Adhesion patterning by a novel air-lock technique enables localization andin-situreal-time imaging of reprogramming events in one-to-one electrofused hybrids.
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 ∼3μm) 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.
Department of Bioengineering, School of Engineering, The University of Tokyo , Tokyo 113-3656, Japan.
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Sakamoto
Department of Mechanical Engineering, School of Engineering, The University of Tokyo , Tokyo 113-3656, Japan.
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0000-0001-8027-8069
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Okeyo
Center for Stem Cell Therapy, The Institute of Medical Science, The University of Tokyo , Tokyo 113-3656, Japan.
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Yamazaki
Department of Bioengineering, School of Engineering, The University of Tokyo , Tokyo 113-3656, Japan.
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Kurosawa
Department of Mechanical Engineering, School of Engineering, The University of Tokyo , Tokyo 113-3656, Japan.
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Oana
Department of Microengineering, School of Engineering, Kyoto University , Kyoto 606-8501, Japan.
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Kotera
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Washizu