained so far to the best of the author's knowledge with the utilization of a shake flask and a defined synthetic medium. Going a step further, the pH stabilization allowed the decrease of the required buffer amount to one-fourth establishing an optimal osmolality range for cultivation. As optimal physiological conditions were implemented with the multi-component release fed-batch cultivation, the supply of 0.2 g glucose in a 10 mL initial culture medium volume with 50 mM MOPS buffer resulted in a twofold higher biomass concentration than in a comparable batch cultivation. CONCLUSIONS: The newly introduced multi-component release with the membrane-based fed-batch shake flask serves a threefold purpose of replenishing depleted substrates in the culture medium, stabilizing the pH throughout the entire cultivation time and minimizing the necessary amount of buffer to maintain an optimal osmolality range. In comparison to a batch cultivation, these settings enable to achieve higher biomass and product concentrations.

Systematic evaluation of characteristics of the membrane-based fed-batch shake flask.

BACKGROUND: The initial part of process development involves extensive screening programs to identify optimal biological systems and cultivation conditions. For a successful scale-up, the operation mode on screening and production scale must be as close as possible. To enable screening under fed-batch conditions, the membrane-based fed-batch shake flask was developed. It is a shake flask mounted with a central feed reservoir with an integrated rotating membrane tip for a controlled substrate release. Building on the previously provided proof of principle for this tool, this work extends its application by constructive modifications and improved methodology to ensure reproducible performance. RESULTS: The previously limited operation window was expanded by a systematic analysis of reservoir set-up variations for cultivations with the fast-growing organism Escherichia coli. Modifying the initial glucose concentration in the reservoir as well as interchanging the built-in membrane, resulted in glucose release rates and oxygen transfer rate levels during the fed-batch phase varying up to a factor of five. The range of utilizable membranes was extended from dialysis membranes to porous microfiltration membranes with the design of an appropriate membrane tip. The alteration of the membrane area, molecular weight cut-off and liquid volume in the reservoir offered additional parameters to fine-tune the duration of the initial batch phase, the oxygen transfer rate level of the fed-batch phase and the duration of feeding. It was shown that a homogeneous composition of the reservoir without a concentration gradient is ensured up to an initial glucose concentration of 750 g/L. Finally, the experimental validity of fed-batch shake flask cultivations was verified with comparable results obtained in a parallel fed-batch cultivation in a laboratory-scale stirred tank reactor. CONCLUSIONS: The membrane-based fed-batch shake flask is a reliable tool for small-scale screening under fed-batch conditions filling the gap between microtiter plates and scaled-down stirred tank reactors. The implemented reservoir system offers various set-up possibilities, which provide a wide range of process settings for diverse biological systems. As a screening tool, it accurately reflects the cultivation conditions in a fed-batch stirred tank reactor and enables a more efficient bioprocess development.

The consequence of an additional NADH dehydrogenase paralog on the growth of Gluconobacter oxydans DSM3504.

Acetic acid bacteria such as Gluconobacter oxydans are used in several biotechnological processes due to their ability to perform rapid incomplete regio- and stereo-selective oxidations of a great variety of carbohydrates, alcohols, and related compounds by their membrane-bound dehydrogenases. In order to understand the growth physiology of industrial strains such as G. oxydans ATCC 621H that has high substrate oxidation rates but poor growth yields, we compared its genome sequence to the genome sequence of strain DSM 3504 that reaches an almost three times higher optical density. Although the genome sequences are very similar, DSM 3504 has additional copies of genes that are absent from ATCC 621H. Most importantly, strain DSM 3504 contains an additional type II NADH dehydrogenase (ndh) gene and an additional triosephosphate isomerase (tpi) gene. We deleted these additional paralogs from DSM 3504, overexpressed NADH dehydrogenase in ATCC 621H, and monitored biomass and the concentration of the representative cell components as well as O2 and CO2 transfer rates in growth experiments on mannitol. The data revealed a clear competition of membrane-bound dehydrogenases and NADH dehydrogenase for channeling electrons in the electron transport chain of Gluconobacter and an important role of the additional NADH dehydrogenase for increased growth yields. The less active the NADH dehydrogenase is, the more active is the membrane-bound polyol dehydrogenase. These results were confirmed by introducing additional ndh genes via plasmid pAJ78 in strain ATCC 621H, which leads to a marked increase of the growth rate.

Quantifying the sensitivity of G. oxydans ATCC 621H and DSM 3504 to osmotic stress triggered by soluble buffers.

In Gluconobacter oxydans cultivations on glucose, CaCO3 is typically used as pH-buffer. This buffer, however, has disadvantages: suspended CaCO3 particles make the medium turbid, thereby, obstructing analysis of microbial growth via optical density and scattered light. Upon searching for alternative soluble pH-buffers, bacterial growth and productivity was inhibited most probably due to osmotic stress. Thus, this study investigates in detail the osmotic sensitivity of G. oxydans ATCC 621H and DSM 3504 using the Respiratory Activity MOnitoring System. The tested soluble pH-buffers and other salts attained osmolalities of 0.32-1.19 osmol kg(-1). This study shows that G. oxydans ATCC 621H and DSM 3504 respond quite sensitively to increased osmolality in comparison to other microbial strains of industrial interest. Osmolality values of >0.5 osmol kg(-1) should not be exceeded to avoid inhibition of growth and product formation. This osmolality threshold needs to be considered when working with soluble pH-buffers.

[Robotic-assisted organ transplantation]. / Transplantation d'organes avec assistance robotique.

Advanced surgical procedures have traditionally been a domain of open surgery. However, minimally invasive approaches are evolving with the development of robotic technology which appears capable to overcome technical limitations of conventional laparoscopy. While traditionally perceived as impossible indications for minimally invasive surgery, reports on robotic organ transplantations have surfaced with promising results.

Oxygen supply controls the onset of pristinamycins production by Streptomyces pristinaespiralis in shaking flasks.

Antibiotics are secondary metabolites, generally produced during stationary phase of growth under different nutritional and hydrodynamic stresses. However, the exact mechanisms of the induction of antibiotics production are still not clearly established. In a previous study, the induction of pristinamycins production by Streptomyces pristinaespiralis as well as product concentrations were correlated with power dissipation per unit of volume (P/V) in shaking flasks. In this study, detailed kinetics of growth, substrate consumption, oxygen transfer rate and pristinamycins production under varying P/V conditions have been obtained and analyzed. Our results showed that higher P/V resulted in a higher concentration of biomass and promoted an earlier nutrient limitation and ultimately an earlier induction of pristinamycins production. The maximal specific growth rate, specific oxygen consumption rate and specific consumption rate of glutamate increased with P/V while influence was less marked with specific consumption rate of glucose, arginine, ammonium ions and phosphate. When oxygen uptake rate (OUR) was limited by free-surface oxygen transfer, pristinamycins production was not detected despite the occurrence of nitrogen and/or phosphate sources limitation. The threshold value for OUR observed was around 25 mmol L(-1) h(-1). This suggested that a limitation in nitrogen and/or phosphate alone was not sufficient to induce pristinamycins production by S. pristinaespiralis pr11. To induce this production, the oxygen transfer had to be non-limiting.

[The viability of kidneys tested by gadolinium-perfusion MRI during ex vivo perfusion]. / La viabilité des reins marginaux testée par perfusion de gadolinium sous IRM pendant leur réanimation.

INTRODUCTION: Marginal kidneys must be reanimated before their transplantation. Reanimation is conducted with hypothermic pulsatile perfusion. The tests used generally to demonstrate the viability is the vascular resistance which is not convenient for everybody. We have developed a magnetic resonance compatible perfusional technology allowing us to test the organs during the perfusion by Gd-perfusion MRI. METHODS AND RESULTS: We have used pigs' kidneys with no warm ischemic time to establish the basis in a normal kidney. After an eight-hour hypothermic pulsatile perfusion, kidneys are submitted to a Gd perfusion. First, we measure the anatomy of the vessels, then the distribution of Gd in the kidney. We obtain simultaneously a dynamic study of the organs where T0 represents the Gd bolus arrival in the cortex and TP the maximum saturation time of Gd. CONCLUSION: We have observed that a normal T0 is inferior to 30s and TP is inferior to one minute. We have compared these values with ATP resynthesis in these organs and found that they correlate. We hope for the future through that predictive use of Gd-MRI to avoid the clinical use of "too" marginal kidneys or the discard of good kidneys but not corresponding with the vascular resistance theory.

Polymer-based controlled-release fed-batch microtiter plate - diminishing the gap between early process development and production conditions.

BACKGROUND: Fed-batch conditions are advantageous for industrial cultivations as they avoid unfavorable phenomena appearing in batch cultivations. Those are for example the formation of overflow metabolites, catabolite repression, oxygen limitation or inhibition due to elevated osmotic concentrations. For both, the early bioprocess development and the optimization of existing bioprocesses, small-scale reaction vessels are applied to ensure high throughput, low costs and prompt results. However, most conventional small-scale procedures work in batch operation mode, which stands in contrast to fed-batch conditions in large-scale bioprocesses. Extensive expenditure for installations and operation accompany almost all cultivation systems in the market allowing fed-batch conditions in small-scale. An alternative, more cost efficient enzymatic glucose release system is strongly influenced by environmental conditions. To overcome these issues, this study investigates a polymer-based fed-batch system for controlled substrate release in microtiter plates. RESULTS: Immobilizing a solid silicone matrix with embedded glucose crystals at the bottom of each well of a microtiter plate is a suitable technique for implementing fed-batch conditions in microtiter plates. The results showed that the glucose release rate depends on the osmotic concentration, the pH and the temperature of the medium. Moreover, the applied nitrogen source proved to influence the glucose release rate. A new developed mathematical tool predicts the glucose release for various media conditions. The two model organisms E. coli and H. polymorpha were cultivated in the fed-batch microtiter plate to investigate the general applicability for microbial systems. Online monitoring of the oxygen transfer rate and offline analysis of substrate, product, biomass and pH confirmed that fed-batch conditions are comparable to large-scale cultivations. Furthermore, due to fed-batch conditions in microtiter plates, product formation could be enhanced by the factor 245 compared to batch cultivations. CONCLUSIONS: The polymer-based fed-batch microtiter plate represents a sophisticated and cost efficient system to mimic typical industrial fed-batch conditions in small-scale. Thus, a more reliable strain screening and early process development can be performed. A systematical scale-down with low expenditure of work, time and money is possible.

Polymer-based ammonium-limited fed-batch cultivation in shake flasks improves lipid productivity of the microalga Chlorella vulgaris.

The aim of this work was to study ammonium-limited fed-batch conditions in heterotrophic C. vulgaris shake flask cultivations. Therefore, an innovative polymer-based ammonium release technique (polymer beads) was developed. Using these beads in shake flasks, C. vulgaris cultivations resulted in simultaneous growth and lipid accumulation. Lipid productivity was increased by 43% compared to batch cultivations. Furthermore, by online monitoring of the metabolic activity (RAMOS technique), unlimited growth and depletion of nutrients could be identified. A previously unknown sulfur limitation was detected in the applied Bold's Basal Medium. Combining the ammonium release beads with the RAMOS technique proved to be an efficient method for microalgae process development.

Respiration activity monitoring system (RAMOS), an efficient tool to study the influence of the oxygen transfer rate on the synthesis of lipopeptide by Bacillus subtilis ATCC6633.

The effect of oxygen transfer rate (OTR) on the synthesis of mycosubtilin, a non ribosomal lipopeptide antifungal biosurfactant, was investigated in the respiration activity monitoring system (RAMOS) for two Bacillus subtilis strains. These cultures were performed under definite oxygen-limited conditions without the adding of any anti-foam in the culture medium. By using four different filling volumes (FV) in the shaken bioreactors, different levels (20, 14, 9 and 7 mmol O(2)l(-1)h(-1)) of oxygen-limited growth could be obtained. A 25-fold increase of the specific productivity of mycosubtilin was observed for B. subtilis ATCC6633 in the case of the most severe oxygen limitation. But nearly no effect could be found with strain BBG100 carrying the constitutive P(repU) promoter instead of the natural P(myc) promoter. Transcript analysis of the fenF gene belonging to the myc operon indicated that the P(myc) promoter regulation could be slightly oxygen sensitive. Additionally, different patterns of the synthetised mycosubtilin homologues were obtained for different level of oxygen-limited growths. At the present state of investigation, oxygen regulation was thus shown to act at different levels suggesting the existence of a complex regulatory system of NRPS lipopeptide synthesis in the natural B. subtilis ATCC6633 strain.

Feasibility of gas/solid carboligation: conversion of benzaldehyde to benzoin using thiamine diphosphate-dependent enzymes.

A carboligation was investigated for the first time as an enzymatic gas phase reaction, where benzaldehyde was converted to benzoin using thiamine diphosphate (ThDP)-dependent enzymes, namely benzaldehyde lyase (BAL) and benzoylformate decarboxylase (BFD). The biocatalyst was immobilized per deposition on non-porous support. Some limitations of the gas/solid biocatalysis are discussed based on this carboligation and it is also demonstrated that the solid/gas system is an interesting tool for more volatile products.

A simple and inexpensive method for investigating microbiological, enzymatic, or inorganic catalysis using standard histology and microbiology laboratory equipment: assembly, mass transfer properties, hydrodynamic conditions and evaluation.

We introduce a generic, simple, and inexpensive method for performing microbiological, enzymatic, or inorganic catalysis with solids using standard histology and microbiology laboratory equipment. Histology cassettes were used to standardize hydrodynamic conditions and to protect the catalysts and their solid supports. Histology cassettes have the following advantages: they are readily available, inexpensive, solvent and acid resistant, automatable, and the slots in the cassette walls allow liquid to circulate freely. Standard Erlenmeyer flasks were used as reaction vessels. We developed a new camera to observe the movement and position of the histology cassettes as well as the liquid in the Erlenmeyer flasks. The camera produces a stable image of the rotating liquid in the Erlenmeyer flask. This visualization method revealed that in a 250 ml Erlenmeyer flask, stable operating conditions are achieved at a shaking frequency of 300 rpm and a fill volume of 30 ml. In vessels with vertical walls, such as beakers or laboratory bottles, the movement of the histology cassette is not reproducible. Mass transfer characterization using a biological model system and the chemical sulfite-oxidation method revealed that the histology cassette does not influence gas-liquid mass transfer.

Parallel use of shake flask and microtiter plate online measuring devices (RAMOS and BioLector) reduces the number of experiments in laboratory-scale stirred tank bioreactors.

BACKGROUND: Conventional experiments in small scale are often performed in a 'Black Box' fashion, analyzing only the product concentration in the final sample. Online monitoring of relevant process characteristics and parameters such as substrate limitation, product inhibition and oxygen supply is lacking. Therefore, fully equipped laboratory-scale stirred tank bioreactors are hitherto required for detailed studies of new microbial systems. However, they are too spacious, laborious and expensive to be operated in larger number in parallel. Thus, the aim of this study is to present a new experimental approach to obtain dense quantitative process information by parallel use of two small-scale culture systems with online monitoring capabilities: Respiration Activity MOnitoring System (RAMOS) and the BioLector device. RESULTS: The same 'mastermix' (medium plus microorganisms) was distributed to the different small-scale culture systems: 1) RAMOS device; 2) 48-well microtiter plate for BioLector device; and 3) separate shake flasks or microtiter plates for offline sampling. By adjusting the same maximum oxygen transfer capacity (OTRmax), the results from the RAMOS and BioLector online monitoring systems supplemented each other very well for all studied microbial systems (E. coli, G. oxydans, K. lactis) and culture conditions (oxygen limitation, diauxic growth, auto-induction, buffer effects). CONCLUSIONS: The parallel use of RAMOS and BioLector devices is a suitable and fast approach to gain comprehensive quantitative data about growth and production behavior of the evaluated microorganisms. These acquired data largely reduce the necessary number of experiments in laboratory-scale stirred tank bioreactors for basic process development. Thus, much more quantitative information is obtained in parallel in shorter time.

Development of an ABO-ELISA for the quantitation of human blood group anti-A and anti-B IgM and IgG antibodies.

An indirect ELISA system was designed for quantitation of human blood group A and B IgM and IgG antibodies. The capturing antigens are blood group substance A or B used to sensitize polystyrol microtiter plates. Bound anti-A or anti-B antibodies are revealed either directly, by development with polyclonal anti-human immunoglobulin class-specific conjugate or with more avid mouse monoclonal anti-human isotype antibodies revealed in turn by goat anti-mouse conjugate. Reproducibly, 100 ng specific anti-A IgG provided for a significant above-background signal of 0.2 at OD405 and 15 serum samples had a mean content of 3.98 +/- 8.74 micrograms (mean +/- 2 SD) (range: 0.305-12.62) of specific anti-A IgG/g total IgG. Thus one molecule specific anti-A IgG is found per 7.9 X 10(4)-3.2 X 10(6) total IgG molecules. Statistical correlations were significant between anti-A IgG levels and agglutination titer (P less than 0.05) but non-significant when the specific anti-A IgG levels of individual serum samples were compared to their total IgG content (P greater than 0.05). Dose-response signals were similar for anti-A and anti-B IgM antibodies. Reproducibility of the assay was excellent. Specificity was ascertained by various approaches involving development of primary antibodies with heterospecific antibody conjugate and adsorption of primary antibody from serum using A and B group erythrocytes or soluble A and B substances. Separation of IgM from IgG anti-A antibodies over sizing gel resulted in fractions that were immunosorbed by mouse monoclonal anti-human IgM and IgG respectively but not vice versa.

Introduction to advantages and problems of shaken cultures.

Shaking bioreactors are the most frequently used reaction vessels in biotechnology and have been so for many decades. In spite of their large practical importance, very little is known about the characteristic properties of shaken cultures from an engineering point of view. The few publications available contain to some extent contradicting statements and conflicting advice concerning the correct operating conditions of shaking bioreactors. Depending on the investigated microbial system, the engineering parameters may more or less significantly influence the experimental results in a quantitative as well as in a qualitative manner. Unfortunately, these kind of interactions are often overlooked or ignored by scientists. Precise knowledge about the controlling hydrodynamic phenomena in shaking bioreactors and quantitative information about the physical parameters influencing the cultures are needed to assure reproducible and meaningful operating conditions. In this introduction, the state of the art of culturing microorganisms in shaking bioreactors is reviewed and some issues of their practical application in screening and process development projects are addressed.

Characterisation of the gas-liquid mass transfer in shaking bioreactors.

The maximum gas-liquid mass transfer capacity of 250ml shaking flasks on orbital shaking machines has been experimentally investigated using the sulphite oxidation method under variation of the shaking frequency, shaking diameter, filling volume and viscosity of the medium. The distribution of the liquid within the flask has been modelled by the intersection between the rotational hyperboloid of the liquid and the inner wall of the shaking flask. This model allows for the calculation of the specific exchange area (a), the mass transfer coefficient (k(L)) and the maximum oxygen transfer capacity (OTR(max)) for given operating conditions and requires no fitting parameters. The model agrees well with the experimental results. It was furthermore shown that the liquid film on the flask wall contributes significantly to the specific mass transfer area (a) and to the oxygen transfer rate (OTR).

Device for sterile online measurement of the oxygen transfer rate in shaking flasks.

The oxygen transfer rate (OTR) is the most suitable measurable parameter to quantify the physiological state of a culture of aerobic microorganisms since most metabolic activities depend on oxygen consumption. Online measurement of the oxygen transfer rate in stirred bioreactors is state of the art although technically difficult. However, the online determination of the oxygen transfer rate in shaking bioreactors under sterile conditions has not been possible until recently. A newly developed measuring device eliminates this deficit. Extremely useful information about cultivating conditions and the physiological state of microorganisms can be gained in early stages of research and bioprocess development from many reactors operated in parallel.

Out-of-phase operating conditions, a hitherto unknown phenomenon in shaking bioreactors.

One of the important parameters in characterising fermentations of aerobic microorganisms is the specific power consumption. A new method has been introduced which enables the accurate determination of the power consumption in shaking bioreactors. It is based on torque measurements in the drive and the appropriate compensation of the friction losses. Measurements of the power consumption revealed the phenomenon of the liquid being 'out-of-phase' for the first time for shaking bioreactors. This occurs at certain operating conditions and is characterised by an increasing amount of liquid not following the rotating movement of the shaker table, thus reducing the specific power consumption, mixing and the gas/liquid mass transfer. With respect to this, different hydrodynamic cases have to be distinguished. All these cases have in common, however, that the probability of 'out-of-phase' conditions increases with lower shaking diameters, lower filling volumes, larger number and sizes of baffles and higher viscosity. For unbaffled flasks with a nominal volume

Mass transfer resistance of sterile plugs in shaking bioreactors.

One of the mass transfer resistances for the gas exchange of shaking flasks is the sterile plug. The gas exchange through the sterile plug is described by an extended model of Henzler and Schedel [Bioprocess Eng. 7 (1991) 123]. Based on this model, a new method was developed to obtain the mass transfer resistance of various sterile closures. It consists of measuring the water evaporation rate of the shaking flask and is therefore very easily applied. Sterile plugs made of cotton, wrapped paper, urethane foam and fibreglass and caps made out of aluminium and silicone have been examined. Instead of the oxygen transfer coefficient (k(O(2))), which is commonly found in the literature, the carbon dioxide diffusion coefficient (D(CO(2))) is used to describe the mass transfer resistance of the sterile plug. The investigation revealed that this resistance is mainly dependent on the neck geometry and to a lesser extent on the plug material and density. The gas exchange of aluminium-caps was not reproducible.