Alginate-encapsulated brain-derived neurotrophic factor-overexpressing mesenchymal stem cells are a promising drug delivery system for protection of auditory neurons.

The cochlear implant outcome is possibly improved by brain-derived neurotrophic factor treatment protecting spiral ganglion neurons. Implantation of genetically modified mesenchymal stem cells may enable the required long-term brain-derived neurotrophic factor administration. Encapsulation of mesenchymal stem cells in ultra-high viscous alginate may protect the mesenchymal stem cells from the recipient's immune system and prevent their uncontrolled migration. Alginate stability and survival of mesenchymal stem cells in alginate were evaluated. Brain-derived neurotrophic factor production was measured and its protective effect was analyzed in dissociated rat spiral ganglion neuron co-culture. Since the cochlear implant is an active electrode, alginate-mesenchymal stem cell samples were electrically stimulated and alginate stability and mesenchymal stem cell survival were investigated. Stability of ultra-high viscous-alginate and alginate-mesenchymal stem cells was proven. Brain-derived neurotrophic factor production was detectable and spiral ganglion neuron survival, bipolar morphology, and neurite outgrowth were increased. Moderate electrical stimulation did not affect the mesenchymal stem cell survival and their viability was good within the investigated time frame. Local drug delivery by ultra-high viscous-alginate-encapsulated brain-derived neurotrophic factor-overexpressing mesenchymal stem cells is a promising strategy to improve the cochlear implant outcome.

Distributed automated manufacturing of pluripotent stem cell products.

Establishing how to effectively manufacture cell therapies is an industry-level problem. Decentralised manufacturing is of increasing importance, and its challenges are recognised by healthcare regulators with deviations and comparability issues receiving specific attention from them. This paper is the first to report the deviations and other risks encountered when implementing the expansion of human pluripotent stem cells (hPSCs) in an automated three international site-decentralised manufacturing setting. An experimental demonstrator project expanded a human embryonal carcinoma cell line (2102Ep) at three development sites in France, Germany and the UK using the CompacT SelecT (Sartorius Stedim, Royston, UK) automated cell culture platform. Anticipated variations between sites spanned material input, features of the process itself and production system details including different quality management systems and personnel. Where possible, these were pre-addressed by implementing strategies including standardisation, cell bank mycoplasma testing and specific engineering and process improvements. However, despite such measures, unexpected deviations occurred between sites including software incompatibility and machine/process errors together with uncharacteristic contaminations. Many only became apparent during process proving or during the process run. Further, parameters including growth rate and viability discrepancies could only be determined post-run, preventing 'live' corrective measures. The work confirms the critical nature of approaches usually taken in Good Manufacturing Practice (GMP) manufacturing settings and especially emphasises the requirement for monitoring steps to be included within the production system. Real-time process monitoring coupled with carefully structured quality systems is essential for multiple site working including clarity of decision-making roles. Additionally, an over-reliance upon post-process visual microscopic comparisons has major limitations; it is difficult for non-experts to detect deleterious culture changes and such detection is slow.

Coating stability and insertion forces of an alginate-cell-based drug delivery implant system for the inner ear.

Long-term drug delivery to the inner ear for neuroprotection might improve the outcome for hearing disabled patients treated with a cochlear implant (CI). Neurotrophic factor (NTF) producing cells encapsulated in an alginate-matrix, to shield them from the host immune system and to avoid migration, and applied as viscose solution or electrode coating could address this requirement. Both application methods were tested for their feasibility in an artificial human cochlea model. Since both strategies potentially influence the electrode implantability, insertion forces and coating stability were analyzed on custom-made electrode arrays. Both, injection of the alginate-cell solution into the model and a manual dip coating of electrode arrays with subsequent insertion into the model were possible. The insertion forces of coated arrays were reduced by 75% of an uncoated reference. In contrast, filling of the model with non-crosslinked alginate-cell solution slightly increased the insertion forces. A good stability of the coating was observed after first insertion (85%) but abrasion increased after multiple insertions (50%). Both application strategies are possible options for cell-induced drug-delivery to the inner ear, but an alginate-cell coating of CI-electrodes has a great potential to combine an endogenous NTF-source with a strong reduction of insertion forces.

Stem Cell Based Drug Delivery for Protection of Auditory Neurons in a Guinea Pig Model of Cochlear Implantation.

Background: The success of a cochlear implant (CI), which is the standard therapy for patients suffering from severe to profound sensorineural hearing loss, depends on the number and excitability of spiral ganglion neurons (SGNs). Brain-derived neurotrophic factor (BDNF) has a protective effect on SGNs but should be applied chronically to guarantee their lifelong survival. Long-term administration of BDNF could be achieved using genetically modified mesenchymal stem cells (MSCs), but these cells should be protected - by ultra-high viscous (UHV-) alginate ('alginate-MSCs') - from the recipient immune system and from uncontrolled migration. Methods: Brain-derived neurotrophic factor-producing MSCs were encapsulated in UHV-alginate. Four experimental groups were investigated using guinea pigs as an animal model. Three of them were systemically deafened and (unilaterally) received one of the following: (I) a CI; (II) an alginate-MSC-coated CI; (III) an injection of alginate-embedded MSCs into the scala tympani followed by CI insertion and alginate polymerization. Group IV was normal hearing, with CI insertion in both ears and a unilateral injection of alginate-MSCs. Using acoustically evoked auditory brainstem response measurements, hearing thresholds were determined before implantation and before sacrificing the animals. Electrode impedance was measured weekly. Four weeks after implantation, the animals were sacrificed and the SGN density and degree of fibrosis were evaluated. Results: The MSCs survived being implanted for 4 weeks in vivo. Neither the alginate-MSC injection nor the coating affected electrode impedance or fibrosis. CI insertion with and without previous alginate injection in normal-hearing animals resulted in increased hearing thresholds within the high-frequency range. Low-frequency hearing loss was additionally observed in the alginate-injected and implanted cochleae, but not in those treated only with a CI. In deafened animals, the alginate-MSC coating of the CI significantly prevented SGN from degeneration, but the injection of alginate-MSCs did not. Conclusion: Brain-derived neurotrophic factor-producing MSCs encapsulated in UHV-alginate prevent SGNs from degeneration in the form of coating on the CI surface, but not in the form of an injection. No increase in fibrosis or impedance was detected. Further research and development aimed at verifying long-term mechanical and biological properties of coated electrodes in vitro and in vivo, in combination with chronic electrical stimulation, is needed before the current concept can be tested in clinical trials.

Vibrational spectroscopic imaging and live cell video microscopy for studying differentiation of primary human alveolar epithelial cells.

Alveolar type II (ATII) cells in the peripheral human lung spontaneously differentiate toward ATI cells, thus enabling air-blood barrier formation. Here, linear Raman and coherent anti-Stokes Raman scattering (CARS) microscopy are applied to study cell differentiation of freshly isolated ATII cells. The Raman spectra can successfully be correlated with gradual morphological and molecular changes during cell differentiation. Alveolar surfactant rich vesicles in ATII cells are identified based on phospholipid vibrations, while ATI-like cells are characterized by the absence of vesicular structures. Complementary, CARS microscopy allows for three-dimensional visualization of lipid vesicles within ATII cells and their secretion, while hyperspectral CARS enables the distinction between cellular proteins and lipids according to their vibrational signatures. This study paves the path for further label-free investigations of lung cells and the role of the pulmonary surfactant, thus also providing a basis for rational development of future lung therapeutics.

Tyramine-conjugated alginate hydrogels as a platform for bioactive scaffolds.

Alginate-based hydrogels represent promising microenvironments for cell culture and tissue engineering, as their mechanical and porous characteristics are adjustable toward in vivo conditions. However, alginate scaffolds are bioinert and thus inhibit cellular interactions. To overcome this disadvantage, bioactive alginate surfaces were produced by conjugating tyramine molecules to high-molecular-weight alginates using the carbodiimide chemistry. Structural elucidation using nuclear magnetic resonance spectroscopy and contact angle measurements revealed a surface chemistry and wettability of tyramine-alginate hydrogels similar to standard cell culture treated polystyrene. In contrast to stiff cell culture plastic, tyramine-alginate scaffolds were found to be soft (60-80 kPa), meeting the elastic moduli of human tissues such as liver and heart. We further demonstrated an enhanced protein adsorption with increasing tyramine conjugation, stable for several weeks. Cell culture studies with human mesenchymal stem cells and human pluripotent stem cell-derived cardiomyocytes qualified tyramine-alginate hydrogels as bioactive platforms enabling cell adhesion and contraction on (structured) 2-D layer and spherical matrices. Due to the alginate functionalization with tyramines, stable cell-matrix interactions were observed beneficial for an implementation in biology, biotechnology, and medicine toward efficient cell culture and tissue substitutes. © 2018 The Authors. Journal of Biomedical Materials Research Part A published by Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 114-121, 2019.

Poly(amidoamine)-alginate hydrogels: directing the behavior of mesenchymal stem cells with charged hydrogel surfaces.

The surface charge of a biomaterial represents a promising tool to direct cellular behavior, which is crucial for therapeutic approaches in regenerative medicine. To expand the understanding of how the material surface charge affects protein adsorption and mesenchymal stem cell behavior, differently charged surfaces with zeta potentials spanning from -25 mV to +15 mV were fabricated by the conjugation of poly(amidoamine) to alginate-based hydrogels. We showed that the increase of the biomaterials surface charge resulted in enhanced quantities of biologically available, surface-attached proteins. Since different surface charges were equalized after protein adsorption, mesenchymal stem cells interacted rather with diverse protein compositions instead of different surface features. Besides an enhanced cell attachment to increasingly positively charged surfaces, the cell spreading area and the expression of adhesion-related genes integrin α5 and tensin 1 were found to be increased after adhesion. Moreover, first results indicate a potential impact of the surface charge on mesenchymal stem cell differentiation towards bone and fat cells. The improved understanding of surface charge-related cell behavior has significant impact on the design of biomedical devices and artificial organs.

Study of SEM preparation artefacts with correlative microscopy: Cell shrinkage of adherent cells by HMDS-drying.

One of the often reported artefacts during cell preparation to scanning electron microscopy (SEM) is the shrinkage of cellular objects, that mostly occurs at a certain time-dependent stage of cell drying. Various methods of drying for SEM, such as critical point drying, freeze-drying, as well as hexamethyldisilazane (HMDS)-drying, were usually used. The latter becomes popular since it is a low cost and fast method. However, the correlation of drying duration and real shrinkage of objects was not investigated yet. In this paper, cell shrinkage at each stage of preparation for SEM was studied. We introduce a shrinkage coefficient using correlative light microscopy (LM) and SEM of the same human mesenchymal stem cells (hMSCs). The influence of HMDS-drying duration on the cell shrinkage is shown: the longer drying duration, the more shrinkage is observed. Furthermore, it was demonstrated that cell shrinkage is inversely proportional to cultivation time: the longer cultivation time, the more cell spreading area and the less cell shrinkage. Our results can be applicable for an exact SEM quantification of cell size and determination of cell spreading area in engineering of artificial cellular environments using biomaterials. SCANNING 38:625-633, 2016. © 2016 Wiley Periodicals, Inc.

Nanoparticle-mediated gene transfer from electrophoretically coated metal surfaces.

The transfer of genetic information into living cells is a powerful tool to manipulate their protein expression by the regulation of protein synthesis. This can be used for the treatment of genetically caused diseases (gene therapy). However, the systemic application of genes is associated with a number of problems, such as a targeted gene delivery and potential side effects. Here we present a method for the spatial application of nanoparticle-based gene therapy. Titanium was electrophoretically coated with DNA-functionalized calcium phosphate nanoparticles. NIH3T3 cells and HeLa cells were transfected with pcDNA3-EGFP. We monitored the transfection in vitro by fluorescence microscopy, flow cytometry, and Western Blot analysis. By coating a transparent substrate, i.e., indium tin oxide (ITO), with nanoparticles, we followed the transfection by live cell imaging.

Dispensing of very low volumes of ultra high viscosity alginate gels: a new tool for encapsulation of adherent cells and rapid prototyping of scaffolds and implants.

We present a tool for dispensing very low volumes (20 nL or more) of ultra high viscosity (UHV) medical-grade alginate hydrogels. It uses a modified piezo-driven micrometering valve, integrated into a versatile system that allows fast prototyping of encapsulation procedures and scaffold production. Valves show excellent dispensing properties for UHV alginate in concentrations of 0.4% and 0.7% and also for aqueous liquids. An optimized process flow provides excellent handling of biological samples under sterile conditions. This technique allows the encapsulation of adherent cells and structuring of substrates for biotechnology and regenerative medicine. A variety of cell lines showed at least 70% viability after encapsulation (including cell lines that are relevant in regenerative medicine like Hep G2), and time-lapse analysis revealed cells proliferating and showing limited motility under alginate spots. Cells show metabolic activity, gene product expression, and physiological function. Encapsulated cells have contact with the substrate and can exchange metabolites while being isolated from macromolecules in the environment. Contactless dispensing allows structuring of substrates with alginate, isolation and transfer of cell-alginate complexes, and the dispensing of biological active hydrogels like extracellular matrix-derived gels.

Determination of hERG channel blockers using a decision tree.

A decision tree approach for the in silico prediction of Torsade de Pointes (TdP)-causing drugs is presented. As TdP is frequently associated with QT-interval prolongation due to inhibition of the rapid activating delayed rectifier potassium channel in the heart (hERG channel), the properties of such blockers were investigated by molecular modeling and semi-empirical AM1 molecular orbital calculations. In addition, we derived a pharmacophoric SMARTS string using structural information from high affinity compounds. A corresponding search in the PubChem database identified several compounds that exhibit QT-interval prolonging activity that were not among our data set. This SMARTS string furthermore showed to be the most significant descriptor in the decision tree approach from which guidelines for the design of safe compounds are suggested.