T Aper1, O E Teebken, G Steinhoff, A Haverich. 1. Division of Thoracic and Cardiovascular Surgery, Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Medical School Hannover, Hannover, Germany.
Abstract
OBJECTIVE: Morphological and functional characterization of cocultured endothelial cells (EC) and myofibroblasts (MFB) seeded on a matrix composed of a fibrin preparation mimicking the microenvironment of a vascular wall. METHODS: MFB and EC were isolated from human saphenous veins and expanded separately in vitro. MFB were seeded on a composite matrix consisting of a fibrin preparation (with or without transforming growth factor-beta2) and a polyglactin-mesh to form a 3-dimensional structure, which was consecutively reseeded with EC. Seeded matrices were incubated in a bioreactor. Characterization was done including fluorescence staining, live-/dead-assay and immunohistochemistry. RESULTS: High density cocultures in hierarchical structure mimicking the formation of a vascular wall were obtained with nearly complete coverage of the surface with EC. Distribution of preseeded MFB in a 519+/-27 microm thick layer (day 14) was achieved. Cell viability was shown in fluorescence staining for at least 19 days. In deeper layers, no viable cells could be detected within the fibrin preparation. EC covered the surface, had uniform morphology, and their preserved viability was shown for at least 5 days. No EC-ingrowth was found into the fibrin preparation. Neoformation of the matrix proteins laminin and collagen IV was observed. CONCLUSION: A structured coculture of MFB and EC was obtained mimicking the formation of a vascular wall with preserved viability utilizing a fibrin preparation. Nutrition problems seem to limit the maximal extent of MFB in the matrix.
OBJECTIVE: Morphological and functional characterization of cocultured endothelial cells (EC) and myofibroblasts (MFB) seeded on a matrix composed of a fibrin preparation mimicking the microenvironment of a vascular wall. METHODS: MFB and EC were isolated from human saphenous veins and expanded separately in vitro. MFB were seeded on a composite matrix consisting of a fibrin preparation (with or without transforming growth factor-beta2) and a polyglactin-mesh to form a 3-dimensional structure, which was consecutively reseeded with EC. Seeded matrices were incubated in a bioreactor. Characterization was done including fluorescence staining, live-/dead-assay and immunohistochemistry. RESULTS: High density cocultures in hierarchical structure mimicking the formation of a vascular wall were obtained with nearly complete coverage of the surface with EC. Distribution of preseeded MFB in a 519+/-27 microm thick layer (day 14) was achieved. Cell viability was shown in fluorescence staining for at least 19 days. In deeper layers, no viable cells could be detected within the fibrin preparation. EC covered the surface, had uniform morphology, and their preserved viability was shown for at least 5 days. No EC-ingrowth was found into the fibrin preparation. Neoformation of the matrix proteins laminin and collagen IV was observed. CONCLUSION: A structured coculture of MFB and EC was obtained mimicking the formation of a vascular wall with preserved viability utilizing a fibrin preparation. Nutrition problems seem to limit the maximal extent of MFB in the matrix.