In vitro construction of a human blood vessel from cultured vascular cells: A morphologic study Nicolas L'Heureux, MSc, Lucie Germain, PhD, Raymond Labbé,

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Presentation transcript:

In vitro construction of a human blood vessel from cultured vascular cells: A morphologic study Nicolas L'Heureux, MSc, Lucie Germain, PhD, Raymond Labbé, MD, FRCSC, François A. Auger, MD, FRCPC Journal of Vascular Surgery Volume 17, Issue 3, Pages 499-509 (March 1993) DOI: 10.1016/0741-5214(93)90150-K Copyright © 1993 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 1 Characterization of HUVEC and HVSMC. Phase contrast micrographs of standard cultures: a, HUVEC (original magnification × 60); b, HVSMC (original magnification × 50). Immunofluorescence labeling: c, HUVEC stained with anti-human factor VIII-Von Willebrand's factor monoclonal antibody (original magnification × 240); e, HUVEC labeled with Ac-LDL-Dil (original magnification × 250); d, HVSMC stained with anti-smooth muscle α-actin monoclonal antibody (original magnification × 240). f, Electron micrograph of HVSMC embedded in collagen gel (original magnification × 11,000). Note discontinuous basement membranelike deposit (arrowheads), dark bodies surrounded by bundles of actin filaments (arrows), and cell junction (open arrowhead). N, Nucleus; RER, rough endoplasmic reticulum. Journal of Vascular Surgery 1993 17, 499-509DOI: (10.1016/0741-5214(93)90150-K) Copyright © 1993 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 2 Morphologic appearance of HVSMC in media-equivalent structure at different times after gel formation (3 × 105 cells/ml), as seen by phase contrast microscopy: (a) just after gel formation; (b) after 2 hours first cells start to spread, and (c) after 48 hours (original magnification × 40). d, A closer view of complex tridimensional network of interconnecting cells (original magnification × 95). Note that the three pictures were taken at slightly different focal levels to show extent of cell ramification. Journal of Vascular Surgery 1993 17, 499-509DOI: (10.1016/0741-5214(93)90150-K) Copyright © 1993 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 3 Longitudinal contraction of media-equivalent structures as function of time. Detachment was induced by mechanical shocks at 24 hours, and release from mandrel occurred at 48 hours. One of three similar experiments. Journal of Vascular Surgery 1993 17, 499-509DOI: (10.1016/0741-5214(93)90150-K) Copyright © 1993 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 4 Reorientation of HVSMC in media-equivalent. Phase contrast micrographs of one end (a) and center (b) of tubular structure (original magnification × 60). Note that tube's axis is in horizontal direction. Obvious change of orientation can be observed from largely longitudinal orientation in middle (b) to exclusively circular one at tube's extremity (a). Transverse (c) and longitudinal (d) histologic sections of 3-week-old reorientated media in which cells are circularly arranged (original magnification × 330). Arrows indicate the longitudinal cell axis. Journal of Vascular Surgery 1993 17, 499-509DOI: (10.1016/0741-5214(93)90150-K) Copyright © 1993 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 5 Phase contrast micrograph of HSF in adventitia-equivalent structure (5 × 105cells/ml). This adventitia was cast on media-equivalent and kept on mandrel for 5 days. HSF displayed stretched aspect but no complex network or particular alignment (original magnification × 65). Journal of Vascular Surgery 1993 17, 499-509DOI: (10.1016/0741-5214(93)90150-K) Copyright © 1993 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 6 Macroscopic (a, b) and microscopic (c, d) aspects of constructed model. a, Five-day-old adventitia-equivalent structure (5 × 105 cells/ml) around well-contracted media-equivalent on its mandrel (3 × 105 cells/ml). Note that adventitia is still relatively uncontracted. b, Media-adventitia-equivalent structure being endothelialized. Note important overall contraction and vessel-like appearance of model. Hematoxylin-eosin staining: c, continuous endothelium grown on media-equivalent; d, junctional area between media (darker staining) and adventitia (original magnification × 210). Junction did not resist very well the fixation process. Note apparent higher collagen density of media, which correlates with different degrees of contraction (a, c, d). Journal of Vascular Surgery 1993 17, 499-509DOI: (10.1016/0741-5214(93)90150-K) Copyright © 1993 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 7 Characterization of endothelium. a, EC on media-equivalent structure as observed by TEM (original magnification × 3130). Note small cell projections into collagen gel (arrows) and typical high nucleus to cytoplasm ratio. Inset, Weibel-Palade bodies (original magnification × 42,000). Three bodies are visible, two in cross section and one cut at an angle. Note one-layer membrane and longitudinal inner small rods. b, Ac-LDL-Dil labeled endothelium as observed by fluorescence microscopy (original magnificaton × 275). Some part of endothelium may have been lost during preparation. c, Phase contrast micrograph of confluent monolayer of EC in lumen of media-equivalent structure (original magnification × 115). Note slightly but unmistakably elongated and parallel morphologic apperance. Cell orientation was parallel to axis of tube. Endothelium was stable for at least 50 days. Journal of Vascular Surgery 1993 17, 499-509DOI: (10.1016/0741-5214(93)90150-K) Copyright © 1993 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions