Alisa Katsen-Globa - Academia.edu (original) (raw)

Papers by Alisa Katsen-Globa

First steps towards the successful surface-based cultivation of human embryonic stem cells in han... more First steps towards the successful surface-based cultivation of human embryonic stem cells in hanging drop systems Miniaturization and parallelization of cell culture procedures are in focus of research in order to develop test platformswith lowmaterial consumption and increased stan-dardization for toxicity and drug screenings. The cultivation in hanging drops (HDs) is a convenient and versatile tool for biological applications and represents an in-teresting model system for the screening applications due to its uniform shape, the advantageous gas supply, and the small volume. However, its application has so far been limited to non-adherent and aggregate forming cells. Here, we describe for the first time the proof-of-principle regarding the adherent cultivation of human embryonic stem cells in HD. For this microcarriers were added to the droplet as dy-namic cultivation surfaces resulting in a maintained pluripotency and proliferation capacity for 10 days. This enables the HD techn...

Bioelectrochemistry, 2022

This study describes the development of a one-pot electrochemical miniaturized system for simulta... more This study describes the development of a one-pot electrochemical miniaturized system for simultaneous cultivation and monitoring of the oxidative status of living cells. This system consisted of screen-printed electrodes modified by electroplated Pd-NPs as an electrocatalyst (i) and living yeast cells (Saccharomyces cerevisiae) (ii) immobilized on the cytocompatible alginate layer (iii). Briefly, during the course of electrochemical investigations a novel electroactive compound methylhydrazine derivative as a secondary metabolite and result of microbial activity was found in yeast cells and used as a signaling molecule for their biochemical profiling. Under the optimized experimental conditions the signal corresponding to the found electroactive secondary metabolite formed in medium of living cells was measured without sample collecting, transport, storage or pre-treatment steps (i.e. extraction, pre-concentration, chemical derivatization or labeling). The electrochemical dependencies, which were derived by a miniaturized electroanalytical system, were fully validated in a conventional three-electrode system under inert atmosphere (Ar) and in the presence of oxygen (air, O2). It is believed that the proposed one-pot nanoreactors serving simultaneously as nanofermenters and amperometric detectors for the quantification of secondary metabolites formed in medium of living cells can significantly enhance the understanding of ongoing fermentation processes in the future and our knowledge on the biochemistry of yeasts.

Acta Ophthalmologica, 2020

To investigate the ultrastructure of the cleavage plane of human cornea after liquid‐bubble‐prepa... more To investigate the ultrastructure of the cleavage plane of human cornea after liquid‐bubble‐prepared tissue for Descemet’s membrane endothelial keratoplasty (DMEK).

Diagnostic Optical Spectroscopy in Biomedicine IV, 2007

Monitoring the functional status of cryo-preserved cells and tissue in-situ, ie. in the frozen st... more Monitoring the functional status of cryo-preserved cells and tissue in-situ, ie. in the frozen state, might allow for optimal adjustment of preservation conditions and might provide the information necessary to predict a functionality recovery rate. Here, an imaging approach with ...

Cellular Physiology and Biochemistry, 2008

Embryonic Stem (ES) cells-derived cardiomyocytes can possibly be applied for cell therapy of dise... more Embryonic Stem (ES) cells-derived cardiomyocytes can possibly be applied for cell therapy of diseases such as heart failure. Biodegradable scaffolds will significantly improve the expansion of sufficient functional ES cell-derived cardiomyocytes and may also increase the survival rate of cardiomyocytes after their transplantation. In the present study, we cultivated cardiomyocytes isolated from a transgenic a-myosin heavy chain (α-MHC) ES cell lineage expressing both puromycin resistance and enhanced green fluorescent protein (EGFP) under the control of the α-MHC promoter in macroporous gelatine microspheres using small-scale bioreactors and proved that cardiomyocytes function after their cultivation in micropsperes. The average number of cultivated cells per microsphere was optimised once the most suitable agitation conditions and the optimal timeframe of cultivation were identified. Our study shows that 72% of CultiSpher-S beads were colonised by cardiomyocytes under optimal conditions. Scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) showed that colonization of the beads was not limited to the surface, but that cells also invaded the inner surfaces of the microspheres. Electrophysiological experiments demonstrated that the action potentials (APs) of α-MHC + cardiomyocytes entrapped in microspheres were identical to action potentials of control cells. This attractive approach for cultivation and expansion of functional cardiomyocytes in biodegradable macroporous may offer a perspective for higher transplantation efficiencies of ES cell-derived cardiomyocytes.

Biomaterials, 2007

We describe the manufacture of highly stable and elastic alginate membranes with good cell adhesi... more We describe the manufacture of highly stable and elastic alginate membranes with good cell adhesivity and adjustable permeability. Clinical grade, ultra-high viscosity alginate is gelled by diffusion of Ba 2+ followed by use of the ''crystal gun'' [Zimmermann H. et al., Fabrication of homogeneously cross-linked, functional alginate microcapsules validated by NMR-, CLSM-and AFM-imaging. Biomaterials 2003;24:2083-96]. Burst pressure of well-hydrated membranes is between 34 and 325 kPa depending on manufacture and storage details. Water flows induced by sorbitol and raffinose (probably diffusional) are lower than those caused by PEG 6000, which may be related to a Hagen-Poiseuille flow. Hydraulic conductivity, L p , from PEG-induced flows ranges between 2.4 Â 10 À12 and 6.5 Â 10 À12 m Pa À1 s À1. Hydraulic conductivity measured with hydrostatic pressure up to 6 kPa is 2-3 orders of magnitude higher and decreases with increasing pressure to about 3 Â 10 À10 m Pa À1 s À1 at 4 kPa. Mechanical introduction of 200 mm-diameter pores increases hydraulic conductivity dramatically without loss of mechanical stability or flexibility. NMR imaging with Cu 2+ as contrast agent shows a layered structure in membranes cross-linked for 2 h. Phase contrast and atomic force microscopy in liquid environment reveals surface protrusions and cavities correlating with steps of the production process. Murine L929 cells adhere strongly to the rough surface of crystal-bombarded membranes. NaCl-mediated membrane swelling can be prevented by partial replacement of salt with sorbitol allowing cell culture on the membranes.

Archives of Virology, 1991

Biological and physicochemical characterization of the major (1.40) and minor (1.45) component of... more Biological and physicochemical characterization of the major (1.40) and minor (1.45) component of infectious avian adeno-associated virus

Biotechnology and Bioengineering, 2007

Recent advances in cell-based therapies require new approaches for cell cryopreservation, capable... more Recent advances in cell-based therapies require new approaches for cell cryopreservation, capable of dealing with large number of samples and providing specific conditions for each cell type. Reduction of sample volume from the commonly used 1 mL to 25 microL in 30-well micro-cryosubstrates improves cryopreservation by allowing automation, data handling and access to individual wells without thawing the whole cryosubstrate. This system was evaluated for the storage of Caco-2 colon adenocarcinoma cells, which differentiate spontaneously after long-term culture. The impact of the cryosample small volume upon post-thawing membrane integrity of the cells and their capacity to proliferate and differentiate was studied. Two different cryoprotectants commonly employed, dimethyl sulfoxide (Me(2)SO) and glycerol, were evaluated as well as the possibility of decreasing their concentration from the 10% concentration, usually used, down to 3% (v/v). The process automation by pipette robotic addition of the cryoprotectant to the micro-cryosubstrates was also evaluated. The micro-cryosubstrates have proven to be at least as efficient as typical 1 mL cryovials for cryopreservation of Caco-2 cells using either Me(2)SO or glycerol. Compared to the manual process, the automatic addition of glycerol to the micro-cryosubstrates allowed higher cell viabilities after thawing while with Me(2)SO no significant changes were observed. Me(2)SO has shown to be more effective than glycerol in maintaining high post-thaw cell membrane integrity, either in micro-cryosubstrates or cryovials, for any of the concentrations tested. The ability of Me(2)SO in maintaining high cell membrane integrity post-thawing was confirmed by long-term (up to 22 days) proliferation and differentiation studies performed with cells cultured immediately after thawing.

Tissue Engineering Part C: Methods, 2009

The commonly applied cryopreservation protocols routinely used in laboratories worldwide were dev... more 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 plate's 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 TM 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 TM 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.

In this study, we prepared hydrogel scaffolds for tissue engineering by computer-assisted extrusi... more In this study, we prepared hydrogel scaffolds for tissue engineering by computer-assisted extrusion three-dimensional (3D) printing with photocured (λ = 445 nm) hyaluronic acid glycidyl methacrylate (HAGM). The developed product was compared with the polylactic-co-glycolic acid (PLGA) scaffolds generated by means of the original antisolvent 3D printing methodology. The cytotoxicity and cytocompatibility of the scaffolds were analyzed in vitro by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide tests, flow cytometry, and scanning electron microscopy. Anti-inflammatory and proangiogenic properties of the scaffolds were evaluated in the dorsal skinfold chamber mouse model by means of intravital fluorescence microscopy, histology, and immunohistochemistry throughout an observation period of 14 days. In vitro, none of the scaffolds revealed cytotoxicity on days 1, 2, and 5 after seeding with umbilical cord-derived multipotent stromal cells, and the primary cell adhesion to th...

Post-thawing characterization: Cell viability was assessed through a membrane integrity assay and... more Post-thawing characterization: Cell viability was assessed through a membrane integrity assay and the metabolic assay alamarBlue TM . The structural integrity and addition over the cells addition over the cells g y g gy y y gy

Problems of Cryobiology and Cryomedicine, 2008

В настоящее время возросла важность развития криобанков стволовых клеток в связи с их расширенным... more В настоящее время возросла важность развития криобанков стволовых клеток в связи с их расширенным изучением и терапевтическим применением. Однако, наряду с другими факторами, вышеуказанная терапия ограничена высокой чувствительностью стволовых клеток к процедурам замораживания-оттаивания. Необходимы как новые подходы к криоконсервированию и связанным с ним методам оценки, так и новые технологии для долгосрочного хранения большого количества стволовых клеток. В настоящей работе мы представляем некоторые улучшенные методы криоконсервирования стволовых клеток, например замораживание эмбриональных стволовых клеток человека с использованием гелеобразного матрикса. Мы представляем результаты применения разработанных на базе микросистемной техники новых криосубстратов и устройств, а также описываем новые методы оценки и результаты изучения циклов температурного стресса.

Cryobiology

The gold standard in cryopreservation is still conventional slow freezing of single cells or smal... more The gold standard in cryopreservation is still conventional slow freezing of single cells or small aggregates in suspension, although major cell loss and limitation to non-specialized cell types in stem cell technology are known drawbacks. The requirement for rapidly available therapeutic and diagnostic cell types is increasing constantly. In the case of human induced pluripotent stem cells (hiPSCs) or their derivates, more sophisticated cryopreservation protocols are needed to address this demand. These should allow a preservation in their physiological, adherent state, an efficient re-cultivation and upscaling upon thawing towards high-throughput applications in cell therapies or disease modelling in drug discovery. Here, we present a novel vitrification-based method for adherent hiPSCs, designed for automated handling by microfluidic approaches and with ready-to-use potential e.g. in suspension-based bioreactors after thawing. Modifiable alginate microcarriers serve as a growth surface for adherent hiPSCs that were cultured in a suspension-based bioreactor and subsequently cryopreserved via droplet-based vitrification in comparison to conventional slow freezing. Soft (0.35 %) versus stiff (0.65 %) alginate microcarriers in concert with adhesion time variation have been examined. Findings revealed specific optimal conditions leading to an adhesion time and growth surface (matrix) elasticity dependent hypothesis on cryo-induced damaging regimes for adherent cell types. Deviations from the found optimum parameters give rise to membrane ruptures assessed via SEM and major cell loss after adherent vitrification. Applying the optimal conditions, droplet-based vitrification was superior to conventional slow freezing. A decreased microcarrier stiffness was found to outperform stiffer material regarding cell recovery, whereas the stemness characteristics of rewarmed hiPSCs were preserved.

ACS Applied Bio Materials

One of the major environmental problems is a global metal contamination. Heavy metals are nonbiod... more One of the major environmental problems is a global metal contamination. Heavy metals are nonbiodegradable and tend to accumulate in living organisms. Therefore, searching for biocompatible materia...

Acta Biomaterialia

Pathophysiological conditions, such as myocardial infarction and mechanical overload affect the m... more Pathophysiological conditions, such as myocardial infarction and mechanical overload affect the mammalian heart integrity, leading to a stiffened fibrotic tissue. With respect to the pathophysiology of cardiac fibrosis but also in the limelight of upcoming approaches of cardiac cell therapy it is of interest to decipher the interaction of cardiomyocytes with fibrotic matrix. Therefore, we designed a hydrogel-based model to engineer fibrotic tissue in vitro as an approach to predict the behavior of cardiomyocytes facing increased matrix rigidity. Here, we generated pure induced pluripotent stem cell-derived cardiomyocytes and cultured them on engineered polyacrylamide hydrogels matching the elasticities of healthy as well as fibrotic cardiac tissue. Only in cardiomyocytes cultured on matrices with fibrotic-like elasticity, transcriptional profiling revealed a substantial up-regulation of a whole panel of cardiac fibrosis-associated transcripts, including collagen I and III, decorin, lumican, and periostin. In addition, matrix metalloproteinases and their inhibitors, known to be essential in cardiac remodeling, were found to be elevated as well as insulin-like growth factor 2. Control experiments with primary cardiac fibroblasts were analyzed and did not show comparable behavior. In conclusion, we do not only present a snapshot on the transcriptomic fingerprint alterations in cardiomyocytes under pathological conditions but also provide a new reproducible approach to study the effects of fibrotic environments to various cell types. STATEMENT OF SIGNIFICANCE: The ageing population in many western countries is faced with an increasing burden of ageing-related diseases such as heart failure which is associated with cardiac fibrosis. A deeper understanding of the interaction of organotypic cells with altered extracellular matrix mechanical properties is of pivotal importance to understand the underlying mechanisms. Here, we present a strategy to combine hydrogel matrices with induced pluripotent stem cell derived cardiomyocytes to study the effect of matrix stiffening on these cells. Our findings suggest an active role of matrix stiffening on cardiomyocyte function and heart failure progression.

International Ophthalmology

The aim of this study was to investigate the clinical outcome after standardized DMEK using a gla... more The aim of this study was to investigate the clinical outcome after standardized DMEK using a glass injector. Methods A total of 254 patients undergoing DMEK surgery using a disposable DMEK borosilicate glass cartridge system were included in this retrospective study. The mean follow-up time was 13.2 months (SD ± 8.1, range 6-36 months). The used glass cartridge system has an aperture diameter of 1.6 mm and a posterior loading orifice of 4.29 mm. Scanning electron microscopy (SEM) was used for estimation of the surface relief of the glass cartridge and comparison with a standard plastic injector cartridge. Results Mean endothelial cell count of donor grafts was 2465 cells/mm 2 (SD ± 199). After 6 weeks of DMEK endothelial cell count decreased by-28.6% to 1759 cells/mm 2 (SD ± 435) (Wilcoxon p = 0.001) and remained stable at the final follow-up at 1735 cells/mm 2 (SD ± 442) (Wilcoxon p = 0.89). SEM showed smoother surface of the glass cartridge in comparison with a plastic cartridge. Conclusion This study showed that this simple and effective DMEK cartridge seems to be a safe and viable device for minimized graft manipulation during DMEK surgery.

Stem cells translational medicine, Jan 19, 2018

Human induced pluripotent stem cells (hiPSCs) are an important tool for research and regenerative... more Human induced pluripotent stem cells (hiPSCs) are an important tool for research and regenerative medicine, but their efficient cryopreservation remains a major challenge. The current gold standard is slow-rate freezing of dissociated colonies in suspension, but low recovery rates limit immediate post-thawing applicability. We tested whether ultrafast cooling by adherent vitrification improves post-thawing survival in a selection of hiPSCs and small molecule neural precursor cells (smNPCs) from Parkinson's disease and controls. In a dual-center study, we compared the results by immunocytochemistry (ICC), fluorescence-activated cell sorting analysis, and RNA-sequencing (RNA-seq). Adherent vitrification was achieved in the so-called TWIST substrate, a device combining cultivation, vitrification, storage, and post-thawing cultivation. Adherent vitrification resulted in preserved confluency and significantly higher cell numbers, and viability at day 1 after thawing, while results we...

Scanning, Jan 16, 2016

One of the often reported artefacts during cell preparation to scanning electron microscopy (SEM)... more 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 ...

Biomaterials, 2014

Cardiomyocytes (CMs) from induced pluripotent stem (iPS) cells mark an important achievement in t... more Cardiomyocytes (CMs) from induced pluripotent stem (iPS) cells mark an important achievement in the development of in vitro pharmacological, toxicological and developmental assays and in the establishment of protocols for cardiac cell replacement therapy. Using CMs generated from murine embryonic stem cells and iPS cells we found increased cell-matrix interaction and more matured embryoid body (EB) structures in iPS cell-derived EBs. However, neither suspension-culture in form of purified cardiac clusters nor adherence-culture on traditional cell culture plastic allowed for extended culture of CMs. CMs grown for five weeks on polystyrene exhibit signs of massive mechanical stress as indicated by α-smooth muscle actin expression and loss of sarcomere integrity. Hydrogels from polyacrylamide allow adapting of the matrix stiffness to that of cardiac tissue. We were able to eliminate the bottleneck of low cell adhesion using 2,5-Dioxopyrrolidin-1-yl-6-acrylamidohexanoate as a crosslinker to immobilize matrix proteins on the gels surface. Finally we present an easy method to generate polyacrylamide gels with a physiological Young&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s modulus of 55 kPa and defined surface ligand, facilitating the culture of murine and human iPS-CMs, removing excess mechanical stresses and reducing the risk of tissue culture artifacts exerted by stiff substrates.