Heiko Zimmermann

Fabrication of homogeneously cross-linked, functional alginate microcapsules validated by NMR-, CLSM- and AFM-imaging

Cross-linked alginate microcapsules of sufficient mechanical strength can immunoisolate cells for the long-term treatment of hormone and other deficiency diseases in human beings. However, gelation of alginate by external Ba(2+) (or other divalent cations) produces non-homogeneous cross-linking of the polymeric mannuronic (M) and guluronic (G) acid chains. The stability of such microcapsules is rather limited. Here, we show that homogeneous cross-linking can be achieved by injecting BaCl(2) crystals into alginate droplets before they come into contact with external BaCl(2). The high effectiveness of this crystal gun method is demonstrated by confocal laser scanning microscopy and by advanced nuclear magnetic resonance imaging. Both techniques gave clear-cut evidence that homogeneous cross-linkage throughout the microcapsule is only obtained with simultaneous internal and external gelation. Atomic force microscopy showed a very smooth surface topography for microcapsules made by the crystal gun method, provided that excess Ba(2+) ions were removed immediately after gelation. In vitro experiments showed greatly suppressed swelling for crystal gun microcapsules. Even alginate extracted from Lessonia nigrescens (highly biocompatible) yielded microcapsules with long-term mechanical stability not hitherto possible. Encapsulation of rat islets, human monoclonal antibodies secreting hybridoma cells and murine mesenchymal stem cells transfected with cDNA encoding for bone morphogenetic protein (BMP-4) revealed that injection of BaCl(2) crystals has no adverse side effects on cell viability and function. However, the release of low-molecular weight factors (such as insulin) may be delayed when using alginate concentrations in the usual range.

Large-area, uniform, high-spatial-frequency ripples generated on silicon using a nanojoule-femtosecond laser at high repetition rate

Large-area high-spatial-frequency patterns (HSFLs) of λ/6 periodicity have been generated by a nanojoule-femtosecond laser scanning technique (80 MHz, 170 fs, 700-950 nm) at the silicon-air interface. The excellent large-area uniformity allowed reproducible and accurate measurements of the periodicity. Variation of experimental parameters as illumination geometry, and pulse energy and number showed no influence on the ripple spacing. A wavelength dependence was observed and compared to current models of HSFL formation. A particular second-harmonic model was found to match the results best but needs to take into account transient changes in the refractive index under laser exposure. A second-harmonic mechanism is further supported by direct spectroscopic observation.

Generation of high spatial frequency ripples on silicon under ultrashort laser pulses irradiation

Periodic high spatial frequency ripples structures have been generated in silicon under femtosecond laser pulses irradiation. The period of the ripples is wavelength dependent. It increases from 110 up to 160 nm when the wavelength varies from 700 to 950 nm, respectively. We propose a refined model of the second harmonic generation ripples spacing theory Λ=λ/2nλ∗ taking into account the modified femtosecond laser excited silicon refractive index n∗ related to the Drude model. Good agreement is found between experimental results and the presented revisited model.

Cryopreservation of Adherent Cells: Strategies to Improve Cell Viability and Function After Thawing

The commonly applied cryopreservation protocols routinely used in laboratories worldwide were developed for simple cell suspensions, and their application to complex systems, such as cell monolayers, tissues, or biosynthetic constructs, is not straightforward. In particular for monolayer cultures, cell detachment and membrane damage are often observed after cryopreservation. In this work, combined strategies for the cryopreservation of cells attached to Matrigel-coated well plates surfaces were investigated based on cell entrapment in clinicalgrade, ultra-high viscosity alginate using two cell lines, neuroblastoma N2a and colon adenocarcinoma Caco-2, with distinct structural and functional characteristics. As the cryopreservation medium, serum-free CryoStor solution was compared with serum-supplemented culture medium, both containing 10% DMSO. Using culture medium, entrapment beneath an alginate layer was needed to improve cell recovery by minimizing membrane damage and cell detachment after thawing; nevertheless, up to 50% cell death still occurred within 24 h after thawing. The use of CryoStor solution represented a considerable improvement of the cryopreservation process for both cell lines, allowing the maintenance of high postthaw membrane integrity as well as full recovery of metabolic activity and differentiation capacity within 24 h postthawing; in this case, entrapment beneath an alginate layer did not confer further protection to cryopreserved Caco-2 cells, but was crucial for maintenance of attachment and integrity of N2a neuronal networks.

Physicochemical features of ultra-high viscosity alginates.

The physicochemical characteristics of the ultra-high viscosity and highly biocompatible alginates extracted from Lessonia nigrescens (UHV(N)) and Lessonia trabeculata (UHV(T)) were analyzed. Fluorescence and (1)H NMR spectroscopies, viscometry, and multi-angle light scattering (MALS) were used for elucidation of the chemical structure, molar mass, and coil size. The sequential structures from NMR spectroscopy showed high guluronate content for UHV(T), but low for UHV(N). Intrinsic viscosity [eta] measurements exhibited unusual high values (up to 2750 mL/g), whereas [eta] of a commercial alginate was only about 970 mL/g. MALS batch measurements of the UHV-alginates yielded ultra-high values of the weight average molar mass (M(w) up to 1.1x10(6) g/mol) and of the z-average gyration radius (R(G)(z) up to 191 nm). The M(w) and R(G)(z) distributions of UHV-alginates and of ultrasonically degraded fractions were determined using size exclusion chromatography combined with MALS and asymmetrical flow-field-flow fractionation. The M(w) dependency of [eta] and R(G)(z) could be described by [eta]=0.059xM(w)(0.78) and R(G)(z)=0.103xM(w)(x). (UHV(N): x=0.52; UHV(T): x=0.53) indicating that the monomer composition has no effect on coil expansion. Therefore, the equations can be used to calculate M(w) and R(G)(z) values of UHV(T)- and UHV(N)-alginate mixtures as used in immunoisolation. Furthermore, the simple and inexpensive capillary viscometry can be used for real-time validation of the extraction and purification process of the UHV-alginates.

Volume regulation of murine T lymphocytes relies on voltage-dependent and two-pore domain potassium channels

A variety of ion channels are supposed to orchestrate the homoeostatic volume regulation in T lymphocytes. However, the relative contribution of different potassium channels to the osmotic volume regulation and in particular to the regulatory volume decrease (RVD) in T cells is far from clear. This study explores a putative role of the newly identified K(2P) channels (TASK1, TASK2, TASK3 and TRESK) along with the voltage-gated potassium channel K(V)1.3 and the calcium-activated potassium channel K(Ca)3.1 in the RVD of murine T lymphocytes, using genetic and pharmacological approaches. K(2P) channel knockouts exerted profound effects on the osmotic properties of murine T lymphocytes, as revealed by reduced water and RVD-related solute permeabilities. Moreover, both genetic and pharmacological data proved a key role of K(V)1.3 and TASK2 channels in the RVD of murine T cells exposed to hypotonic saline. Our experiments demonstrate a leading role of potassium channels in the osmoregulation of T lymphocytes under different conditions. In summary, the present study sheds new light on the complex and partially redundant network of potassium channels involved in the basic physiological process of the cellular volume homeostasis and extends the repertoire of potassium channels by the family of K(2P) channels.

Biological and Physicochemical Characterization of a Serum-and Xeno-Free Chemically Defined Cryopreservation Procedure for Adult Human Progenitor Cells:

While therapeutic cell transplantations using progenitor cells are increasingly evolving towards phase I and II clinical trials and chemically defined cell culture is established, standardization in biobanking is still in the stage of infancy. In this study, the EU FP6-funded CRYSTAL (CRYo-banking of Stem cells for human Therapeutic AppLication) consortium aimed to validate novel Standard Operating Procedures (SOPs) to perform and validate xeno-free and chemically defined cryopreservation of human progenitor cells and to reduce the amount of the potentially toxic cryoprotectant additive (CPA) dimethyl sulfoxide (DMSO). To achieve this goal, three human adult progenitor and stem cell populations—umbilical cord blood (UCB)-derived erythroid cells (UCB-ECs), UCB-derived endothelial colony forming cells (UCB-ECFCs), and adipose tissue (AT)-derived mesenchymal stromal cells (AT-MSCs)—were cryopreserved in chemically defined medium supplemented with 10% or 5% DMSO. Cell recovery, cell repopulation, and functionality were evaluated postthaw in comparison to cryopreservation in standard fetal bovine serum (FBS)-containing freezing medium. Even with a reduction of the DMSO CPA to 5%, postthaw cell count and viability assays indicated no overall significant difference versus standard cryomedium. Additionally, to compare cellular morphology/membrane integrity and ice crystal formation during cryopreservation, multiphoton laser-scanning cryomicroscopy (cryo-MPLSM) and scanning electron microscopy (SEM) were used. Neither cryo-MPLSM nor SEM indicated differences in membrane integrity for the tested cell populations under various conditions. Moreover, no influence was observed on functional properties of the cells following cryopreservation in chemically defined freezing medium, except for UCB-ECs, which showed a significantly reduced differentiation capacity after cryopreservation in chemically defined medium supplemented with 5% DMSO. In summary, these results demonstrate the feasibility and robustness of standardized xeno-free cryopreservation of different human progenitor cells and encourage their use even more in the field of tissue-engineering and regenerative medicine.

Formation mechanism of femtosecond laser-induced high spatial frequency ripples on semiconductors at low fluence and high repetition rate

Periodic high spatial frequency ripples structures (HSFL) have been generated in silicon (Si) and in germanium (Ge) at very low fluence below or close to the melting fluence threshold, at different wavelengths and at high repetition rate femtosecond laser pulses (80 MHz, 700–950 nm, 170 fs). HSFL initiation, formation, and arrangement combine structural modification of the surface initiated by heat accumulation of successive pulses with second harmonic generation. HSFL are wavelength dependent and the refractive index plays a central role on their periodicities. HSFL spacing follows quite well a law of Λ=λ/2nλ*, where nλ* is the modified femtosecond laser excited refractive index as a function of the wavelength for Si and Ge.

Cell traces--footprints of individual cells during locomotion and adhesion.

Animal cells release traces of material onto glass or silicon surfaces during adhesion and migration. This little studied phenomenon is a widespread and normal concomitant of cell migration. The paper introduces the study of such material. The traces can be visualised by different microscopic techniques (e.g. TIRF, IRM, CLSM, AFM, SEM). Cell traces typical for different cell lines (NIH 3T3 and L929 mouse fibroblasts, mouse macrophages, mouse sarcoma cells and human osteosarcoma cells) are shown and discussed. There are well organised structures such as different linear and nodular elements as well as patches. Traces can extend up to some hundred micrometers from the cell, but the dimensions of the linear elements are in the submicron range. Cell traces are not identical with focal contacts but can include them. A first classification of basic elements is proposed. It allows an estimation of the total volume and surface in comparison to the donor cell. Higher order structures are discussed and a first insight into the protein composition of traces produced by mouse fibroblasts is given. Our observations, together with the cell adhesion literature suggest that the amount of released material, its extent and chemical and structural properties depend on cell type and physiology as well as other external influences. Cell traces in combination with the adhesion pattern of the donor cell should give information about the activity and physiological status of individual cells, the mechanisms of cell locomotion and the molecular composition of the donor cell membrane. The traces might possibly be used as submicron elements for passive electric characterisation and biotechnological applications.

Temperature fluctuations during deep temperature cryopreservation reduce PBMC recovery, viability and T-cell function.

The ability to analyze cryopreserved peripheral blood mononuclear cell (PBMC) from biobanks for antigen-specific immunity is necessary to evaluate response to immune-based therapies. To ensure comparable assay results, collaborative research in multicenter trials needs reliable and reproducible cryopreservation that maintains cell viability and functionality. A standardized cryopreservation procedure is comprised of not only sample collection, preparation and freezing but also low temperature storage in liquid nitrogen without any temperature fluctuations, to avoid cell damage. Therefore, we have developed a storage approach to minimize suboptimal storage conditions in order to maximize cell viability, recovery and T-cell functionality. We compared the influence of repeated temperature fluctuations on cell health from sample storage, sample sorting and removal in comparison to sample storage without temperature rises. We found that cyclical temperature shifts during low temperature storage reduce cell viability, recovery and immune response against specific-antigens. We showed that samples handled under a protective hood system, to avoid or minimize such repeated temperature rises, have comparable cell viability and cell recovery rates to samples stored without any temperature fluctuations. Also T-cell functionality could be considerably increased with the use of the protective hood system compared to sample handling without such a protection system. This data suggests that the impact of temperature fluctuation on cell integrity should be carefully considered in future clinical vaccine trials and consideration should be given to optimal sample storage conditions.