Christiane Wetzel

Quality assessment of corneal storage media and their components

BackgroundTo keep the loss of endothelial cell density in donor corneas to a minimum, a storage medium which is adjusted to their nutritional needs is necessary. Different media, used either serum-supplemented or serum-free, are available. The quality of medium- and serum-batches as well as support of endothelial cell viability by the medium are to be tested with a quality assured screening system that allows routine examination.MethodsA screening system was developed which is based on cell-culture tests with the well-established human corneal endothelial cell line HCEC-12, and therefore can be performed without the need for donor corneas. The cells are plated at a defined density in cell-culture dishes, and are cultured for a defined period of time in the test media. Evaluation is carried out by assaying cell count, activity of cell metabolism (resazurin conversion), and determining the number of apoptotic and necrotic cells (combined vital staining with YO-PRO®-1/propidium iodide and subsequent flow cytometry).ResultsHuman corneal endothelial cells that are cultured in a medium which is adjusted to their nutritional needs achieve higher cell numbers and show a higher metabolic rate. Simultaneously, the percentage of apoptotic and necrotic cells is lower. The screening system developed in this study allows for easy and reliable detection of slightest differences between different media, different processing steps for same media, and different supplements, as well as different serum batches.ConclusionsThe differentiated results show that the screening system is sensitive enough to show even minor quality differences. Therefore, it is more suitable than the hitherto commonly used growth assay with primary, mostly porcine, corneal endothelial cells.

Nondestructive testing of electron beam sterilization by means of an optically active marker material

Secure proof of sterilization processes on packaging materials is an important issue in many economic sectors. In this context, electron beam sterilization is a highly effective low temperature technique. However, verifying the application of a sufficient electron dose is still difficult - especially on products with complex geometry. Here we report on an optical, hence fast and contactless approach which gives reliable evidence of a successful e-beam treatment. The technique is based on placing a suitable marker material (rare-earth based particles) inside or as a coating on the packaging material. By electron irradiation these particles change their optical properties and thus indicate the successful application of the electron beam.

Pathogens Inactivated by Low-Energy-Electron Irradiation Maintain Antigenic Properties and Induce Protective Immune Responses

Inactivated vaccines are commonly produced by incubating pathogens with chemicals such as formaldehyde or β-propiolactone. This is a time-consuming process, the inactivation efficiency displays high variability and extensive downstream procedures are often required. Moreover, application of chemicals alters the antigenic components of the viruses or bacteria, resulting in reduced antibody specificity and therefore stimulation of a less effective immune response. An alternative method for inactivation of pathogens is ionizing radiation. It acts very fast and predominantly damages nucleic acids, conserving most of the antigenic structures. However, currently used irradiation technologies (mostly gamma-rays and high energy electrons) require large and complex shielding constructions to protect the environment from radioactivity or X-rays generated during the process. This excludes them from direct integration into biological production facilities. Here, low-energy electron irradiation (LEEI) is presented as an alternative inactivation method for pathogens in liquid solutions. LEEI can be used in normal laboratories, including good manufacturing practice (GMP)- or high biosafety level (BSL)-environments, as only minor shielding is necessary. We show that LEEI efficiently inactivates different viruses (influenza A (H3N8), porcine reproductive and respiratory syndrome virus (PRRSV), equine herpesvirus 1 (EHV-1)) and bacteria (Escherichia coli) and maintains their antigenicity. Moreover, LEEI-inactivated influenza A viruses elicit protective immune responses in animals, as analyzed by virus neutralization assays and viral load determination upon challenge. These results have implications for novel ways of developing and manufacturing inactivated vaccines with improved efficacy.