In Vitro Simulated Neuronal Environmental Conditions Qualify Umbilical Cord Derived Highly Potent Stem Cells for Neuronal Differentiation.

The healing of neuronal injuries is still an unachieved goal. Medicine-based therapies can only extend the survival of patients, but not finally lead to a healing process. Currently, a variety of stem cell-based tissue engineering developments are the subject of many research projects to bridge this gap. As yet, neuronal differentiation of induced pluripotent stem cells (iPS), embryonic cell lines, or neuronal stem cells could be accomplished and produce functional neuronally differentiated cells. However, clinical application of cells from these sources is hampered by ethical considerations. To overcome these hurdles numerous studies investigated the potential of adult mesenchymal stem cells (MSCs) as a potential stem cell source. Adult MSCs have been approved as cellular therapeutical products due to their regenerative potential and immunomodulatory properties. Only a few of these studies could demonstrate the capacity to differentiate MSCs into active firing neuron like cells. With this study we investigated the potential of Wharton's Jelly (WJ) derived stem cells and focused on the intrinsic pluripotent stem cell pool and their potential to differentiate into active neurons. With a comprehensive neuronal differentiation protocol comprised of mechanical and biochemical inductive cues, we investigated the capacity of spontaneously forming stem cell spheroids (SCS) from cultured WJ stromal cells in regard to their neuronal differentiation potential and compared them to undifferentiated spheroids or adherent MSCs. Spontaneously formed SCSs show pluripotent and neuroectodermal lineage markers, meeting the pre-condition for neuronal differentiation and contain a higher amount of cells which can be differentiated into cells whose functional phenotypes in calcium and voltage responsive electrical activity are similar to neurons. In conclusion we show that up-concentration of stem cells from WJ with pluripotent characteristics is a tool to generate neuronal cell replacement.

Optimized polymer-based glucose release in microtiter plates for small-scale E. coli fed-batch cultivations.

BACKGROUND: Small-scale cultivation vessels, which allow fed-batch operation mode, become more and more important for fast and reliable early process development. Recently, the polymer-based feeding system was introduced to allow fed-batch conditions in microtiter plates. Maximum glucose release rates of 0.35 mg/h per well (48-well-plate) at 37 °C can be achieved with these plates, depending on the media properties. The fed-batch cultivation of fluorescent protein-expressing E. coli at oxygen transfer rate levels of 5 mmol/L/h proved to be superior compared to simple batch cultivations. However, literature suggests that higher glucose release rates than achieved with the currently available fed-batch microtiter plate are beneficial, especially for fast-growing microorganisms. During the fed-batch phase of the cultivation, a resulting oxygen transfer rate level of 28 mmol/L/h should be achieved. RESULTS: Customization of the polymer matrix enabled a considerable increase in the glucose release rate of more than 250% to up to 0.90 mg/h per well. Therefore, the molecular weight of the prepolymer and the addition of a hydrophilic PDMS-PEG copolymer allowed for the individual adjustment of a targeted glucose release rate. The newly developed polymer matrix was additionally invariant to medium properties like the osmotic concentration or the pH-value. The glucose release rate of the optimized matrix was constant in various synthetic and complex media. Fed-batch cultivations of E. coli in microtiter plates with the optimized matrix revealed elevated oxygen transfer rates during the fed-batch phase of approximately 28 mmol/L/h. However, these increased glucose release rates resulted in a prolonged initial batch phase and oxygen limitations. The newly developed polymer-based feeding system provides options to manufacture individual feed rates in a range from 0.24-0.90 mg/h per well. CONCLUSIONS: The optimized polymer-based fed-batch microtiter plate allows higher reproducibility of fed-batch experiments since cultivation media properties have almost no influence on the release rate. The adjustment of individual feeding rates in a wide range supports the early process development for slow, average and fast-growing microorganisms in microtiter plates. The study underlines the importance of a detailed understanding of the metabolic behavior (through online monitoring techniques) to identify optimal feed rates.

Precultures Grown under Fed-Batch Conditions Increase the Reliability and Reproducibility of High-Throughput Screening Results.

One essential task in bioprocess development is strain selection. A common screening procedure consists of three steps: first, the picking of colonies; second, the execution of a batch preculture and main culture, e.g., in microtiter plates (MTPs); and third, the evaluation of product formation. Especially during the picking step, unintended variations occur due to undefined amounts and varying viability of transferred cells. The aim of this study is to demonstrate that the application of polymer-based controlled-release fed-batch MTPs during preculture eliminates these variations. The concept of equalizing growth through fed-batch conditions during preculture is theoretically discussed and then tested in a model system, namely, a cellulase-producing Escherichia coli clone bank containing 32 strains. Preculture is conducted once in the batch mode and once in the fed-batch mode. By applying the fed-batch mode, equalized growth is observed in the subsequent main culture. Furthermore, the standard deviation of cellulase activity is reduced compared to that observed in the conventional approach. Compared with the strains in the batch preculture process, the first-ranked strain in the fed-batch preculture process is the superior cellulase producer. These findings recommend the application of the fed-batch MTPs during preculture in high-throughput screening processes to achieve accurate and reliable results.

Zwitterionic Functionalizable Scaffolds with Gyroid Pore Architecture for Tissue Engineering.

Stereolithography-assisted fabrication of hydrogels of carboxybetaine methacrylamide (CBMAA) and a α,ω-methacrylate poly(d,l-lactide-block-ethylene glycol-block- d,l-lactide) (MA-PDLLA-PEG-PDLLA-MA) telechelic triblock macromer is presented. This technique allows printing complex structures with gyroid interconnected porosity possessing extremely high specific area. Hydrogels are characterized by infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), and laser scanning confocal microscopy (LSCM). The copolymerization with zwitterionic comonomer leads hydrogels with high equilibrium water content (EWC), up to 700% while maintaining mechanical robustness. The introduction of carboxybetaine yields excellent resistance to nonspecific protein adsorption while providing a facile way for specific biofunctionalization with a model protein, fluorescein isothiocyanate labeled bovine serum albumin (BSA). The homogeneous protein immobilization across the hydrogel pores prove the accessibility to the innermost pore volumes. The remarkably low protein adsorption combined with the interconnected nature of the porosity allowing fast diffusion of nutrient and waste product and the mimicry of bone trabecular, makes the hydrogels presented here highly attractive for tissue engineering.

Effect of shear stress on the reduction of bacterial adhesion to antifouling polymers.

In this work, two antifouling polymer brushes were tested at different shear stress conditions to evaluate their performance in reducing the initial adhesion of Escherichia coli. Assays were performed using a parallel plate flow chamber and a shear stress range between 0.005 and 0.056 Pa. These shear stress values are found in different locations in the human body where biomedical devices are placed. The poly(MeOEGMA) and poly(HPMA) brushes were characterized and it was shown that they can reduce initial adhesion up to 90% when compared to glass. Importantly, the performance of these surfaces was not affected by the shear stress, which is an indication that they do not collapse under this shear stress range. The brushes displayed a similar behavior despite the differences in their chemical composition and surface energy. Both surfaces have shown ultra-low adsorption of macromolecules from the medium when tested with relevant biological fluids (urine and serum). This indicates that these surfaces can potentially be used in biomedical devices to reduce initial bacterial colonization and eventually reduce biofilm formation on these devices.

Intraocular silicone implant to treat chronic ocular hypotony-preliminary feasibility data.

PURPOSE: Ocular hypotony secondary to proliferative vitreoretinopathy-related retinal detachment, trauma or inflammation is difficult to treat. Besides endotamponades such as silicone oil, vitreous implants such as iris diaphragms or balloons have been developed to stabilize the eye and to prevent phthisis of the globe. Vitreous implants tested thus far exhibit a seam at the attachment site of the hemispheres, or micropores. This manuscript reports the development of a seamless silicone balloon implant without micropores, which can be filled with silicone oil and surface-modified to improve its biocompatibility. Developed for intraocular placement in the management of chronic hypotony and phthisis prevention, it may also be suitable for tamponading retinal detachments. METHODS: Silicone was used as the basic structure for the fabrication of a seamless balloon-shaped intraocular implant, which was coated by employing a six-arm star-shaped (sP) macromer of a copolymer of 80 % ethylene oxide (EO) and 20 % propylene oxide (PO) with conjugated functional terminal isocyanate groups, NCO-sP(EO-stat-PO), with and without heparin. Three variants of implants, which differ in their surfaces, were manufactured: uncoated silicone, NCO-sP (EO-stat-PO) coated silicone and heparin-NCO-sP (EO-stat-PO) coated silicone implants. To exert a tamponade effect, the implant was filled with silicone oil and its properties were studied. RESULTS: Seamless thin balloon implants made of silicone, which are considered biocompatible and intrinsically resistant to biological attacks in vivo, could be fabricated in different sizes. The silicone oil-filled implant can mimic the mechanism of buoyant force and high surface tension of silicone oil, which is the only long-term vitreous substitute currently available. The silicone oil-filled implant can also mimic the natural vitreous body by occupying the entire posterior segment. CONCLUSIONS: The intraocular silicone implant as an alternative long-term treatment of chronic ocular hypotony might offer a new option for clinical ophthalmological practice. In vivo studies need to be performed to collect more data on the implant's long-term mechanical and optical properties, as well as long-term biocompatibility.

Hydrogel coated and dexamethasone releasing cochlear implants: quantification of fibrosis in guinea pigs and evaluation of insertion forces in a human cochlea model.

The insertion of cochlear implants (CIs) often causes fibrous tissue growth around the electrode, which leads to attenuation of function of CIs. Inhibition of fibrosis in vivo using dexamethasone (Dex) released from the implant base material (polydimethylsiloxane [PDMS]) coated with a protein repelling hydrogel (star-shaped polyethylene glycol prepolymer, sPEG) was, therefore, the aim of the study. PDMS filaments with Dex or sPEG were implanted into guinea pigs. The hearing status after implantation did not differ significantly in the treated groups. Using confocal laser scanning microscopy in transparent whole mount preparations, Dex, Dex/sPEG, as well as sPEG showed a tendency toward reduced formation of connective tissue around the implant. To apply such coatings for glass fibers for optical stimulation of the inner ear, insertion forces were measured into a human scala tympani model using fibers with sPEG coating. The results show that the hydrogel did not reduce insertion forces compared to the uncoated samples. However, PDMS-embedded fibers provide comparable insertion forces and depth to those measured with conventional CI electrodes, demonstrating the suitability of laser fibers for a minimal traumatic cochlear implantation.

Dexamethasone released from cochlear implant coatings combined with a protein repellent hydrogel layer inhibits fibroblast proliferation.

The insertion of cochlear implants into the inner ear often causes inflammation and fibrosis inside the scala tympani and thus growth of fibrous tissue on the implant surface. This deposition leads to the loss of function in both electrical and laser-based implants. The design of this study was to realize fibroblast growth inhibition by dexamethasone (Dex) released from the base material of the implant [polydimethylsiloxane (PDMS)]. To prevent cell and protein adhesion, the PDMS was coated with a hydrogel layer [star-shaped polyethylene glycol prepolymer (sPEG)]. Drug release rates were studied over 3 months, and surface characterization was performed. It was observed that the hydrogel slightly smoothened the surface roughened by the Dex crystals. The hydrogel coating reduced and prolonged the release of the drug over several months. Unmodified, sPEG-coated, Dex-loaded, and Dex/sPEG-equipped PDMS filaments were cocultivated in vitro with fluorescent fibroblasts, analyzed by fluorescent microscopy, and quantified by cell counting. Compared to the unmodified PDMS, cell growth on all modified filaments was averagely 95% ±standard deviation (SD) less, while cell growth on the bottom of the culture dishes containing Dex-loaded filaments was reduced by 70% ±SD. Both, Dex and sPEG prevented direct cell growth on the filament surfaces, while drug delivery was maintained for the duration of several months.

Controlling pH in shake flasks using polymer-based controlled-release discs with pre-determined release kinetics.

BACKGROUND: There are significant differences in the culture conditions between small-scale screenings and large-scale fermentation processes. Production processes are usually conducted in fed-batch cultivation mode with active pH-monitoring and control. In contrast, screening experiments in shake flasks are usually conducted in batch mode without active pH-control, but with high buffer concentrations to prevent excessive pH-drifts. These differences make it difficult to compare results from screening experiments and laboratory and technical scale cultivations and, thus, complicate rational process development. In particular, the pH-value plays an important role in fermentation processes due to the narrow physiological or optimal pH-range of microorganisms. To reduce the differences between the scales and to establish a pH-control in shake flasks, a newly developed easy to use polymer-based controlled-release system is presented in this paper. This system consists of bio-compatible silicone discs embedding the alkaline reagent Na2CO3. Since the sodium carbonate is gradually released from the discs in pre-determined kinetics, it will ultimately compensate the decrease in pH caused by the biological activity of microorganisms. RESULTS: The controlled-release discs presented here were successfully used to cultivate E. coli K12 and E. coli BL21 pRSET eYFP-IL6 in mineral media with glucose and glycerol as carbon (C) sources, respectively. With glucose as the C-source it was possible to reduce the required buffer concentration in shake flask cultures by 50%. Moreover, with glycerol as the C-source, no buffer was needed at all. CONCLUSIONS: These novel polymer-based controlled-release discs allowed buffer concentrations in shake flask media to be substantially reduced or omitted, while the pH remains in the physiological range of the microorganisms during the whole cultivation time. Therefore, the controlled-release discs allow a better control of the pH, than merely using high buffer concentrations. The conditions applied here, i.e. with significantly reduced buffer concentrations, enhance the comparability of the culture conditions used in screening experiments and large-scale fermentation processes.

High-throughput screening of Hansenula polymorpha clones in the batch compared with the controlled-release fed-batch mode on a small scale.

Most large-scale production processes in biotechnology are performed in fed-batch operational mode. In contrast, the screenings for microbial production strains are run in batch mode, which results in the microorganisms being subjected to different physiological conditions. This significantly affects strain selection. To demonstrate differences in ranking during strain selection depending on the operational mode, screenings were performed in batch and fed-batch modes. Two model populations of the methylotrophic yeast Hansenula polymorpha RB11 with vector pC10-FMD (P(FMD)-GFP) (220 clones) and vector pC10-MOX (P(MOX)-GFP) (224 clones) were applied. For fed-batch cultivations in deep-well microtiter plates, a controlled-release system made of silicone elastomer discs containing glucose was used. Three experimental set-ups were investigated: batch cultivation with (1) glucose as a substrate, which catabolite represses product formation, and (2) glycerol as a carbon source, which is partially repressing, respectively, and (3) fed-batch cultivation with glucose as a limiting substrate using the controlled-release system. These three experimental set-ups showed significant variations in green fluorescent protein (GFP) yield. Interestingly, screenings in fed-batch mode with glucose as a substrate resulted in the selection of yeast strains different from those cultivated in batch mode with glycerol or glucose. Ultimately, fed-batch screening is considerably better than screening in batch mode for fed-batch production processes with glucose as a carbon source.

Equalizing growth in high-throughput small scale cultivations via precultures operated in fed-batch mode.

An often underestimated problem when working with different clones in microtiter plates and shake flask screenings is the non-parallel and non-equal growth of batch cultures. These growth differences are caused by variances of individual clones regarding initial biomass concentration, lag-phase or specific growth rate. Problems arising from unequal growth kinetics are different induction points in expression studies or uneven cultivation periods at the time of harvest. Screening for the best producing clones of a library under comparable conditions is thus often impractical or even impossible. A new approach to circumvent the problem of unequal growth kinetics of main cultures is the application of fed-batch mode in precultures in microtiter plates and shake flasks. Fed-batch operation in precultures is realized through a slow-release system for glucose. After differently growing cultures turn to glucose-limited growth, they all consume the same amount of glucose due to the fixed feed profile of glucose provided by the slow-release system. This leads to equalized growth. Inherent advantages of this method are that it is easy to use and requires no additional equipment like pumps. This new technique for growth equalization in high-throughput cultivations is simulated and verified experimentally. The growth of distinctly inoculated precultures in microtiter plates and shake flasks could be equalized for different microorganisms such as Escherichia coli and Hansenula polymorpha.

Process development in Hansenula polymorpha and Arxula adeninivorans, a re-assessment.

A range of industrial H. polymorpha-based processes exist, most of them for the production of pharmaceuticals. The established industrial processes lean on the use of promoters derived from MOX and FMD, genes of the methanol metabolism pathway. In Hansenula polymorpha these promoters are de-repressed upon depletion of a range of carbon sources like glucose and glycerol instead of being induced by methanol as reported for other methylotrophs. Due to these characteristics screening and fermentation modes have been defined for strains harbouring such expression control elements that lean on a limited supplementation of glycerol or glucose to a culture medium. For fermentation of H. polymorpha a synthetic minimal medium (SYN6) has been developed. No industrial processes have been developed so far based on Arxula adeninivorans and only a limited range of strong promoter elements exists, suitable for heterologous gene expression. SYN6 originally designed for H. polymorpha provided a suitable basis for the initial definition of fermentation conditions for this dimorphic yeast. Characteristics like osmo- and thermotolerance can be addressed for the definition of culture conditions.