Ursula Anderer

In vitro engineering of human autogenous cartilage.

A challenge in tissue engineering is the in vitro generation of human cartilage. To meet standards for in vitro‐engineered cartilage, such as prevention of immune response and structural as well as functional integration to surrounding tissue, we established a three‐dimensional cell culture system without adding exogenous growth factors or scaffolds. Human chondrocytes were cultured as spheroids. Tissue morphology and protein expression was analyzed using histological and immunohistochemical investigations on spheroid cryosections. A cartilage‐like tissue similar to naturally occurring cartilage was generated when spheroids were cultured in medium supplemented only with human serum. This in vitro tissue was characterized by the synthesis of the hyaline‐specific proteins collagen type II and S‐100, as well as the synthesis of hyaline‐specific mucopolysaccharides that increased with prolonged culture time. After 3 months, cell number in the interior of in vitro tissues was diminished and was only twice as much as in native cartilage. Additionally, spheroids quickly adhered to and migrated on glass slides and on human condyle cartilage. The addition of antibiotics to autologous spheroid cultures inhibited the synthesis of matrix proteins. Remarkably, replacing human serum by fetal calf serum resulted in the destruction of the inner part of the spheroids and only a viable rim of cells remained on the surface. These results show that the spheroid culture allows for the first time the autogenous in vitro engineering of human cartilage‐like tissue where medium supplements were restricted to human serum.

Automatic Analysis of Immunofluorescence Patterns of HEp-2 Cells

Abstract:  The standard screening test for the recognition of autoimmune diseases is the proof of autoantibodies in serum of patients by indirect immunofluorescence (IIF) based on HEp‐2 cells. Manual evaluation of this test is very subjective, slow, and there are no objective parameters as guidelines available. Interlaboratory tests showed occasionally large deviations in the test evaluation resulting in a high variance of results. The aim of this project is fast, objective, safe, and economical automatic analysis of HEp‐2 IIF patterns. Images of IIF patterns were completely and automatically captured using an inverse motorized fluorescence microscope. Thereby, device‐specific parameters were controlled automatically, too. For fast analysis of IIF patterns new algorithms of image processing were developed. Artifacts were recognized and excluded from analysis by the developed software. Analysis of more than 80,000 images clearly demonstrated full automatization and fast processing of IIF patterns. Additionally serum‐specific fluorescence could be easily distinguished from background. Even very weak but positive patterns can be recognized and used for diagnosis. A detailed separation into different basic patterns is possible. Objective, fast, and disease‐related economical analysis of HEp‐2 immunofluorescence patterns is feasible. The implemented software algorithms allowed a mathematical way of describing IIF patterns and can therefore be a useful tool for the needed standardization process.

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.

Three-dimensional scaffold-free fusion culture: the way to enhance chondrogenesis of in vitro propagated human articular chondrocytes.

Cartilage regeneration based on isolated and culture-expanded chondrocytes has been studied in various in vitro models, but the quality varies with respect to the morphology and the physiology of the synthesized tissues. The aim of our study was to promote in vitro chondrogenesis of human articular chondrocytes using a novel three-dimensional (3-D) cultivation system in combination with the chondrogenic differentiation factors transforming growth factor beta 2 (TGF-b2) and L-ascorbic acid. Articular chondrocytes isolated from six elderly patients were expanded in monolayer culture. A single-cell suspension of the dedifferentiated chondrocytes was then added to agar-coated dishes without using any scaffold material, in the presence, or absence of TGF-b2 and/or L-ascorbic acid. Three-dimensional cartilage-like constructs, called single spheroids, and microtissues consisting of several spheroids fused together, named as fusions, were formed. Generated tissues were mainly characterized using histological and immunohistochemical techniques. The morphology of the in vitro tissues shared some similarities to native hyaline cartilage in regard to differentiated S100-positive chondrocytes within a cartilaginous matrix, with strong collagen type II expression and increased synthesis of proteoglycans. Finally, our innovative scaffold-free fusion culture technique supported enhanced chondrogenesis of human articular chondrocytes in vitro. These 3-D hyaline cartilage-like microtissues will be useful for in vitro studies of cartilage differentiation and regeneration, enabling optimization of functional tissue engineering and possibly contributing to the development of new approaches to treat traumatic cartilage defects or osteoarthritis.

Establishment of HEp‐2 cell preparation for automated analysis of ANA fluorescence pattern

Identification of antinuclear antibodies (ANAs) has large clinical importance for the assessment of autoimmune diseases. HEp‐2 cell preparations on microscopic slides are commonly used as antigenic substrate. Methods used for cell preparation are important for ANA pattern analysis; however, these methods differ widely and are mostly not specified.

Primary‐like Human Hepatocytes Genetically Engineered to Obtain Proliferation Competence Display Hepatic Differentiation Characteristics in Monolayer and Organotypical Spheroid Cultures

Primary human hepatocytes are in great demand during drug development and in hepatology. However, both scarcity of tissue supply and donor variability of primary cells create a need for the development of alternative hepatocyte systems. By using a lentivirus vector system to transfer coding sequences of Upcyte® proliferation genes, we generated non‐transformed stable hepatocyte cultures from human liver tissue samples. Here, we show data on newly generated proliferation‐competent HepaFH3 cells investigated as conventional two‐dimensional monolayer and as organotypical three‐dimensional (3D) spheroid culture. In monolayer culture, HepaFH3 cells show typical cobblestone‐like hepatocyte morphology and anchorage‐dependent growth for at least 20 passages. Immunofluorescence staining revealed that characteristic hepatocyte marker proteins cytokeratin 8, human serum albumin, and cytochrome P450 (CYP) 3A4 were expressed. Quantitative real‐time PCR analyses showed that expression levels of analyzed phase I CYP enzymes were at similar levels compared to those of cultured primary human hepatocytes and considerably higher than in the liver carcinoma cell line HepG2. Additionally, transcripts for phase II liver enzymes and transporter proteins OATP‐C, MRP2, Oct1, and BSEP were present in HepaFH3. The cells produced urea and converted model compounds such as testosterone, diclofenac, and 7‐OH‐coumarin into phases I and II metabolites. Interestingly, phases I and II enzymes were expressed at about the same levels in convenient monolayer cultures and complex 3D spheroids. In conclusion, HepaFH3 cells and related primary‐like hepatocyte lines seem to be promising tools for in vitro research of liver functions and as test system in drug development and toxicology analysis.

Biphasic influence of PGE2 on the resorption activity of osteoclast-like cells derived from human peripheral blood monocytes and mouse RAW264.7 cells

Osteoclasts are large bone-resorbing cells of hematopoietic origin. Their main function is to dissolve the inorganic component hydroxyapatite and to degrade the organic bone matrix. Prostaglandin E2 (PGE2) indirectly affects osteoclasts by stimulating osteoblasts to release factors that influence osteoclast activity. The direct effect of PGE2 on osteoclasts is still controversial. To study the influence of PGE2 on osteoclast activity, human peripheral blood monocytes (hPBMC) and mouse RAW264.7 cells were cultured on osteoblast-derived extracellular matrix. hPBMC and RAW264.7 cells were differentiated by the addition of macrophage colony-stimulation factor and receptor activator of NFκB ligand and treated with PGE2 before and after differentiation induction. The pit area, an indicator of resorption activity, and the activity of tartrate-resistant acid phosphatase were dose-dependently inhibited when PGE2 was present ab initio, whereas the resorption activity remained unchanged when the cells were exposed to PGE2 from day 4 of culture. These results lead to the conclusion that PGE2 treatment inhibits only the differentiation of precursor osteoclasts whereas differentiated osteoclasts are not affected.

Featured Article: In vitro 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 statement A 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.

Applying XTT, WST-1, and WST-8 to human chondrocytes: A comparison of membrane-impermeable tetrazolium salts in 2D and 3D cultures

BACKGROUND Tetrazolium-based assays are optimized to assess proliferation/toxicity of monolayer or suspension cells in microtiter plates. With regard to tissue engineering and regenerative medicine the need for in vivo like 3D microtissues has an increasing relevance. Applying tetrazolium-based assays to 3D culture systems is technically more challenging. The composed microenvironment may influence the assay standards, e.g. equal distribution of tetrazolium. OBJECTIVE Evaluation of membrane-impermeable tetrazolium salt-based assays with regard to spheroid culture (3D) of human chondrocytes. METHODS Chondrocytes were isolated from human articular cartilage. XTT, WST-1, and WST-8 were applied to monolayer cells (2D, varying cell numbers) and spheroids (3D, different sizes) in 96well plates. Formazan formation was measured spectrophotometrically after different incubation periods. Evaluation was done using phase contrast microsopy (toxicity), analyzing the correlation of cell number and absorbance signals (Gompertz function), and document signal over background ratio. RESULTS In monolayer culture the assays showed a correlation between seeded cell numbers and absorption data. Spheroid sizes are directly related to the starting cell number. A correlation between size and absorbance was only detectable starting from 10,000 cells/aggregate. Phase contrast microscopy of monolayer cells revealed strong toxicity effects of the WST-1 (4 h) and XTT (8 h) assay and no signs of toxicity using WST-8. CONCLUSION The WST-8 assay is non-toxic and revealed the highest sensitivity in comparison to the XTT or WST-1 assay. There is evidence, that only cells of the outer rim of spheroids are able to convert membrane-impermeable tetrazolium salts to formazans.

Correction: Simultaneous Automated Screening and Confirmatory Testing for Vasculitis-Specific ANCA

The second affiliation for the tenth author is not indicated. Pier Luigi Meroni is also affiliated with: IRCCS Istituto Auxologico Italiano, Milan, Italy.