Automated Closed-System Expansion of Pluripotent Stem Cell Aggregates in a Rocking-Motion Bioreactor.

Pluripotent stem cell suspension aggregates have proven to be an efficient and phenotypically stable means for expansion and directed differentiation. Bioreactor systems with automation of perfusion, fluidization, and gas exchange are essential for scaling up pluripotent stem cell cultures. Since stem cell pluripotency and differentiation are affected by both chemical and physical signals, we investigated a low-shear method for the expansion of cells in a rocking-motion bioreactor. The rocking motion drives continual mixing and aeration, and the single-use disposable bioreactors avoid issues around contamination during seeding, medium exchange, passage, and cell harvest. Serial passaging from a 150 mL to a 1 L scale was demonstrated, achieving cell densities of up to 4 million cells/mL. In an average of 13 experiments, pluripotent stem cell aggregates expanded 5.7-fold (with maximal 9.5-fold expansion) and maintained 97% viability over 4 days in a rocking bioreactor culture. In seven experiments with improved culture conditions, the average expansion was 6.8-fold. Maintenance of pluripotency was confirmed by differentiation to all three germ layers and surface marker expression, and the expanded aggregates maintained a stable normal karyotype. The automation associated with the rocking platform bioreactor required no user intervention during the 4-day culture, providing hands-off expansion of pluripotent stem cells.

Plasma membrane temperature gradients and multiple cell permeabilization induced by low peak power density femtosecond lasers.

Calculations indicate that selectively heating the extracellular media induces membrane temperature gradients that combine with electric fields and a temperature-induced reduction in the electropermeabilization threshold to potentially facilitate exogenous molecular delivery. Experiments by a wide-field, pulsed femtosecond laser with peak power density far below typical single cell optical delivery systems confirmed this hypothesis. Operating this laser in continuous wave mode at the same average power permeabilized many fewer cells, suggesting that bulk heating alone is insufficient and temperature gradients are crucial for permeabilization. This work suggests promising opportunities for a high throughput, low cost, contactless method for laser mediated exogenous molecule delivery without the complex optics of typical single cell optoinjection, for potential integration into microscope imaging and microfluidic systems.