Development of a High-Throughput Screening Assay Based on the 3-Dimensional Pannus Model for Rheumatoid Arthritis

Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG geförderten) Allianz- bzw. Nationallizenz frei zugänglich.This publication is with permission of the rights owner freely accessible due to an Alliance licence and a national licence (funded by the DFG, German Research Foundation) respectively.The 3-dimensional (3-D) pannus model for rheumatoid arthritis (RA) is based on the interactive co-culture of cartilage and synovial fibroblasts (SFs). Besides the investigation of the pathogenesis of RA, it can be used to analyze the active profiles of antirheumatic pharmaceuticals and other bioactive substances under in vitro conditions. For a potential application in the industrial drug-screening process as a transitional step between 2-dimensional (2-D) cell-based assays and in vivo animal studies, the pannus model was developed into an in vitro high-throughput screening (HTS) assay. Using the CyBi™-Disk workstation for parallel liquid handling, the main cell culture steps of cell seeding and cultivation were automated. Chondrocytes were isolated from articular cartilage and seeded directly into 96-well microplates in high-density pellets to ensure formation of cartilage-specific extracellular matrix (ECM). Cell seeding was performed automatically and manually to compare both processes regarding accuracy, reproducibility, consistency, and handling time. For automated cultivation of the chondrocyte pellet cultures, a sequential program was developed using the CyBio Control software to minimize shear forces and handling time. After 14 days of cultivation, the pannus model was completed by coating the cartilage pellets with a layer of human SFs. The effects due to automation in comparison to manual handling were analyzed by optical analysis of the pellets, histological and immunohistochemical staining, and real-time PCR. Automation of this in vitro model was successfully achieved and resulted in an improved quality of the generated pannus cultures by enhancing the formation of cartilage-specific ECM. In addition, automated cell seeding and media exchange increased the efficiency due to a reduction of labor intensity and handling time. (Journal of Biomolecular Screening 2007:956-965)BMBF, 0313604A, Verbundprojekt: Evaluierung eines interagierenden 3D Testsystems als Krankheitsmodell der rheumatoiden Arthritis (in vitro Pannus Modell) zur effektiven Prüfung von Wirkstoffen, Teilprojekt 1BMBF, 0313604B, Verbundprojekt: Entwicklung eines interagierenden 3D Testsystems als Krankheitsmodell der rheumatoiden Arthritis (in vitro Pannus Modell) zur effektiven Prüfung von Wirkstoffen, Teilprojekt

Microcarrier-based expansion of primary cells for tissue engineering in the bioreactor

Die vorgestellte Arbeit liefert neue Erkenntnisse zur mikroträgerbasierten Expansion von primären mesenchymalen Stammzellen (MSC) und Chondrozyten im Spinnerflaschen-Bioreaktor. Diese Expansionsmethode stellt eine viel versprechende Alternative zur herkömmlichen 2-dimensionalen Monolayerkultur von Primärzellen im Rahmen des Tissue Engineering dar. Zusätzliche Vorteile verspricht die Verwendung von bioresorbierbaren Mikroträgern, welche zusammen mit den expandierten Zellen in die Transplantatherstellung eingehen können. Hierzu war es notwendig, Mikroträger aus PLGA selbst herzustellen und entsprechend ihrer Form und Oberflächenbeschaffenheit, Größe und Größenverteilung, sowie anhand ihres Degradationsverhaltens zu charakterisieren. Zur Etablierung der Methode wurden auch kommerzielle Cytodex Mikroträger verwendet. Neben der erstmaligen Isolierung von MSC mittels Mikroträgern sollte die Adhärenz und Proliferation der Zellen auf den Trägern gezeigt werden. Anschließend wurde das Differenzierungspotential der 3-dimensional expandierten Zellen in osteogene und chondrogene Richtung überprüft. Die PLGA Mikroträger konnten erfolgreich als Kugeln mit glatter Oberfläche in der gewünschten Größe von 100-200µm hergestellt werden. Das Degradationsverhalten entsprach insbesondere beim PLGA 0500 Material, welches aus einem 85:15 Verhältnis an PLA zu PGA besteht, den gewünschten Kriterien. Eine sehr gute Adhärenz der Zellen konnte bereits nach 3h auf allen Mikroträgern nachgewiesen werden, wobei Cytodex 1 die höchsten Werte erreichte. Die direkte Isolierung der in der Zellzahl limitierten MSC mit Hilfe von Mikroträgern konnte erstmals gezeigt werden. Die Zellspezies hatte wenig Einfluss auf die Adhärenz, während eine Adaption an Monolayerbedingungen eine Steigerung der adhärenten Zellen bewirkte. Eine Proliferation war für alle untersuchten Zelltypen nachweisbar, blieb jedoch hinter der Monolayerkultur zurück. Eine Optimierung der mikroträgerbasierten Methode könnte die Wachstumsraten entsprechend verbessern. Das Expansionspotential der mikroträgerbasierten Kultur war durch die gezeigte Rekolonisation frischer Carrier ohne Limitierung und stellt somit einen entscheidenden Vorteil dieser Methode dar. Das Differenzierungspotential der Zellen blieb während der 3D-Expansion erhalten. Die grundsätzliche Verwendbarkeit von resorbierbaren PLGA Mikroträgern konnte gezeigt werden. Insgesamt ist die mikroträgerbasierte Expansion von Zellen im Spinnerflaschen-Bioreaktor eine Erfolg versprechende Methode zur in vitro Vermehrung von primären Zellen.This work provides new insights into the microcarrier-based expansion of primary mesenchymal stem cells (MSC) and chondrocytes in a spinner flask bioreactor. This expansion method displays a promising alternative to the conventional 2-dimensional monolayer culture in the context of tissue engineering. The application of bioresorbable microcarriers, which, combined with the expanded cells, can be used for transplantation, promises additional advantages. For this reason, it was necessary to produce microcarriers from a PLGA polymer and to characterise them in terms of shape and surface structure, size and size distribution, and degradation behaviour. For implementation of the new expansion method, commercially available cytodex microcarriers were used. Next to the isolation of MSC with carriers, the adhesion and proliferation of the cells on the microcarriers was shown. Subsequently, the differentiation potential of the 3-dimensionally expanded cells was evaluated for the osteogenic and chondrogenic lineage. The PLGA microcarriers could be produced successfully as spheres with a plane surface structure in the desired size of 100-200µm diameter. The degradation behaviour was satisfying especially for the PLGA 0500 material, which consists of 85% PLA. Very good adhesion of the cells to the carriers was distinguished after 3h of incubation whereas cytodex type 1 showed the highest number of adherent cells. Additionally, the isolation of MSC via microcarriers from bone marrow could be demonstrated. Porcine and human cell types behaved the same on behalf of adhesion, whereas an additional passage caused an increase of adherent cells. Proliferation was detectable for all cell types but revealed higher generation times compared to the monolayer culture. An optimisation of the microcarrier-based method could improve the expansion rate. One advantage of this method is the unlimited expansion potential due to recolonisation of freshly added microcarriers, which could be presented clearly. The differentiation potential of the cells was maintained during the 3-dimensional expansion. The usability of biodegradable PLGA microcarriers for these purposes could be shown. Totalling the results, the microcarrier-based expansion in spinner flasks is a promising method for the in vitro progeny of primary cells

Toward biomimetic materials in bone regeneration : functional behavior of mesenchymal stem cells on a broad spectrum of extracellular matrix components

Bone defect treatments can be augmented by mesenchymal stem cell (MSC) based therapies. MSC interaction with the extracellular matrix (ECM) of the surrounding tissue regulates their functional behavior. Understanding of these specific regulatory mechanisms is essential for the therapeutic stimulation of MSC in vivo. However, these interactions are presently only partially understood. This study examined in parallel, for the first time, the effects on the functional behavior of MSCs of 13 ECM components from bone, cartilage and hematoma compared to a control protein, and hence draws conclusions for rational biomaterial design. ECM components specifically modulated MSC adhesion, migration, proliferation, and osteogenic differentiation, for example, fibronectin facilitated migration, adhesion, and proliferation, but not osteogenic differentiation, whereas fibrinogen enhanced adhesion and proliferation, but not migration. Subsequently, the integrin expression pattern of MSCs was determined and related to the cell behavior on specific ECM components. Finally, on this basis, peptide sequences are reported for the potential stimulation of MSC functions. Based on the results of this study, ECM component coatings could be designed to specifically guide cell functions