Volume 9, Issue 4, Pages 607-616 (April 2004) Electric Field-Induced Molecular Vibration for Noninvasive, High-Efficiency DNA Transfection  Lin Song, Lillian Chau, Yoshitaka Sakamoto, Juichiro Nakashima, Masafumi Koide, Rocky S Tuan  Molecular Therapy  Volume 9, Issue 4, Pages 607-616 (April 2004) DOI: 10.1016/j.ymthe.2004.01.017 Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 1 Electric field-induced molecular vibration using the Gene Symphonizer. (A) The equipment consists of the controller unit (left) and the ultra-high-voltage exciter unit (right). (B) A glass dish containing the cell suspension is placed between the terminal probes mounted inside the insulated chamber of the high-voltage exciter unit. The ionized nitrogen flare (purple luminescent field, arrow) denotes the generation of the instantaneous, vigorous electric field that provides high electrical energy to molecules and produces the resonating vibration between cells and molecules, allowing for the entry of exogenous molecules into cells. Molecular Therapy 2004 9, 607-616DOI: (10.1016/j.ymthe.2004.01.017) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 2 Fluorescence microscopy detection of GFP-positive cells transfected using the Gene Symphonizer. hMSCs (A–D) and chick limb mesenchymal cells (E–H) were incubated with pCMS-EGFP; transfected at 12 V, 120 Hz, and 300 wave ratio for 10 s; and placed into culture. At 48 h posttransfection, cell cultures were photographed under phase contrast (A, C, E, G) and epifluorescence (B, D, F, H) microscopy. (A, B, E, and F) Transfection in the presence of pCMS-EGFP; (C, D, G, and H) same procedure in the absence of plasmid DNA. Fluorescent, GFP-positive cells are seen in both (B) hMSCs and (H) chick limb mesenchymal cells only when transfected in the presence of 1 μg/μl pCMS-EGFP. For chick limb mesenchyme micromass cultures, images were acquired in the center of the culture to ensure representation of the high-density culture. Scale bar, 100 μm. Molecular Therapy 2004 9, 607-616DOI: (10.1016/j.ymthe.2004.01.017) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 3 Representative results of flow cytometric analysis of human mesenchymal stem cells and chick embryonic limb mesenchymal cells transfected using the Gene Symphonizer. Cells were transfected and analyzed after 48 h of culture as described under Materials and Methods. (A) hMSCs; (B) limb mesenchymal cells. Forward (FSC-H)- and side (SSC-H)-scattering lights were used to detect single viable cells in the cell population. Fluorescent light emitted from pCMS-EGFP-transfected cells was captured at 530 nm in the FL1-H channel (green) and at 570 nm in the FL2-H channel (red). Molecular Therapy 2004 9, 607-616DOI: (10.1016/j.ymthe.2004.01.017) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 4 Mesenchymal progenitor cells maintained their differentiation potential after Gene Symphonizer-mediated transfection. (A–C) hMSCs and (D–F) chick limb mesenchymal cells in micromass culture were transfected as described for Fig. 2. At 48 h posttransfection, GFP-expressing hMSCs were selected by FACS and cultured in the presence of differentiation supplements in the medium. Osteogenic differentiation was confirmed on the basis of (A) histochemically detectable alkaline phosphatase activity and (B) the presence of mineralized matrix by Alizarin red staining. (C) Oil Red O staining (counterstaining with hematoxylin) detected the presence of lipid droplets in the cytoplasm for cells cultured under adipogenic conditions. Chick limb bud mesenchymal cells were placed into high-density micromass cultures to assess the ability of transfected cells to participate in chondrogenic differentiation. (D) Phase-contrast image of day 3 cartilage nodules in high-density micromass cultures. (E) Chondrogenesis in the micromass cultures was detected by Alcian blue staining of cartilage nodules, and (F) the presence of GFP-expressing cells in the nodules indicates their ability to participate in cartilage formation. (D), (E), and (F) are sequential phase-contrast, bright-field, and fluorescence images of the same specimen. Scale bar, 100 μm. Molecular Therapy 2004 9, 607-616DOI: (10.1016/j.ymthe.2004.01.017) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 5 Gene Symphonizer-mediated cell transfection in chick embryonic tissue explants. Transfection with pCMS-EGFP was carried out as described under Materials and Methods. Embryonic sternum was obtained from 11-day-old chick embryo. At 24 h posttransfection, sections were immunostained with collagen type II antibody and viewed under (B) phase contrast and epifluorescence for (C) DAPI nuclear staining, (D) GFP, and (E) immunostaining. (F) A merged image of (D) and (E). (A) Untransfected sternum explants. GFP-positive cells were observed embedded in collagen type II extracellular matrix. Scale bar, 20 μm. Muscle explants were obtained from 11-day-old chick embryos. At 24 h posttransfection, muscle sections were immunostained with sarcomeric myosin heavy chain antibody and viewed under (H) phase contrast and epifluorescence for (I) DAPI nuclear staining, (J) GFP, and (K) immunostaining. (L) A merged image of (J) and (K). (G) Untransfected muscle explants. GFP expression was detected in cells positive for sarcomeric myosin heavy chain. Scale bar, 10 μm. Molecular Therapy 2004 9, 607-616DOI: (10.1016/j.ymthe.2004.01.017) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions