Journal: The FASEB Journal

Article Title: Fabrication of 3-dimensional multicellular microvascular structures

doi: 10.1096/fj.14-263343

Figure Lengend Snippet: Deposition of Col IV, Fn, and Lmn by ECFCs in 3D vs. 2D. Confocal Z-stack image projections are shown of ECM in (A) 3D fibrin microfibers and (B) 2D fibrin-coated surfaces. Red indicates corresponding ECM, blue shows nuclei, and green indicates F-actin. Scale bars, 100 μm. Inset scale bars, 25 μm. C) RT-PCR analysis of expression of ECM genes by ECFCs cultured on 2D vs. 3D. Error bars represent SEM. Significance levels in the distribution are represented by *P < 0.05 and **P < 0.01 (n ≥ 3).

Article Snippet: Plasmin treatment Fibrin microfibers with or without cells were treated with 15, 1, 0.25, and 0.1 CU/ml plasmin from human plasma (Athens Research and Technology, Athens, GA, USA) in DMEM (Life Technologies for 1, 6, 12, and 24 hours, respectively, in a humidified incubator at 37°C in a 5% CO 2 atmosphere.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Cell Culture

Journal: The FASEB Journal

Article Title: Fabrication of 3-dimensional multicellular microvascular structures

doi: 10.1096/fj.14-263343

Figure Lengend Snippet: Deposition of Col I, III, and IV, Fn, and Lmn by pericytes in 3D vs. 2D. Confocal Z-stack image projections are shown of ECM in (A) 3D fibrin microfibers and (B) 2D fibrin-coated surfaces. Red indicates corresponding ECM, blue shows nuclei, and green indicates F-actin. Arrows and arrowheads point to randomly deposited and nonpolymerized ECM proteins, respectively. Double-headed arrows show alignment direction of organized ECM deposition. Scale bars, 100 μm. Inset scale bars, 25 μm. C) RT-PCR analysis of expression of ECM genes by SMCs cultured on 2D vs. 3D. ND, not detected. Error bars represent SEM. D) Orthogonal view of pericytes grown on microfibers. Arrowheads point to cells underneath the outer cell layer; arrows point to extracellularly deposited Lmn. Scale bar, 50 μm.

Article Snippet: Plasmin treatment Fibrin microfibers with or without cells were treated with 15, 1, 0.25, and 0.1 CU/ml plasmin from human plasma (Athens Research and Technology, Athens, GA, USA) in DMEM (Life Technologies for 1, 6, 12, and 24 hours, respectively, in a humidified incubator at 37°C in a 5% CO 2 atmosphere.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Cell Culture

Journal: The FASEB Journal

Article Title: Fabrication of 3-dimensional multicellular microvascular structures

doi: 10.1096/fj.14-263343

Figure Lengend Snippet: Deposition of Col I, III, and IV, Eln, Fn, and Lmn by SMCs in 3D vs. 2D. Confocal Z-stack image reconstructions are shown of ECM in (A) 3D fibrin fibers and (B) 2D fibrin-coated surfaces. Red indicates corresponding ECM, blue shows nuclei, and green indicates F-actin. Arrows and arrowheads point to randomly deposited and nonpolymerized ECM proteins, respectively. Double-headed arrows show alignment direction of organized ECM deposition. Scale bars, 100 μm. Inset scale bars, 25 μm. C) RT-PCR analysis of expression of ECM genes by pericytes cultured on 2D vs. 3D. Error bars represent SEM (n ≥ 2). Significance levels in the distribution are represented by *P < 0.05 and **P < 0.01. D) Orthogonal view of pericytes grown on microfibers. Arrowheads point to cells underneath the outer cell layer; arrows point to extracellularly deposited Lmn. Scale bar, 20 μm.

Article Snippet: Plasmin treatment Fibrin microfibers with or without cells were treated with 15, 1, 0.25, and 0.1 CU/ml plasmin from human plasma (Athens Research and Technology, Athens, GA, USA) in DMEM (Life Technologies for 1, 6, 12, and 24 hours, respectively, in a humidified incubator at 37°C in a 5% CO 2 atmosphere.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Cell Culture

Journal: The FASEB Journal

Article Title: Fabrication of 3-dimensional multicellular microvascular structures

doi: 10.1096/fj.14-263343

Figure Lengend Snippet: Fiber degradation and viability of cells and ECM after plasmin treatment. A) Light-microscopy images of fibrin microfibers treated with plasmin. Scale bars, 100 μm. B) Immunofluorescence images of viability assays of ECFCs after 12- and 24-hour treatments with plasmin. Red indicates dead cells; green shows live cells. Scale bars, 500 μm. C) Confocal Z-stack image projections of fibrin fibers with ECFCs for 5 days and treated with plasmin for (I) 12 and (II) 24 hours. Scale bars, 100 μm (left) and 50 μm (right). D) Orthogonal view of structures after plasmin treatments. Green indicates F-actin, blue shows nuclei, red indicates Col IV, and magenta shows Fn (I) and Lmn (II) (n ≥ 2). Scale bars, 50 μm.

Article Snippet: Plasmin treatment Fibrin microfibers with or without cells were treated with 15, 1, 0.25, and 0.1 CU/ml plasmin from human plasma (Athens Research and Technology, Athens, GA, USA) in DMEM (Life Technologies for 1, 6, 12, and 24 hours, respectively, in a humidified incubator at 37°C in a 5% CO 2 atmosphere.

Techniques: Light Microscopy, Immunofluorescence

Journal: The FASEB Journal

Article Title: Fabrication of 3-dimensional multicellular microvascular structures

doi: 10.1096/fj.14-263343

Figure Lengend Snippet: ECFC and perivascular cell cocultures on fibrin microfibers. Confocal Z-stack image projections are shown of ECFCs grown for 5 days on fibrin microfibers followed by 5 days more of (A) pericyte coculture. Red indicates (I) VEcad, (II) SM22, and (III) Col III, blue shows nuclei, and green indicates F-actin. B) SMC coculture. Red indicates (I) VEcad, (II) SM22, and (III) Eln, blue shows nuclei, and green indicates F-actin. Confocal Z-stack image 3D reconstructions are shown of structures cultured for 5 days with ECFCs followed by 5 more days with perivascular cells and then treated for 12 hours with 0.25 CU/ml plasmin. C) Pericyte coculture. Red indicates (I and III) VEcad and (II and IV) Col III, magenta shows (I and III) SM22 and (II and IV) Col IV, blue indicates nuclei, and green shows F-actin. D) SMC coculture. Red indicates (I and III) VEcad and (II and IV) Eln, magenta shows (I and III) SM22 and (II and IV) Fn, blue indicates nuclei, and green shows F-actin. Scale bars, 50 μm (n ≥ 2).

Article Snippet: Plasmin treatment Fibrin microfibers with or without cells were treated with 15, 1, 0.25, and 0.1 CU/ml plasmin from human plasma (Athens Research and Technology, Athens, GA, USA) in DMEM (Life Technologies for 1, 6, 12, and 24 hours, respectively, in a humidified incubator at 37°C in a 5% CO 2 atmosphere.

Techniques: Cell Culture