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Leipzig, Germany

Georgiev V.,Florida A&M University | Schumann A.,Vita 34 AG | Pavlov A.,University of Food Technologies | Pavlov A.,Bulgarian Academy of Science | Bley T.,TU Dresden
Engineering in Life Sciences | Year: 2014

Plant tissue and organ cultures in vitro usually face technological challenges. When submerged cultivation of plant cells in a controlled environment is desired, the characteristic growth morphology and physiology of differentiated organ cultures present a problem in process scale-up. Temporary immersion systems (TIS) were developed several decades ago. These systems are providing the most natural environment for in vitro culture of plant shoots and seedlings. Over the past few years, TIS have been recognized as a perspective technology for plant micropropagation, production of plant-derived secondary metabolites, expression of foreign proteins, and potential solutions in phytoremediation. Nowadays, several TIS, operating on similar or divergent technological principles, have been developed and successfully applied in the cultivation of various plant in vitro systems, including somatic embryos and transformed root cultures. In this article, the operational principle and technological design of the most popular TIS are reviewed. In addition, recent examples of the application of temporary immersion technology for in vitro cultivation of plant tissue and organ cultures at laboratory and pilot scales are discussed. Finally, future prospects and challenges to the industrial realization of that fast-developing technique are outlined. © 2014 WILEY-VCH Verlag GmbH & Co. Source

Boltze J.,Fraunhofer Institute for Cell Therapy and Immunology | Boltze J.,University of Leipzig | Reich D.M.,Fraunhofer Institute for Cell Therapy and Immunology | Hau S.,Fraunhofer Institute for Cell Therapy and Immunology | And 7 more authors.
Cell Transplantation | Year: 2012

Experimental transplantation of human umbilical cord blood (hUCB) mononuclear cells (MNCs) in rodent stroke models revealed the therapeutic potential of these cells. However, effective cells within the heterogeneous MNC population and their modes of action are still under discussion. MNCs and MNC fractions enriched (CD34+) or depleted (CD34-) for CD34-expressing stem/progenitor cells were isolated from hUCB. Cells were transplanted intravenously following middle cerebral artery occlusion in spontaneously hypertensive rats and directly or indirectly cocultivated with hippocampal slices previously subjected to oxygen and glucose deprivation. Application of saline solution or a human T-cell line served as controls. In vivo, MNCs, CD34+ and CD34- cells reduced neurofunctional deficits and diminished lesion volume as determined by magnetic resonance imaging. MNCs were superior to other fractions. However, human cells could not be identified in brain tissue 29 days after stroke induction. Following direct application on postischemic hippocampal slices, MNCs reduced neural damage throughout a 3-day observation period. CD34+ cells provided transient protection for 2 days. The CD34- fraction, in contrast to in vivo results, failed to reduce neural damage. Direct cocultivation of MNCs was superior to indirect cocultivation of equal cell numbers. Indirect application of up to 10-fold MNC concentrations enhanced neuroprotection to a level comparable to direct cocultivation. After direct application, MNCs migrated into the slices. Flow cytometric analysis of migrated cells revealed that the CD34+ cells within MNCs were preferably attracted by damaged hippocampal tissue. Our study suggests that MNCs provide the most prominent neuroprotective effect, with CD34+ cells seeming to be particularly involved in the protective action of MNCs. CD34+ cells preferentially home to neural tissue in vitro, but are not superior concerning the overall effect, implying that there is another, still undiscovered, protective cell population. Furthermore, MNCs did not survive in the ischemic brain for longer periods without immunosuppression. © 2012 Cognizant Comm. Corp. Source

Sygnecka K.,University of Leipzig | Heider A.,University of Leipzig | Scherf N.,Carl Gustav Carus Institute | Alt R.,University of Leipzig | And 3 more authors.
Stem Cells and Development | Year: 2015

Mesenchymal stem cells (MSCs) have been identified as promising candidates for neuroregenerative cell therapies. However, the impact of different isolation procedures on the functional and regenerative characteristics of MSC populations has not been studied thoroughly. To quantify these differences, we directly compared classically isolated bulk bone marrow-derived MSCs (bulk BM-MSCs) to the subpopulation Sca-1+Lin-CD45--derived MSCs- (SL45-MSCs), isolated by fluorescence-activated cell sorting from bulk BM-cell suspensions. Both populations were analyzed with respect to functional readouts, that are, frequency of fibroblast colony forming units (CFU-f), general morphology, and expression of stem cell markers. The SL45-MSC population is characterized by greater morphological homogeneity, higher CFU-f frequency, and significantly increased nestin expression compared with bulk BM-MSCs. We further quantified the potential of both cell populations to enhance neuronal fiber growth, using an ex vivo model of organotypic brain slice co-cultures of the mesocortical dopaminergic projection system. The MSC populations were cultivated underneath the slice co-cultures without direct contact using a transwell system. After cultivation, the fiber density in the border region between the two brain slices was quantified. While both populations significantly enhanced fiber outgrowth as compared with controls, purified SL45-MSCs stimulated fiber growth to a larger degree. Subsequently, we analyzed the expression of different growth factors in both cell populations. The results show a significantly higher expression of brain-derived neurotrophic factor (BDNF) and basic fibroblast growth factor in the SL45-MSCs population. Altogether, we conclude that MSC preparations enriched for primary MSCs promote neuronal regeneration and axonal regrowth, more effectively than bulk BM-MSCs, an effect that may be mediated by a higher BDNF secretion. © Mary Ann Liebert, Inc. Source

Herzog N.,Sudan University of Science and Technology | Hansen M.,Sudan University of Science and Technology | Hansen M.,Vita 34 AG | Miethbauer S.,Sudan University of Science and Technology | And 9 more authors.
Cell Biology International | Year: 2016

Primary human hepatocytes are in great demand during drug development and in hepatology. However, both scarcity of tissue supply and donor variability of primary cells create a need for the development of alternative hepatocyte systems. By using a lentivirus vector system to transfer coding sequences of Upcyte® proliferation genes, we generated non-transformed stable hepatocyte cultures from human liver tissue samples. Here, we show data on newly generated proliferation-competent HepaFH3 cells investigated as conventional two-dimensional monolayer and as organotypical three-dimensional (3D) spheroid culture. In monolayer culture, HepaFH3 cells show typical cobblestone-like hepatocyte morphology and anchorage-dependent growth for at least 20 passages. Immunofluorescence staining revealed that characteristic hepatocyte marker proteins cytokeratin 8, human serum albumin, and cytochrome P450 (CYP) 3A4 were expressed. Quantitative real-time PCR analyses showed that expression levels of analyzed phase I CYP enzymes were at similar levels compared to those of cultured primary human hepatocytes and considerably higher than in the liver carcinoma cell line HepG2. Additionally, transcripts for phase II liver enzymes and transporter proteins OATP-C, MRP2, Oct1, and BSEP were present in HepaFH3. The cells produced urea and converted model compounds such as testosterone, diclofenac, and 7-OH-coumarin into phases I and II metabolites. Interestingly, phases I and II enzymes were expressed at about the same levels in convenient monolayer cultures and complex 3D spheroids. In conclusion, HepaFH3 cells and related primary-like hepatocyte lines seem to be promising tools for in vitro research of liver functions and as test system in drug development and toxicology analysis. © 2016 International Federation for Cell Biology. Source

Scholbach J.,University of Leipzig | Schulz A.,Fraunhofer Institute for Cell Therapy and Immunology | Westphal F.,University of Leipzig | Egger D.,Vita 34 AG | And 5 more authors.
PLoS ONE | Year: 2012

To study the function and maturation of the human hematopoietic and immune system without endangering individuals, translational human-like animal models are needed. We compare the efficiency of CD34+ stem cells isolated from cryopreserved cord blood from a blood bank (CCB) and fresh cord blood (FCB) in generating highly engrafted humanized mice in NOD-SCID IL2Rγnull (NSG) rodents. Interestingly, the isolation of CD34+ cells from CCB results in a lower yield and purity compared to FCB. The purity of CD34+ isolation from CCB decreases with an increasing number of mononuclear cells that is not evident in FCB. Despite the lower yield and purity of CD34+ stem cell isolation from CCB compared to FCB, the overall reconstitution with human immune cells (CD45) and the differentiation of its subpopulations e.g., B cells, T cells or monocytes is comparable between both sources. In addition, independent of the cord blood origin, human B cells are able to produce high amounts of human IgM antibodies and human T cells are able to proliferate after stimulation with anti-CD3 antibodies. Nevertheless, T cells generated from FCB showed increased response to restimulation with anti-CD3. Our study reveals that the application of CCB samples for the engraftment of humanized mice does not result in less engraftment or a loss of differentiation and function of its subpopulations. Therefore, CCB is a reasonable alternative to FCB and allows the selection of specific genotypes (or any other criteria), which allows scientists to be independent from the daily changing birth rate. © 2012 Scholbach et al. Source

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