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.