Scalable GMP compliant suspension culture system for human ES cells.

Suspension bioreactors are an attractive alternative to static culture of human embryonic stem cells (hESCs) for the generation of clinically relevant cell numbers in a controlled system. In this study, we have developed a scalable suspension culture system using serum-free defined media with spinner flasks for hESC expansion as cell aggregates. With optimized cell seeding density and splitting interval, we demonstrate prolonged passaging and expansion of several hESC lines with overall expansion, yield, viability and maintenance of pluripotency equivalent to adherent culture. Human ESCs maintained in suspension as aggregates can be passaged at least 20 times to achieve over 1×10(13) fold calculated expansion with high undifferentiation rate and normal karyotype. Furthermore, the aggregates are able to differentiate to cardiomyocytes in a directed fashion. Finally, we show that the cells can be cryopreserved in serum-free medium and thawed into adherent or suspension cultures to continue passaging and expansion. We have successfully used this method under cGMP or cGMP-equivalent conditions to generate cell banks of several hESC lines. Taken together, our suspension culture system provides a powerful approach for scale-up expansion of hESCs under defined and serum-free conditions for clinical and research applications.

Effective generation of transgenic pigs and mice by linker based sperm-mediated gene transfer.

BACKGROUND: Transgenic animals have become valuable tools for both research and applied purposes. The current method of gene transfer, microinjection, which is widely used in transgenic mouse production, has only had limited success in producing transgenic animals of larger or higher species. Here, we report a linker based sperm-mediated gene transfer method (LB-SMGT) that greatly improves the production efficiency of large transgenic animals. RESULTS: The linker protein, a monoclonal antibody (mAb C), is reactive to a surface antigen on sperm of all tested species including pig, mouse, chicken, cow, goat, sheep, and human. mAb C is a basic protein that binds to DNA through ionic interaction allowing exogenous DNA to be linked specifically to sperm. After fertilization of the egg, the DNA is shown to be successfully integrated into the genome of viable pig and mouse offspring with germ-line transfer to the F1 generation at a highly efficient rate: 37.5% of pigs and 33% of mice. The integration is demonstrated again by FISH analysis and F2 transmission in pigs. Furthermore, expression of the transgene is demonstrated in 61% (35/57) of transgenic pigs (F0 generation). CONCLUSIONS: Our data suggests that LB-SMGT could be used to generate transgenic animals efficiently in many different species.