Three-dimensional perfusion imaging of microcirculatory networks within axially vascularized artificial tissue: study protocol
Evaluating the complex, three-dimensional (3D) architecture of de novo angiogenesis in artificially engineered tissue remains a significant challenge, as conventional methods like 2D histology and microimaging techniques are limited. For axial vascularization techniques, a reproducible method for co...
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| Main Authors: | , , , , , , |
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| Format: | Article (Journal) |
| Language: | English |
| Published: |
February 1, 2026
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| In: |
Tissue engineering. Part C, Methods
Year: 2026, Volume: 32, Issue: 2, Pages: 56-65 |
| ISSN: | 1937-3392 |
| DOI: | 10.1177/19373384261422709 |
| Online Access: | Verlag, lizenzpflichtig, Volltext: https://doi.org/10.1177/19373384261422709
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| Author Notes: | Christoph Koepple, MD, Lukas Pollmann, MD, Nicola Sariye Pollmann, MD, Ulrich Kneser, MD, Dirk J. Schaefer, MD, Norbert Gretz, MD, and Volker J. Schmidt, MD |
| Summary: | Evaluating the complex, three-dimensional (3D) architecture of de novo angiogenesis in artificially engineered tissue remains a significant challenge, as conventional methods like 2D histology and microimaging techniques are limited. For axial vascularization techniques, a reproducible method for complete visualization of the microcirculatory system is needed. We present an integrated workflow for high-resolution 3D visualization of neovascularization within arteriovenous (AV) loop-based tissue constructs in a rat model. An intravascular perfusion with a cationic near-infrared fluorescent dye, MHI148-polyethylenimine, was used to 3D label the patent vasculature. Following perfusion-fixation and explantation, the construct was rendered optically transparent using an ethyl cinnamate?based clearing protocol. The fluorescent signal was then imaged using confocal and light-sheet fluorescence microscopy at 7 and 28 days postimplantation. Our workflow successfully achieved high-contrast, 3D visualization of the microvascular network, allowing for whole-mount and segmental analysis of the vascular tree. At day 7, imaging delineated solely the AV loop axis while by day 28, a dense and complex, interconnected capillary plexus from the central axis demonstrated a progressive neovascularization. Downstream processing compatibility was confirmed through successful rehydration and 3D nuclear counterstaining. This workflow offers a powerful and reproducible method for detailed structural assessment of microvascular networks in large engineered constructs, overcoming key limitations of existing techniques.Impact Statement Visualizing the complete 3D microvascular network in engineered tissue is a major challenge, as current methods are limited and often destructive. We introduce a novel workflow combining intravascular fluorescent perfusion, optical tissue clearing, and 3D microscopy in an AV loop model. This provides unprecedented high-resolution, whole-mount imaging of the entire patent vascular tree. Crucially, our aqueous-based protocol preserves endothelial integrity, allowing for subsequent rehydration and molecular analysis?a major advantage over traditional casting techniques. This reproducible tool accelerates the study of neovascularization and the development of clinically relevant engineered tissues. |
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| Item Description: | Erstmals veröffentlicht: 10. Februar 2026 Gesehen am 05.03.2026 |
| Physical Description: | Online Resource |
| ISSN: | 1937-3392 |
| DOI: | 10.1177/19373384261422709 |