Yield increases in intact influenza vaccine virus from chicken allantoic fluid through isolation from insoluble allantoic debris.

A yield enhancement technology for use in influenza vaccine manufacturing has been developed to maximize the recovery of influenza virus from allantoic fluid of virus-infected chick embryos; the standard raw material for influenza vaccine. Virus associated with amorphous debris in the allantoic fluid can be dissociated from the debris and recovered, thereby increasing viral yield. Dissociation can be achieved by subjecting the virus-debris complex to conditions of increased ionic strength at defined pH. Multifold increases in viral yield per ml of allantoic fluid were observed. The degree of yield enhancement is strain-specific, however, increases were observed in all type A and type B influenza strains tested. The heightened influenza virus recoveries can facilitate rapid vaccine manufacture, with increased numbers of doses produced, and may become essential at a time of influenza pandemic.

A small stress protein acts synergistically with trehalose to confer desiccation tolerance on mammalian cells.

The ability to desiccate mammalian cells while maintaining a high degree of viability would be very important in many areas of biological science, including tissue engineering, cell transplantation, and biosensor technologies. Certain proteins and sugars found in animals capable of surviving desiccation might aid this process. We report here that human embryonic kidney (293H) cells transfected with the gene for the stress protein p26 from Artemia and loaded with trehalose showed a sharp increase in survival during air-drying. Further, we find vacuum-drying greatly improved the ability of the cells to survive, and that the physical shape and structure of the cellular sample had a large influence on recovery following rehydration. Cells suspended in a rounded droplet survived desiccation markedly better than those spread as a thin film. Finally, we used alamarBlue to monitor cellular metabolism and Hema 3 to assess colony formation after vacuum-drying. AlamarBlue fluorescence indicated that the transfected 293H cells expressing p26 (E11'L) grew much better than the control 293H cells. In fact, immediate survival and colony formation in E11'L cells increased as much as 34-fold compared with control cells when the samples were dried to a water content of 0.2 g H2O/g dry weight, as measured by gravimetric analysis. These results indicate that p26 improves cell survival following drying and rehydration, and suggest that dry storage of mammalian cells is a likely possibility in the future.

High-density seeding of myocyte cells for cardiac tissue engineering.

Tissue engineering of 1- to 5-mm-thick, functional constructs based on cells that cannot tolerate hypoxia for prolonged time periods (e.g., cardiac myocytes) critically depends on our ability to seed the cells at a high and spatially uniform initial density and to maintain their viability and function. We hypothesized that rapid gel-cell inoculation in conjunction with direct medium perfusion through the seeded scaffold would increase the rate, yield, viability, and uniformity of cell seeding. Two cell types were studied: neonatal rat cardiomyocytes for feasibility studies of seeding and cultivation with direct medium perfusion, and C2C12 cells (a murine myoblast cell line) for detailed seeding studies. Cells were seeded at densities corresponding to those normally present in the adult rat heart ([0.5-1] x 10(8) cells/cm(3)), into collagen sponges (13 mm x 3 mm discs), using Matrigel as a vehicle for rapid cell delivery. Scaffolds inoculated with cell-gel suspension were seeded either in perfused cartridges with alternating medium flow or in orbitally mixed Petri dishes. The effects of seeding time (1.5 or 4.5 h), initial cell number (6 or 12 million cells per scaffold), and seeding set-up (medium perfusion at 0.5 and 1.5 mL/min; orbitally mixed dishes) were investigated using a randomized three-factor factorial experimental design with two or three levels and three replicates. The seeding cell yield was consistently high (over 80%), and it appeared to be determined by the rapid gel inoculation. The decrease in cell viability was markedly lower for perfused cartridges than for orbitally mixed dishes (e.g., 8.8 +/- 0.8% and 56.3 +/- 4%, respectively, for 12 million cells at 4.5 h post-seeding). Spatially uniform cell distributions were observed in perfused constructs, whereas cells were mainly located within a thin (100-200 microm) surface layer in dish seeded constructs. Over 7 days of cultivation, medium perfusion maintained the viability and differentiated function of cardiac myocytes, and the constructs contracted synchronously in response to electrical stimulation. Direct perfusion can thus enable seeding of hypoxia-sensitive cells at physiologically high and spatially uniform initial densities and maintain cell viability and function.