Production of retroviral vectors for gene therapy with the human packaging cell line FLYRD18.

The development of gene therapy is hampered by the difficulty of producing large stocks of retroviral vectors at high titer. This study aimed to improve culture conditions and to intensify the production of retroviruses by FLYRD18, a packaging cell line derived from the HT1080 human fibrosarcoma line. Batch virus production proved to be feasible in unsupplemented basal medium and provided significantly higher titers and productivities than medium supplemented with 10% serum. For longer-term production, however, AIM-V complete serum-free medium and basal medium supplemented with 2% serum gave superior results. Serum supplementation should nevertheless be optimized to take into account the presence of inhibitors of viral production. In monolayer cultures with 0.2 mL/cm(2), the cell concentration was increased up to 2 x 10(6) cells/mL without loss of cell productivity. A semicontinuous production process, which enables the collection of larger amounts of viruses from the same culture, has also been successfully used. Suspension culture processes were prevented by the anchorage dependency of the FLYRD18 cell line. Microcarrier cultures were able to produce viruses but will require further investigation and optimization for their performance to become competitive with monolayer cultures. In the course of this study, more than a 10-fold increase of titer has been achieved.

Direct probing of the surface ultrastructure and molecular interactions of dormant and germinating spores of Phanerochaete chrysosporium.

Atomic force microscopy (AFM) has been used to probe, under physiological conditions, the surface ultrastructure and molecular interactions of spores of the filamentous fungus Phanerochaete chrysosporium. High-resolution images revealed that the surface of dormant spores was uniformly covered with rodlets having a periodicity of 10 +/- 1 nm, which is in agreement with earlier freeze-etching measurements. In contrast, germinating spores had a very smooth surface partially covered with rough granular structures. Force-distance curve measurements demonstrated that the changes in spore surface ultrastructure during germination are correlated with profound modifications of molecular interactions: while dormant spores showed no adhesion with the AFM probe, germinating spores exhibited strong adhesion forces, of 9 +/- 2 nN magnitude. These forces are attributed to polysaccharide binding and suggested to be responsible for spore aggregation. This study represents the first direct characterization of the surface ultrastructure and molecular interactions of living fungal spores at the nanometer scale and offers new prospects for mapping microbial cell surface properties under native conditions.

Improved titers of retroviral vectors from the human FLYRD18 packaging cell line in serum- and protein-free medium.

The influence of serum on the production of retroviral vectors by the HT1080 human fibrosarcoma-derived packaging cell line FLYRD18 was investigated. A fourfold increase in virus titer was observed under serum-free conditions, as compared with medium supplemented with 10% fetal calf serum. A similar improvement was also seen for bulk transduction efficiency. Serum had a negative and dose-dependent effect on titer without affecting cell growth, virus stability, or infectivity. In contrast to virus from NIH 3T3-derived packaging cells [Hanenberg, H., et al. (1996). Nature Med. 2, 876-882], the FLYRD18-derived virus did not adhere to fibronectin or serum proteins adsorbed at the surface of culture flasks. Electron microscopy supports the conclusion that the effect of serum is at the level of virus production by the cells. Addition of soybean trypsin inhibitor had an inhibitory effect on virus production, while pretreatment of serum with trypsin was found to enhance the retroviral titer. These results suggest that protease inhibitors present in serum may be responsible for the inhibition of virus production. The exact mechanism remains, however, to be determined. As compared with medium supplemented with 10% serum, the combination of increased virus titer and absence of exogenous protein under serum-free conditions resulted in a 300-fold increase in the virus:total protein ratio in the supernatants harvested from the FLYRD18 packaging line. This improvement enhances prospects for further concentration and purification of the virus.

Detection of rodlets in the outer wall region of conidiospores of Phanerochaete chrysosporium.

The surface morphology of the conidiospores of Phanerochaete chrysosporium was investigated using freeze-etching. A multilayered structure composed of rodlets was detected. The rodlets had a diameter of 10.2 +/- 0.5 nm and were organised as long parallel fibres. Granules, smooth materials, and bark-like structures were found to cover part of this rodlet layer. During germination, the outer pellicle of the spore wall became fragmented and residual aggregates with rodlets were disseminated on the new conidiospore surface. The latter, as well as the germ tube, was composed of fibrillar material.

Surface properties of the conidiospores of Phanerochaete chrysosporium and their relevance to pellet formation.

The conidiospores of the white rot basidiomycete Phanerochaete chrysosporium tend to aggregate during swelling and germination in agitated liquid medium; as time passes, the initial aggregates tend to associate together and to capture conidiospores that remain isolated. The surface chemical compositions of the conidiospores and of developed hyphae were analyzed by X-ray photoelectron spectroscopy. The data were interpreted by modelling the surface in terms of proteins, polysaccharides and hydrocarbonlike compounds. The surface molecular composition of the dormant conidiospores was estimated to be about 45% proteins, 20% carbohydrates, and 35% hydrocarbonlike compounds. There was an increase in the polysaccharide content during germination. Later, when the hyphae were developed, the polysaccharide content became still higher, and the protein content dropped. The initial step of aggregation is attributed to polysaccharide bridging; its occurrence cannot be explained by a change of the overall hydrophobicity or electrical properties of the conidiospores.