Adhesion patterning by a novel air-lock technique enables localization and in-situ real-time imaging of reprogramming events in one-to-one electrofused hybrids

Abstract

Although fusion of somatic cells with embryonic stem (ES) cells has been shown to induce reprogramming, single-cell level details of the transitory phenotypic changes that occur during fusion-based reprogramming are still lacking. Our group previously reported on the technique of one-to-one electrofusion via micro-slits in a microfluidic platform. In this study, we focused on developing a novel air-lock patterning technique for creating localized adhesion zones around the micro-slits for cell localization and real-time imaging of post fusion events with a single-cell resolution. Mouse embryonic fibroblasts (MEF) were fused individually with mouse ES cells using a polydimethylsiloxane (PDMS) fusion chip consisting of two feeder channels with a separating wall containing an array of micro-slits (slit width ∼3 μm) at a regular spacing. ES cells and MEFs were introduced separately into the channels, juxtaposed on the micro-slits by dielectrophoresis and fused one-to-one by a pulse voltage. To localize fused cells for on-chip culture and time-lapse microscopy, we implemented a two-step approach of air-lock bovine serum albumin patterning and Matrigel coating to create localized adhesion areas around the micro-slits. As a result of time-lapse imaging, we could determine that cell division occurs within 24 h after fusion, much earlier than the 2–3 days reported by earlier studies. Remarkably, Oct4-GFP (Green Fluorescent Protein) was confirmed after 25 h of fusion and thereafter stably expressed by daughter cells of fused cells. Thus, integrated into our high-yield electrofusion platform, the technique of air-lock assisted adhesion patterning enables a single-cell level tracking of fused cells to highlight cell-level dynamics during fusion-based reprogramming.

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

“…One-to-one electrofusion, which essentially involves application of a potential to cause a reversible breakdown of the cell membrane, is attractive since it enables cell fusion easily and without chemical or genetic contaminations. In fact, in our previous study, we demonstrated the possibility of reprogramming mouse embryonic fibroblasts (MEFs) by one-to-one electrofusion with mouse ES cells 19 . Cell division and OCT4-GFP (green fluorescent protein) expression by MEF was confirmed within 25 h of fusion 19 .…”

“…In fact, in our previous study, we demonstrated the possibility of reprogramming mouse embryonic fibroblasts (MEFs) by one-to-one electrofusion with mouse ES cells 19 . Cell division and OCT4-GFP (green fluorescent protein) expression by MEF was confirmed within 25 h of fusion 19 . However, in this case, the nuclei of the hybrids/fusants could slip through the microslit and form heterokaryons during the M-phase, when the nuclear membrane breaks down.…”

Recent Advances in Microscale Electroporation

“…(D) Schematic process of one-to-one cell pairing at microslits for fusion using a channel constriction. Reproduced with permission from ref . Copyright 2016 AIP publishing.…”

Advances in hybridoma preparation using electrofusion technology

“…Moreover, the chip solves the problem of separating fused cells from unfused cells and realizes the visual monitoring of the cell electrofusion process. In 2016, the same group improved the separating step of fused cells from unfused cells by developing a new "air-lock patterning technique", which creates an "adhesion zone" on the micro-orifice bottom [20] . The advantage of this technique is that the fused cells will be located in the "adhesion zone" (Figure .…”

“…Part B is reprinted with permission from Gel et al [19]. Part C is reprinted with permission from Sakamono et al [20].…”