A Novel Xenogeneic Co-Culture System to Examine Neuronal Differentiation Capability of Various Adult Human Stem Cells

Background: Targeted differentiation of stem cells is mainly achieved by the sequential administration of defined growth factors and cytokines, although these approaches are quite artificial, cost-intensive and time-consuming. We now present a simple xenogeneic rat brain co-culture system which supports neuronal differentiation of adult human stem cells under more in vivo-like conditions. Methods and Findings: This system was applied to well-characterized stem cell populations isolated from human skin, parotid gland and pancreas. In addition to general multi-lineage differentiation potential, these cells tend to differentiate spontaneously into neuronal cell types in vitro and are thus ideal candidates for the introduced co-culture system. Consequently, after two days of co-culture up to 12% of the cells showed neuronal morphology and expressed corresponding markers on the mRNA and protein level. Additionally, growth factors with the ability to induce neuronal different iation in stem cells could be found in the media supernatants of the co-cultures. Conclusions: The co-culture system described here is suitable for testing neuronal differentiation capability of numerous types of stem cells. Especially in the case of human cells, it may be of clinical relevance for future cell-based therapeutic applications

A922 Sequential measurement of 1 hour creatinine clearance (1-CRCL) in critically ill patients at risk of acute kidney injury (AKI)

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A novel fully automated incubation, manipulation and documentation system for the avian embryogenesis

Scientifically the avian egg is a well-established and increasingly used animal model predominantly represented by the chicken (gallus gallus domesticus). Apart from developmental biology studies, chicken eggs are used in angiogenesis, tumor and irritation studies. For all those experimental setups a culture and incubation system is required providing excellent documentation of environmental parameters as temperature and relative humidity and optical accessibility, flexible manipulation during the complete embryonic development of the chicken (usually 21 days) and stable survival rate. Here we present an avian egg incubator with a modular lid system comprising a glass window with condensation prevention and a parallel access for manipulation. We show the influence of light exposure as well as turning speed and frequency on the embryo survival rate for optimized culture conditions. Incubator and lid system were evaluated successfully up to the hatch of normally developed chicken, while automatically documenting their embryogenesis. The new incubation system holds benefits like automated high quality documentation, online manipulation and adjustable incubation and opens up completely new applications in the field of avian embryo culture

Stirred bioreactors for the expansion of adult pancreatic stem cells

Adult pluripotent stem cells are a cellular resource representing unprecedented potential for cell. therapy and tissue engineering. Complementary to this promise, there is a need for efficient bioprocesses for their large scale expansion and/or differentiation. With this goat in mind, our work focused on the development of three-dimensional (3-D) culture systems for controlled expansion of adult pancreatic stem cells (PSCs). For this purpose, two different culturing strategies were evaluated, using spinner vessels: cell aggregated cultures versus microcarrier technology. The use of microcarrier supports (Cytodex 1 and Cytodex 3) rendered expanded cell populations which retained their self-renewal ability, cell marker, and the potential to differentiate into adipocytes. This strategy surmounted the drawbacks of aggregates in culture which were demonstrably unfeasible as cells clumped together did not proliferate and lost PSC marker expression. Furthermore, the results obtained showed that although both microcarriers tested here were suitable for sustaining cell. expansion, Cytodex 3 provided a better substrate for the promotion of cell adherence and growth. For the latter approach, the potential of bioreactor technology was combined with the efficient Cytodex 3 strategy under controlled environmental conditions (pH-7.2, pO(2)-30% and temperature-37 degrees C); cell growth was more efficient, as shown by faster doubling time, higher growth rate and higher fold increase in cell concentration, when compared to spinner cultures. This study describes a robust bioprocess for the controlled expansion of adult PSC, representing an efficient starting point for the development of novel technologies for cell therapy

Low-energy electron beam sterilization for medical technical applications

New and highly functionalized medical products cannot be sterilized by standard sterilization methods like hot steam or sterilizing gases, as they are temperature sensitive, contain electronic parts like microchips or consist of polymeric materials. The use of gamma irradiation for the sterilization of such products is also problematic due to long exposure times under radical process conditions, which lead to an increased degradation and therefore a loss of functionality or product stability. Using low-energy electron beam irradiation (eB) enables sterilization of medical surfaces within seconds as this technology offers high dose rates in comparison to gamma irradiation. Therefore, degradation processes can be prevented. Electron beam irradiation complies with international standards (ISO 11137) and is a worldwide accepted sterilization method. In addition, by using low-energy electron beam irradiation it is possible to define the penetration depth in order to prevent electronic parts damage. We investigated whether complex 3D geometries can be sterilized using a mini-eB source and low-energy eB treatment with a source smaller than the packed medical product. Therefore, a process for 3D handling using a small eB source was developed and investigated in terms of efficacy and safety. Furthermore, a suitable packaging material was evaluated for sterile handling of the medical products. We demonstrated that B. pumilus and P. aeruginosa surface-contaminated test specimens were sterilized reproducibly by the process developed, while the eB treatment had no negative influence on the biocompatibility, form and function of the sterilized test specimens and the selected packaging material

Effect of gold nanoparticles on adipogenic differentiation of human mesenchymal stem cells

Gold nanoparticles are very attractive for biomedical products. However, there is a serious lack of information concerning the biological activity of nanosized gold in human tissue cells. An influence of nanoparticles on stem cells might lead to unforeseen consequences to organ and tissue functions as long as all cells arising from the initial stem cell might be subsequently damaged. Therefore the effect of negatively charged gold nanoparticles (9 and 95 nm), which are certified as reference material for preclinical biomedical research, on the adipogenic differentiation of human mesenchymal stem cells (hMSCs) is investigated here. Bone marrow hMSCs are chosen as differentiation model since bone marrow hMSCs are well characterized and their differentiation into the adipogenic lineage shows clear and easily detectable differentiation. In this study effects of gold nanoparticles on adipogenic differentiation are analyzed regarding fat storage and mitochondrial activity aft er different exposure times (4-21 days). Using time lapse microscopy the differentiation progress under chronically gold nanoparticle treatment is continuously investigated. In this preliminary study, chronically treatment of adipogenic differentiating hMSCs with gold nanoparticles resulted in a reduced number and size of lipid vacuoles and reduced mitochondrial activity depending on the applied concentration and the surface charge of the particles

Low-energy electron-beam treatment as alternative for on-site sterilization of highly functionalized medical products - a feasibility study

Over the last decades, the medical device industry has grown significantly. Complex and highly functionalized medical devices and implants are being developed to improve patient treatment and to enhance their health-related quality of life. However, medical devices from this new generation often cannot be sterilized by standard methods such as autoclaving or sterilizing gases, as they are temperature sensitive, containing electronic com-ponents like sensors and microchips, or consist of polymers. Gamma irradiation for sterilization of such products is also problematic due to long processing times under highly reactive conditions resulting in material de-gradation or loss of functionality. Low-energy electron-beam treatment could enable irradiation sterilization of medical surfaces within seconds. This method is very fast in comparison to gamma irradiation because of its high dose rate and therefore degradation processes of polymers can be reduced or even prevented. Additionally, electron penetration depth can be precisely controlled to prevent damage of sensitive components like elec-tronics and semiconductors.The presented study focuses on two key aspects: 1.) Can new and highly functionalized medical products in future be sterilized using low-energy electron-beam irradiation; and 2.) Is the low-energy electron-beam tech-nology suitable to be set up on-site to speed up sterilization processing or make it available “just-in-time”.To address these questions, different test specimens were chosen with complex geometry or electronic functional parts to gather information about the limitations and chances for this new approach. The test specimens were inoculated with clinical relevant test organisms (Pseudomonas aeruginosa) as well as with approved radiation resistant organisms (Deinococcus radiodurans and Bacillus pumilus) to prove the suitability of low-energy electron-beam treatment for the above-mentioned medical products. The calculation of the D10 value for B. pumilus revealed equal efficacy when compared to standard high-energy irradiation sterilization. All of the above-mentioned germs were successfully inactivated by low-energy electron-beam treatment when test specimens were inoculated with a germ load > 106 CFU and treated with doses ≥ 10 kGy (for B. pumilus and P. aerugi-nosa) and > 300 kGy (for D. radiodurans) respectively. As an example, for specialized electronic components to be sterilized, an impedance sensor for cell culture applications was sterilized and unimpaired functionality was demonstrated even after five repeated sterilization cycles to a total dose of 50 kGy. To address the second aspect of on-site suitability of this technology, the product handling for low-energy electron-beam treatment had to be adapted to minimize the size of the electron-beam facility. Therefore, a mini electron-beam source was used and a specialized sample holder and 3D-handling regime were developed to allow reproducible surface treatment for complex product geometries. Inactivation of B. pumilus inoculated medical screws (> 106 CFU) was successful using the developed handling procedure. In addition, a packaging material (PET12/PE50) for medical products was investigated for its suitability for low-energy irradiation sterilization. Biocompatibility assessment revealed the material to be eligible for this application as even overdoses did not impair the biocompatibility of the material