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

Abstract

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 × 10 cells ml 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.

Bioengineering Considerations for a Nurturing Way to Enhance Scalable Expansion of Human Pluripotent Stem Cells

“…This method stimulates proliferation through disruption of cell‐cell contact within compacted aggregation and achieves higher hiPSC yields in well‐preserved pluripotency. A recent study reported that temporary disruption of E‐cadherin‐mediated cell–cell adhesion using HA is a simple in situ method for hiPSC aggregate break‐up in bioreactors (Figure ) . HA‐mediated break‐up of hiPSC aggregates showed a higher number of live cells and higher expansion fold compared with aggregates dissociated using enzymatic digestion.…”

“…Another recent study demonstrated that spontaneous deviation in hiPSC colony center was suppressed by relaxation of E‐cadherin‐mediated adherens junctions via addition of botulinum hemagglutinin (HA), an E‐cadherin function‐blocking agent. It was found that cell adhesion and cytoskeletal rearrangements occurred in hiPSC colonies by promoting cell migration through HA‐mediated relaxation of cell–cell junctions . The coordinated regulation of balance between cell–cell and cell–substrate interactions through cell migration leads to the prevention of nuclear laminA/C accumulation in hiPSC colony center .…”

“…iii) Maintenance of an undifferentiated state of hiPSCs through botulinum hemagglutinin (HA)‐mediated regulation of cell behaviors . B) Large‐scale expansion culture of hPSCs through HA‐mediated regulation of cell behavior . The temporal E‐cadherin disruption by HA has a key role in establishing a simple in situ method for hiPSC aggregate break‐up in bioreactors, enabling high‐density culture.…”

Integrated Biologics Manufacturing in Stirred-Suspension Bioreactor: A Stem Cell Perspective

“…Non-enzymatic detachment is also available by changing temperature or pH [18][19][20]. Aggregate culture in bioreactors may not necessarily need a detachment step for harvesting [21][22][23][24][25]. Next steps are washing and volume reduction, which can be done by centrifugation or tangential flow filtration on a large scale by using automated commercial devices (kSep systems and Terumo BCT).…”

“…Bioreactor expansion of hPSCs on microcarrier is troublesome for clinical application because it needs an extra step for microcarrier separation from the final cell harvest. On the other hand, aggregate culture in bioreactors may not necessarily need a detachment step for harvesting [21][22][23][24][25] and clinically relevant numbers of cells can be produced in a single bioreactor as aggregate [21,[106][107][108].…”