Human interferon-alpha 2c (IFN-alpha 2c) was produced in Escherichia coli under the control of the alkaline phosphatase promoter using a periplasmic expression system. Compared with other leader sequences, the heat-stable enterotoxin II leader of E. coli (STII) resulted in the highest rate of correct processing as judged by Western-blot analysis. The fermentation was designed as a batch-fed process in order to obtain a high yield of biomass. The processing rate of IFN-alpha 2c could be increased from 25% to more than 50% by shifting the fermentation pH from 7.0 to 6.7. IFN-alpha 2c extracted from the periplasm was purified by a new four-step chromatographic procedure. Whereas cytoplasmically produced IFN-alpha 2c does not have its full native structure, IFN-alpha 2c extracted from the periplasm was found to be correctly folded, as shown by c.d. spectroscopy. Peptide-map analysis in combination with m.s. revealed the correct formation of disulphide bridges. N-terminal sequence analysis showed complete removal of the leader sequence, creating the authentic N-terminus starting with cysteine.
In cooperation with BAXTER Vaccine AG, which supplies incubated special pathogen-free chicken eggs (including a full veterinary record), a permanent hen's egg chorio-allantoic membrane test (HET-CAM) unit has been established, where angiogenesis testing, cell culture, and digital and histological analyses are performed. At the Core Unit for Biomedical Research, the location of the animal testing facility of the Medical University Vienna, cell–scaffold constructs must be evaluated in vitro and in ovo prior to eventual in vivo tissue engineering experiments. The animal testing advisory committee requires that new test proposals are first evaluated by using cell culture and HET-CAM models. Approvals for in vivo experiments are postponed and not issued prior to in vitro/in ovo evaluation. Examples are presented of protocols planned for in vivo studies on cell seeded scaffolds, which were refined after in vitro/in ovo evaluations.
The implantation of new biomedical devices into living animals without any previous toxicity or biocompatibility evaluation is possible under current legislation. The HET–CAM (Hen Egg Test–Chorionallantoic Membrane) test offers a partially immunodeficient, borderline in vitro/in vivo test system that allows the simulation of transplantation experiments to obtain biocompatibility data prior to animal testing. A collagen type I/III scaffold, designed for tissue regeneration, was tested for angiogenetic properties and biocompatibility patterns. A significant angiogenetic stimulus caused by the collagen scaffold material was observed. Altering biocompatibility patterns by incubation with the potentially hazardous chemicals acridine orange and ethidium bromide led to severe vessel thrombosis and a foreign body tissue response. CAM testing of biomaterials and tissue engineered products allows selection of the most suitable biomaterial and the elimination of unsuitable materials from animal experiments, leading to a refinement of testing procedures and a reduction in the number of animals required for biocompatibility testing.
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