Cell Monitoring and Manipulation Systems (CMMSs) based on Glass Cell-Culture Chips (GC³s).

We developed different types of glass cell-culture chips (GC³s) for culturing cells for microscopic observation in open media-containing troughs or in microfluidic structures. Platinum sensor and manipulation structures were used to monitor physiological parameters and to allocate and permeabilize cells. Electro-thermal micro pumps distributed chemical compounds in the microfluidic systems. The integrated temperature sensors showed a linear, Pt1000-like behavior. Cell adhesion and proliferation were monitored using interdigitated electrode structures (IDESs). The cell-doubling times of primary murine embryonic neuronal cells (PNCs) were determined based on the IDES capacitance-peak shifts. The electrical activity of PNC networks was detected using multi-electrode arrays (MEAs). During seeding, the cells were dielectrophoretically allocated to individual MEAs to improve network structures. MEA pads with diameters of 15, 20, 25, and 35 µm were tested. After 3 weeks, the magnitudes of the determined action potentials were highest for pads of 25 µm in diameter and did not differ when the inter-pad distances were 100 or 170 µm. Using 25-µm diameter circular oxygen electrodes, the signal currents in the cell-culture media were found to range from approximately -0.08 nA (0% O2) to -2.35 nA (21% O2). It was observed that 60-nm thick silicon nitride-sensor layers were stable potentiometric pH sensors under cell-culture conditions for periods of days. Their sensitivity between pH 5 and 9 was as high as 45 mV per pH step. We concluded that sensorized GC³s are potential animal replacement systems for purposes such as toxicity pre-screening. For example, the effect of mefloquine, a medication used to treat malaria, on the electrical activity of neuronal cells was determined in this study using a GC³ system.

Cellular Nutrition in Complex Three-Dimensional Scaffolds: A Comparison between Experiments and Computer Simulations.

Studies on bone cell ingrowth into synthetic, porous three-dimensional (3D) implants showed difficulties arising from impaired cellular proliferation and differentiation in the core region of these scaffolds with increasing scaffold volume in vitro. Therefore, we developed an in vitro perfusion cell culture module, which allows the analysis of cells in the interior of scaffolds under different medium flow rates. For each flow rate the cell viability was measured and compared with results from computer simulations that predict the local oxygen supply and shear stress inside the scaffold based on the finite element method. We found that the local cell viability correlates with the local oxygen concentration and the local shear stress. On the one hand the oxygen supply of the cells in the core becomes optimal with a higher perfusion flow. On the other hand shear stress caused by high flow rates impedes cell vitality, especially at the surface of the scaffold. Our results demonstrate that both parameters must be considered to derive an optimal nutrient flow rate.

Design and Characterization of a Sensorized Microfluidic Cell-Culture System with Electro-Thermal Micro-Pumps and Sensors for Cell Adhesion, Oxygen, and pH on a Glass Chip.

We combined a multi-sensor glass-chip with a microfluidic channel grid for the characterization of cellular behavior. The grid was imprinted in poly-dimethyl-siloxane. Mouse-embryonal/fetal calvaria fibroblasts (MC3T3-E1) were used as a model system. Thin-film platinum (Pt) sensors for respiration (amperometric oxygen electrode), acidification (potentiometric pH electrodes) and cell adhesion (interdigitated-electrodes structures, IDES) allowed us to monitor cell-physiological parameters as well as the cell-spreading behavior. Two on-chip electro-thermal micro-pumps (ETµPs) permitted the induction of medium flow in the system, e.g., for medium mixing and drug delivery. The glass-wafer technology ensured the microscopic observability of the on-chip cell culture. Connecting Pt structures were passivated by a 1.2 µm layer of silicon nitride (Si3N4). Thin Si3N4 layers (20 nm or 60 nm) were used as the sensitive material of the pH electrodes. These electrodes showed a linear behavior in the pH range from 4 to 9, with a sensitivity of up to 39 mV per pH step. The oxygen sensors were circular Pt electrodes with a sensor area of 78.5 µm(2). Their sensitivity was 100 pA per 1% oxygen increase in the range from 0% to 21% oxygen (air saturated). Two different IDES geometries with 30- and 50-µm finger spacings showed comparable sensitivities in detecting the proliferation rate of MC3T3 cells. These cells were cultured for 11 days in vitro to test the biocompatibility, microfluidics and electric sensors of our system under standard laboratory conditions.

Generation of physiological parameter sets for hip joint motions and loads during daily life activities for application in wear simulators of the artificial hip joint.

At present, wear investigations of total hip replacements are performed in accordance with the ISO standard 14242, which is based on simplified kinematic and force data of the gait cycle. The aim of this analytical study was to generate parameter sets of daily life activities in order to replicate more realistic joint load situations in wear testing. Hence, published in vivo motion and force data of daily life activities were evaluated and adjusted using analytical techniques. The created kinematically and dynamically consistent parameter sets comprised time trajectories of three Cardan angles to describe the motion of the femur with respect to the pelvis and time trajectories of three force components, representing the hip joint contact force. The parameter sets include the activities of walking, knee bending, stair climbing and a combined load case of sitting down and standing up. Additionally, a motion sequence following the frequency of daily life activities was presented. Differences of the evaluated angular motions and joint contact forces in comparison to the ISO standard 14242-1 were pointed out. The results of this study offer the possibility to extend the kinematics and dynamics of the ISO standard test protocol and to support the loading conditions of hip wear simulators with a comprehensive set of motions and loads close to reality.

A novel modular device for 3-D bone cell culture and non-destructive cell analysis.

Synthetic materials have emerged as bone substitutes for filling bone defects of critical sizes. Because bone healing requires a mechanically resistant matrix (scaffold) attractive to osteogenic cells and must allow revascularization for nutrient and oxygen supply, scaffold-based strategies focus on the further development of chemical and physical qualities of the material. Cellular ingrowth towards the scaffold center is critical; therefore selective information from inner regions, in particular from the central part, is essential. In this paper we introduce a novel modular in vitro system for three-dimensional (3-D) in vitro bone cell cultures. This 3-D system is developed exclusively for in vitro research purposes, with special emphasis on the geometrical scaffold design (pore size, pore design). The system is composed of a stack of titanium slices which are mounted on a clamp and which enable the separate monitoring of cell growth patterns on every single slice of the slide stack. In this way we are able to gain selective information about the regulation of the cell physiology in the inner part of the 3-D construct which can be used for the development of an optimized scaffold design for orthopedic implants.

Calcium phosphate surfaces promote osteogenic differentiation of mesenchymal stem cells.

Although studies in vivo revealed promising results in bone regeneration after implantation of scaffolds together with osteogenic progenitor cells, basic questions remain how material surfaces control the biology of mesenchymal stem cells (MSC). We used human MSC derived from bone marrow and studied the osteogenic differentiation on calcium phosphate surfaces. In osteogenic differentiation medium MSC differentiated to osteoblasts on hydroxyapatite and BONITmatrix, a degradable xerogel composite, within 14 days. Cells revealed a higher alkaline phosphatase (ALP) activity and increased RNA expression of collagen I and osteocalcin using real-time RTPCR compared with cells on tissue culture plastic. To test whether material surface characteristics alone are able to stimulate osteogenic differentiation, MSC were cultured on the materials in expansion medium without soluble additives for osteogenic differentiation. Indeed, cells on calcium phosphate without osteogenic differentiation additives developed to osteoblasts as shown by increased ALP activity and expression of osteogenic genes, which was not the case on tissue culture plastic. Because we reasoned that the stimulating effect on osteogenesis by calcium phosphate surfaces depends on an altered cell-extracellular matrix interaction we studied the dynamic behaviour of focal adhesions using cells transfected with GFP labelled vinculin. On BONITmatrix, an increased mobility of focal adhesions was observed compared with cells on tissue culture plastic. In conclusion, calcium phosphate surfaces are able to drive MSC to osteoblasts in the absence of osteogenic differentiation supplements in the medium. An altered dynamic behaviour of focal adhesions on calcium phosphate surfaces might be involved in the molecular mechanisms which promote osteogenic differentiation.