New Platform Technology for Comprehensive Serological Diagnostics of Autoimmune Diseases

Antibody assessment is an essential part in the serological diagnosis of autoimmune diseases. However, different diagnostic strategies have been proposed for the work up of sera in particular from patients with systemic autoimmune rheumatic disease (SARD). In general, screening for SARD-associated antibodies by indirect immunofluorescence (IIF) is followed by confirmatory testing covering different assay techniques. Due to lacking automation, standardization, modern data management, and human bias in IIF screening, this two-stage approach has recently been challenged by multiplex techniques particularly in laboratories with high workload. However, detection of antinuclear antibodies by IIF is still recommended to be the gold standard method for antibody screening in sera from patients with suspected SARD. To address the limitations of IIF and to meet the demand for cost-efficient autoantibody screening, automated IIF methods employing novel pattern recognition algorithms for image analysis have been introduced recently. In this respect, the AKLIDES technology has been the first commercially available platform for automated interpretation of cell-based IIF testing and provides multiplexing by addressable microbead immunoassays for confirmatory testing. This paper gives an overview of recently published studies demonstrating the advantages of this new technology for SARD serology

Generation of Scaffold Free 3-D Cartilage-Like Microtissues from Human Chondrocytes

Since our society is characterized by an increasing age of its people on the one hand and a high number of persons dealing with sports on the other hand, the number of patients suffering from traumatic defects or osteoarthritis is growing. In combination with the articular cartilage specific limited capacity to regenerate, a need for suitable therapies is obvious. Thereby, cell-based therapies are of major interest. This type of clinical intervention was introduced to patients at the beginning of the 1990s. During the last years, a technological shift from simple cell suspensions to more complex 3D structures was performed. In order to optimize the scaffold free generation of cartilage, such as microtissues from human chondrocytes, the authors examine the influence of a static or spinner flask culture with respect to differentiation and architecture of the engineered microtissues. Additionally, the impact of the soluble factors TGF-ß2 and ascorbic acid on this process are investigated. The results demonstrate a positive impact of TGF-ß2 and ascorbic acid supplementation with respect to general Type II Collagen and proteoglycan expression for both the static and spinner flask culture.</jats:p

Featured Article:<i>In vitro</i>development of personalized cartilage microtissues uncovers an individualized differentiation capacity of human chondrocytes

Personalized features in the treatment of knee injuries and articular replacement therapies play an important role in modern life with increasing demand. Therefore, cell-based therapeutic approaches for the regeneration of traumatic defects of cartilage tissue were developed. However, great variations in the quality of repair tissue or therapeutic outcome were observed. The aim of the study was to capture and visualize individual differentiation capacities of chondrocytes derived from different donors with regard to a possible personal regeneration capacity using a cell-based therapy. The redifferentiation potential of monolayer cultured cells was analyzed in a scaffold-free three-dimensional tissue model. Furthermore, stimulating options using cartilage maturation factors such as L-ascorbic acid and transforming growth factor beta 2 (TGF-β2) on this process were of special interest. Cells and tissues were analyzed via histological and immunohistochemical methods. Gene expression was measured by quantitative real-time polymerase chain reaction. In monolayer culture, cells from all donors showed an almost identical differentiation profile. In contrast, the differentiation state of cartilage-like three-dimensional microtissues revealed clear differences with respect to individual donors. Analyses at the protein and mRNA levels showed high variations regarding cartilage-typical matrix components (e.g. proteoglycans, collagen type II) and intracellular proteins (e.g. S100). Interestingly, only donor chondrocytes with a basic tendency to re-differentiate in a three-dimensional environment were able to increase this tissue-specific maturation when exposed to L-ascorbic acid and/or TGF-β2. Our approach revealed clear-cut possibilities for classification of individual donors into responders or non-responders. On the basis of these results an in vitro platform could be designed to discriminate responders from non-responders. This in vitro three-dimensional test system may be a suitable basis to establish a “personalized diagnostic tool” with the opportunity to assess the capacity of expanded chondrocytes to respond to an autologous cell-based therapy.Impact statementA challenge in cell-based cartilage regeneration therapies is the identification of a “personalized diagnostic tool” to predict the chondrogenic potency of cells from patients who are going to be treated with autologous cells. Comparing the phenotype of isolated chondrocytes from different donors in vitro revealed an individual cartilage-specific differentiation capacity. These personalized features are not detectable in vitro until the monolayer cells have the possibility to rearrange in 3D tissues. Cells from articular cartilage in monolayer culture may not be a suitable basis to discriminate responders from non-responders with respect to a personalized cell-based therapy to treat cartilage defects. A more physiological 3D (micro-)environment enable the cells to present their individual differentiation capacity. The here described microtissue model might be the basis for an in vitro platform to predict the therapeutic outcome of autologous cell-based cartilage repair and/or a suitable tool to identify early biomarkers to classify the patients.</jats:sec

Die HEp-2-Zelle als Target für multiparametrische Autoantikörperanalytik – Automatisierung und Standardisierung/The HEp-2 cell as target for multiparametric autoantibody analyses: automation and standardisation

Zusammenfassung Trotz Einführung von Multiplex-Assays ist die Bestimmung von Autoantikörpern (AAK) mittels indirekter Immunfluoreszenz (IIF) nach wie vor ein wesentlicher Bestandteil der Autoimmundiagnostik. Das Screening auf nichtorganspezifische AAK wird in der Routinediagnostik von autoimmunen rheumatischen Erkrankungen mittels indirekter Immunfluoreszenz an HEp-2-Zellen durchgeführt. Leider differieren die mit diesem Test erhaltenen Befunde z. T. recht erheblich auf Grund von objektiven (z. B. HEp-2-Zellfixierung) und subjektiven (z. B. Erfahrungswerte) Faktoren. Die Intra- und Interlaborvarianzen sind daher relativ hoch. Erste Ergebnisse von Evaluierungsstudien eines neu entwickelten Systems zur automatischen Bildanalyse (AKLIDES®) zeigen einen Weg in Richtung Standardisierung (bzw. Harmonisierung) des HEp-2-Zell-Assays auf, in dem zunächst subjektive Fehlinterpretationen vermieden werden können. Eine weitere Harmonisierung erfordert die Weiterentwicklung der Software-Algorithmen zur Mustererkennung sowie neue Kalibrationssysteme.</jats:p

Tissue Specific Differentiation of Human Chondrocytes Depends on Cell Microenvironment and Serum Selection

Therapeutic options to cure osteoarthritis (OA) are not yet available, although cell-based therapies for the treatment of traumatic defects of cartilage have already been developed using, e.g., articular chondrocytes. In order to adapt cell-based therapies to treat OA, appropriate cell culture conditions are necessary. Chondrocytes require a 3-dimensional (3D) environment for redifferentiation after 2-dimensional (2D) expansion. Fetal bovine serum (FBS) is commonly used as a medium supplement, although the usage of a xenogeneic serum could mask the intrinsic behavior of human cells in vitro. The aim of this study was to compare human articular chondrocytes cultivated as monolayers (2D) and the development of microtissues (3D) in the presence of FBS with those cultivated with human serum (HS). Evaluation of the expression of various markers via immunocytochemistry on monolayer cells revealed a higher dedifferentiation degree of chondrocytes cultivated with HS. Scaffold-free microtissues were generated using the agar overlay technique, and their differentiation level was evaluated via histochemistry and immunohistochemistry. Microtissues cultivated in the medium with FBS showed a higher redifferentiation level. This was evidenced by bigger microtissues and a more cartilage-like composition of the matrix with not any/less positivity for cartilage-specific markers in HS versus moderate-to-high positivity in FBS-cultured microtissues. The present study showed that the differentiation degree of chondrocytes depends both on the microenvironment of the cells and the serum type with FBS achieving the best results. However, HS should be preferred for the engineering of cartilage-like microtissues, as it rather enables a "human-based" situation in vitro. Hence, cultivation conditions might be further optimized to gain an even more adequate and donor-independent redifferentiation of chondrocytes in microtissues, e.g., designing a suitable chemically-defined serum supplement