Models and Assays
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Barrier Models

Respiratory barrier

The assay uses respiratory cells (Calu-3, A549) on transwell membranes cultured in an air-liquid interface. Cells can be exposed to liquids, to nebulized aerosols in the VITROCELL® system and to manually generated aerosols using MicroSprayerÒ IA-1C Aerosolizer and DP-4 Dry Powder Insufflator™. The models are used for Papp testing, uptake and transport rates, and cytotoxicity studies.

Blood-brain barrier

In addition to the existing static model based on transwell membranes a dynamic system using hollow fibre CELLMAX DUO® bioreactor with dynamic TEER measurement is being developed. The system uses hcMED/D3 microvascular epithelial cells in co-culture with primary astrocytes.

 

     

 

 

 

Cytotoxicity: Acute and Chronic and Mode of Action Studies

Interference with cell function can be identified by cytotoxicity screening assays, such as MTS, MTT, WST-1, ATP-content, sulforhodamine B, neutral red, as well as by microscopy-based techniques (cell monitoring).

For testing of medical products detection of endotoxin and testing of cytotoxicity following ISO 10993-5 guidelines is offered.

More detailed information on the mode of interference with cell function is studied by assays for necrosis, apoptosis, and proliferation. These assays include LDH, AK, Calcein AM/PI, caspase 3/7 activation, TUNEL, M30, cleaved caspase 3, 3H-thymidine, BrdU, and cell cycle analysis.

Alterations of cell metabolism can also be due to interference with organelle function, which can be identified by Mitotracker, TMRM, JC-1, LysoTracker, LysoSensor, acridine orange, Lyso-ID, lysosomal sulfatase, and cathepsin B staining.

Oxidative stress as frequent cause for cell dysfunction is identified by using dihydroethidium, dihydrochlorofluorescein acetate, heme oxigenase-1 staining and depletion of GSH as indicators.

 

Effects of prolonged exposure over 14-28d can be studied by culture on microcarriers in the benchtop bioreactor BioLevitator™ 3D cell culture system. This system is also suitable to propagate cells to high densities.

Membrane-based transwell systems are available for long-term observation of respiratory cells in air-liquid interface culture.

 

     

 

 

Hemocompatibility and Immunotoxicity Testing

Adverse effects on blood, hemolysis and changes in hemostasis, can be identified using Hb-release and interference with plasmatic clotting and platelet activation is identified by F1.2, TAT, D-dimer, CD62P/CD42b, b-thromboglobulin detection and platelet aggregation.

For identification of interaction with the cellular (monocytes, macrophages, neutrophilic granulocytes) and the acellular unspecific immune system complement 3a and 5a, NO-detection, oxidative burst, E. coli K12-bioparticle phagocytosis, phagoburst, CD11b/CD15 and chemotaxis is performed. Mitogen-activated lymphocyte proliferation is used to assess lymphocyte function and cytokine release, either in conventional ELISA or as multiplex assays to study inflammation.