Implantation of ultrathin, biofunctionalized polyimide membranes into the subretinal space of rats.

Subretinal implants aim to replace the photoreceptor function in patients suffering from degenerative retinal disease by topically applying electrical stimuli in the subretinal space. Critical obstacles in the design of high-resolution subretinal implants include the proximity of stimulating electrodes to the target cells and enabling nutrient flow between the retina and the choroid. The present work evaluates the adhesion, migration and survival of retinal cells on an ultrathin (5 µm), highly porous (Ø 1 µm spaced 3 µm), gelatin-coated polyimide (PI) membrane. The biocompatibility was examined in mice indicating a good tolerance upon subcutaneous implantation with only a mild inflammatory response. In addition, organotypic cultures of rat retina evidenced that the porous membrane allowed the necessary nutrient flow for the retinal cell survival and maintenance. A transscleral implantation technique was applied to position the membrane into the subretinal space of rats. The effect on the obtained retinal integration was investigated in vivo using scanning laser ophthalmoscopy (SLO) and optical coherence tomography (OCT). In 12 out of 18 rat eyes, the implant was successfully placed subretinally. SLO and OCT demonstrated complete retinal attachment and fluorescein angiography showed no retinal vascular abnormalities over and around the implant, immediately after and up to four weeks after the implantation. Histological examination of the eyes showed a close attachment of a thin fibrocyte layer to the implant, the occlusion of the pores by living cells and the survival of some photoreceptors at the implantation site.

Chip-based impedance measurement on single cells for monitoring sub-toxic effects on cell membranes.

There is a lack of methods suitable for generation of data about the dynamics of effects on cell membranes with a high sensitivity. Such methods are urgently needed to support the optimisation of interaction of substances, particles or materials with cell. The goal of this article is to use an improved microhole chip system to monitor the alterations of cells due to the interactions of polymer-DNA complexes. This should demonstrate exemplarily that subtoxic effect of biological relevant particles or substances at relevant concentrations can be monitored for several hours. By using a microhole cell chip and a microfluidic unit single cells can be electrically interfaced via microholes and the use of small electrodes with high impedances is not necessary. For separation and positioning of the cells onto the hole negative pressure is applied on the reverse side of the chip. Under cell culture conditions the cell starts to spread on the biocompatible insulating chip membrane resulting in a stable interface to an adherent growing cell. After the spreading process is finished, the polymer/polyplex solution is added and the impedance is measured with respect to time. To illustrate the cellular parameter which can affect the measured impedance a simple simulation based on the finite element method (FEM) is performed. It was shown for the first time that the impedance-based method predicated on the microhole chip can be used for biological relevant substances at relevant concentrations and that it is more sensitive than the well-established biological marker.

A scaffold-free in vitro model for osteogenesis of human mesenchymal stem cells.

For studying cellular processes three-dimensional (3D) in vitro models are of a high importance. For tissue engineering approaches osseous differentiation is performed on 3D scaffolds, but material depending influences promote cellular processes like adhesion, proliferation and differentiation. To investigate developmental processes of mesenchymal stem cells without cell-substrate interactions, self-contained in vitro models mimicking physiological condition are required. However, with respect to scientific investigations and pharmaceutical tests, it is essential that these tissue models are well characterised and are of a high reproducibility. In order to establish an appropriate in vitro model for bone formation, different protocols are compared and optimised regarding their aggregate formation efficiency, homogeneity of the aggregates, the viability and their ability to induce differentiation into the osteogenic lineage. The protocols for the generation of 3D cell models are based on rotation culture, hanging drop technique, and the cultivation in non adhesive culture vessels (single vessels as well as 96 well plates). To conclude, the cultivation of hMSCs in 96 well non adhesive plates facilitates an easy way to cultivate homogenous cellular aggregates with high performance efficiency in parallel. The size can be controlled by the initial cell density per well and within this spheroids, bone formation has been induced.

Detection of the osteogenic differentiation of mesenchymal stem cells in 2D and 3D cultures by electrochemical impedance spectroscopy.

Human mesenchymal stem cells are promising candidates for cell-based therapies since they have the capacity to differentiate into a variety of cell types. However, the acceptance of hMSCs for clinical applications as well as in vitro tissue models will depend on strategies for standard characterisations. Impedance spectroscopy is a proven and powerful tool for non-invasive monitoring of cellular processes. The aim of this study was to prove the hypothesis, that the process of osteogenic differentiation can be monitored non-invasively and time-continuously by using impedance spectroscopy. This hypothesis was examined for 2D cell layers of hMSCs by continuous impedance spectroscopy employing a planar electrode-based chip and for 3D aggregates of hMSCs after 21 and 25 days of osteogenic treatment by using a capillary measurement system. The impedance spectra of osteogenic treated hMSCs reported a significant increase of the magnitude of impedance compared to controls cultivated in normal growth medium. The osteogenic status of the cells was determined by alkaline phosphatase expression and von Kossa staining. In respect to that finding it is concluded that impedance spectroscopy is an appropriate method for non-invasive characterisation of osteogenic differentiation of hMSCs, which is relevant for quality control of cell-based implants and cell-based test systems for drug development.

Influence of cell culture media conditions on the osteogenic differentiation of cord blood-derived mesenchymal stem cells.

In this study the critical parameters directing osteogenic differentiation of umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) were investigated, key factors and conditions identified and improved protocols for a more cell-type adapted differentiation developed. Today only little information about the specific conditions directing osteogenic development is available and current protocols for cultivation and differentiation of UCB-MSCs are based mainly on experience with bone marrow-derived MSCs (BM-MSCs) without further adaptation. Thus, protocols for improved osteoinduction are of particular interest. The goal of this study was to investigate the influence of three different culture media (A) alpha MEM, 15% FBS, (B) DMEM, 15% FBS and (C) MSCGM, 10% SingleQuot growth supplement on the osteogenic differentiation of UCB-MSCs. Moreover, a systematic analysis of two concentrations of dexamethasone (10(-8)M/10(-7)M) in combination with or without BMP-2 (10(-7)M) was carried out by detecting the expression of alkaline phosphatase (ALP), collagen-1 and the mineralization of ECM. We found that MSCGM, 10% SingleQuot had a supportive effect on the osteogenic differentiation of UCB-MSCs. In case of treatment with 10(-8)M dexamethasone, mineralization occurred in combination with BMP-2 exclusively, while a concentration of 10(-7)M dexamethasone led to a high amount of mineralized ECM and the expression of collagen-1 independent of BMP-2 addition. According to this data dexamethasone is the leading osteoinductive factor, but BMP-2 seems to have supportive properties in UCB-MSCs. In conclusion, MSCGM supplemented with 10% SingleQuot and 10(-7)M dexamethasone was the condition identified to be best for inducing the osteogenic differentiation of UCB-MSCs.

Cathepsin B is essential for regeneration of scratch-wounded normal human epidermal keratinocytes.

Migration, proliferation and differentiation of keratinocytes are important processes during tissue regeneration and wound healing of the skin. Here, we focussed on proteases that contribute to extracellular matrix (ECM) remodeling as a prerequisite of keratinocyte migration. In particular, we assessed the significance of the mammalian cysteine peptidase cathepsin B for human keratinocytes during regeneration from scratch wounding. We describe the construction of a scratch apparatus that allows applying scratches of defined length, width and depth to cultured cells in a reproducible fashion. The rationale for our approach derived from our previous work where we have shown that HaCaT keratinocytes secrete cathepsin B into the extracellular space during spontaneous and induced migration. Here, we observed rapid removal of type IV collagen from underneath lamellipodial extensions of keratinocytes at the advancing fronts of regenerating monolayers, indicating that proteolytic ECM remodeling starts upon initiation of keratinocyte migration. Furthermore, we verified our previous results with HaCaT cells by using normal human epidermal keratinocytes (NHEK) and show that non-cell-permeant cathepsin B-specific inhibitors delayed full regeneration of the monolayers from scratch wounding in both cell systems, HaCaT and NHEK. Application of a single dose of cathepsin B inhibitor directly after scratch wounding of keratinocytes demonstrated that cathepsin B is essential during initial stages of wound healing, while its contribution to the subsequent processes of proliferation and differentiation of keratinocytes was of less significance. This notion was supported by our observation that the cathepsin B inhibitors used in this study did not affect proliferation rates of keratinocytes of regenerating cultures. Thus, we conclude that cathepsin B is indeed involved in ECM remodeling after its secretion from migrating keratinocytes. Cathepsin B might directly cleave ECM constituents or it may initiate proteolytic cascades that involve other proteases with the ability to degrade ECM components. Because cathepsin B is important for enabling migration of both, HaCaT cells and NHEK, our results support the notion that HaCaT keratinocytes represent an excellent cell culture model for analysis of human epidermal skin keratinocyte migration.

HaCaT keratinocytes secrete lysosomal cysteine proteinases during migration.

Cathepsin B, a lysosomal cysteine proteinase, was detected within vesicles of cellular protrusions forming cell-cell contact sites between keratinocytes of the stratum spinosum of human skin. This observation suggested the possibility that secretion of the protease into the pericellular spaces could be involved in the dissociation of cell-cell contacts to enable intraepidermal keratinocyte migration. To determine whether cathepsin B is indeed secreted from migrating keratinocytes, we first used subconfluent HaCaT cells as a culture model to study spontaneous keratinocyte migration. A cathepsin B-specific fluorescent affinity label proved the association of mature cathepsin B with the surfaces of HaCaT cells at the leading edges of growing cells. Second, we used scratch-wounds of confluent HaCaT monolayers as a model of induced keratinocyte migration. Cathepsin B was detected within lysosomes, i.e. vesicles within the perinuclear region of non-wounded cells. Expression of cathepsin B was up-regulated and cathepsin B-positive vesicles showed a redistribution from perinuclear to peripheral regions of keratinocytes at the wound margins within 4 h after wounding. Enzyme cytochemistry further showed that cell surface-associated cathepsin B was proteolytically active at the leading fronts of migrating keratinocytes. In addition, increased amounts of mature forms of cathepsin B were detected within the conditioned media of HaCaT cells during the first 4 h after scratch-wounding. In contrast, and as a control, the activity of the cytosolic enzyme lactate dehydrogenase was not significantly higher in media of wounded cells as compared with non-wounded controls, arguing for a specific induction of cathepsin B secretion upon wounding and migration of the cells. This was further substantiated by applying various cathepsin B-specific inhibitors after wounding. These experiments showed that the migration ability of keratinocytes was reduced due to the blockage of functional cathepsin B. Thus, our results strongly suggest that cell surface-associated cathepsin B is a protease that contributes to the remodelling of the extracellular matrix and thereby promotes keratinocyte migration during wound healing.