Nuclear transplantation between allogeneic cells through topological reconnection of plasma membrane in a microfluidic system

Abstract

Previous studies have demonstrated that somatic cells fused with pluripotent stem cells can be reprogrammed on the basis of reprogramming factors acquired from the latter. However, fusion-reprogrammed cells are deemed unsuitable for therapeutic applications mainly because conventional fusion techniques often yield tetraploid fusants that contain exogenous genes acquired from the fusion partners. Here, we present a novel cell–cell topological reconnection technique and demonstrate its application to nuclear transplantation between a somatic cell and a stem cell without nuclei mixing. As a proof of concept, a microfluidic fusion chip embodied with a microslit (4 μ m in width) to prevent nuclei mixing was developed and used to perform one-to-one electrofusion of a target somatic cell (Jurkat cell) with an induced pluripotent stem (iPS) cell. To extract its cytoplasm, the target cell was first topologically connected to a sacrificial iPS cell by electrofusion via a microslit, followed by shear flow removal of the latter to obtain a cytoplasm-depleted nucleus of the target cell. Then, to replace the lost cytoplasm, topological reconnection to a second iPS cell was performed similarly by electrofusion, followed by shear flow separation of the target cell to enable it acquire most of the iPS cytoplasm, but without nuclei mixing. Microscopic observation of target cells harvested and cultured post hoc in a microwell confirmed that they manifested cell division. Taken together, these results demonstrate the potential application of the cell–cell topological reconnection technique to somatic cell nuclear transplantation for the generation of autologous pluripotent stem cells.

A single‐cell surgery microfluidic device for transplanting tumor cytoplasm into dendritic cells without nuclei mixing

“…Overall, consistent with our previous studies. [ 29,33 ] fusion efficiency was determined to be ∼ 80%, based on the ratio of successfully fused cells (exhibiting both green and orange fluorescence) to the number of pairs formed at the microorifices.…”

“…Overall, consistent with our previous studies. [29,33] narrower compared to the size of the cell nucleus (> 5 μm), the nuclei of the fused cells could not pass through even during shear flow reseparation. [25,28] Indeed, Jurkat cells remained trapped at the microorifices after reseparation, as depicted in Figure 2C.…”

“…Cell fusion was performed essentially as described previously. [29,33] Briefly, labelled cells suspended in fusion buffer were drawn into the PDMS chip by a micropipette. The PDMS device was precoated with BSA to prevent cell adhesion during loading.…”