Botulinum hemagglutinin-mediated in situ break-up of human induced pluripotent stem cell aggregates for high-density suspension culture.

Large numbers of human induced pluripotent stem cells (hiPSCs) are required for making stable cell bank. Although suspension culture yields high cell numbers, there remain unresolved challenges for obtaining high-density of hiPSCs because large size aggregates exhibit low growth rates. Here, we established a simple method for hiPSC aggregate break-up using botulinum hemagglutinin (HA), which specifically bound with E-cadherin and disrupted cell-cell connections in hiPSC aggregates. HA showed temporary activity for disrupting the E-cadherin-mediated cell-cell connections to facilitate the break-up of aggregates into small sizes only 9 hr after HA addition. The transportation of HA into the aggregates was mediated by transcellular and paracellular way after HA addition to the culture medium. hiPSC aggregates broken up by HA showed a higher number of live cells, higher cell density, and higher expansion fold compared to those of aggregates dissociated with enzymatic digestion. Moreover, a maximum cell density of 4.5 ± 0.2 × 106 cells ml-1 was obtained by aggregate break-up into small ones, which was three times higher than that with the conventional culture without aggregate break-up. Therefore, the temporary activity of HA for disrupting E-cadherin-mediated cell-cell connection was key to establishing a simple in situ method for hiPSC aggregate break-up in bioreactors, leading to high cell density in suspension culture.

Mesoscale modelling of polymer aggregate digestion.

We use mesoscale simulations to gain insight into the digestion of biopolymers by studying the break-up dynamics of polymer aggregates (boluses) bound by physical cross-links. We investigate aggregate evolution, establishing that the linking bead fraction and the interaction energy are the main parameters controlling stability with respect to diffusion. We show via a simplified model that chemical breakdown of the constituent molecules causes aggregates that would otherwise be stable to disperse. We further investigate breakdown of biopolymer aggregates in the presence of fluid flow. Shear flow in the absence of chemical breakdown induces three different regimes depending on the flow Weissenberg number ( W i ). i) At W i ≪ 1 , shear flow has a negligible effect on the aggregates. ii) At W i ∼ 1 , the aggregates behave approximately as solid bodies and move and rotate with the flow. iii) At W i ≫ 1 , the energy input due to shear overcomes the attractive cross-linking interactions and the boluses are broken up. Finally, we study bolus evolution under the combined action of shear flow and chemical breakdown, demonstrating a synergistic effect between the two at high reaction rates.