Surface Coverage of Vascular Grafts with Cultured Human Endothelial Cells from Subcutaneous Fat Tissue Obtained with a Biopsy Needle

Abstract

SummarySurface coverage with autogeneous endothelial cells is effective in reducing thrombogenicity of an artificial vascular graft, but procedure for obtaining the cells is invasive for patients. The purpose of this study was to establish cultures of human endothelial cells separated from a small piece of subcutaneous fat tissue. A piece of tissue weighing about 10 mg was obtained from subcutaneous fat using a biopsy needle, and treated with collagenase and dispase. Microvascular endothelial cells were selected and other types of cells contaminating the cultures were eliminated by scraping with a needle under a microscope. The yield of the cells was 8362 ± 4264/10 mg of subcutaneous fat (n = 7). The cultures reached confluence in about 2 weeks. The cells were positive for von Willebrand factor, P-selectin, and uptake of acetylated low density lipoprotein. The cells produced 15.9 ± 3.3 ng/mg cell protein/h of 6-ketoprostaglandin F1α (n = 5) when stimulated with thrombin. Thrombin also stimulated the production of platelet-activating factor: 7653 ± 4297 dpm/106 cells (n = 5). Endothelin-1 accumulation in the medium of unstimulated endothelial cells was 0.54 ± 0.16 ng/mg cell protein/10 h (n = 8). As a preliminary experiment for graft seeding, the cells were also cultured on pieces of a gelatin-coated Dacron graft, and scanning electron microscopy revealed the surface coverage of the graft. We herein described about successful culture of human microvascular endothelial cells from subcutaneous fat tissue obtained using a biopsy needle. The cultured cells may be applicable to a seeded vascular graft.

Expression of Tumor Necrosis Factor-α in Cultured Human Endothelial Cells Stimulated With Lipopolysaccharide or Interleukin-1α

“…LPS from Escherichia coli serotype 0III:B4, human serum albumin, and protease inhibitor cocktail were from Sigma. M199, DMEM, FBS, primer oligo(dT) [12][13][14][15][16][17][18] , and M-Mulv reversetranscriptase were from Gibco-BRL. Ficoll-Paque Plus was from Amersham Pharmacia Biotech.…”

“…Total RNA was isolated from the cells by use of an RNeasy total RNA isolation kit. Single-strand cDNA for a PCR template was synthesized from 1 g of total RNA by use of primer oligo(dT) [12][13][14][15][16][17][18] and M-Mulv reverse-transcriptase under conditions indicated by the manufacturer. Specific primers were designed from cDNA sequences for TNF-␣, TACE, and GAPDH, and each cDNA was amplified by PCR with Taq DNA polymerase.…”

“…Cells were fixed either with 5% paraformaldehyde or ethanol/methanol (1:1) and subjected to immunofluorescent staining for TNF-␣ as described previously. 12 Cells were incubated with Superblock and then with a 1:100 dilution of monoclonal anti-TNF-␣ antibody. After they were washed with PBS, cells were incubated with anti-mouse IgG labeled with biotin followed by incubation with streptavidin-FITC.…”

Expression of Vascular Endothelial Growth Factor in Human Umbilical Vein Endothelial Cells Stimulated with Interleukin-1α

“…HUVEC were stimulated with 1 ng/ml IL-1␣ for 16 h. The cells were fixed with ethanol/methanol (1 : 1), and subjected to immunofluorescent staining for VEGF as described previously (29). The cells were incubated with Superblock ® and then with a 1 : 100 dilution of a monoclonal anti-VEGF antibody.…”

Tissue-engineered cardiovascular grafts and novel applications of tissue engineering by self-assembly (TESA™)

“…Many groups in the fi eld have proposed seeding patient endothelial cells onto the surface of synthetic grafts as a way of preventing thrombus formation (Fasol et al , 1989;Wigod and Klitzman, 1993;Griesser et al , 1994;Herring et al , 1994;Mazzucotelli et al , 1994;Thompson et al , 1994;Zilla et al , 1994;Bull et al , 1995;Ott and Ballermann, 1995;Burmeister et al , 1996;Koyama et al , 1996;Meinhart et al , 1997). However, it is the work of Zilla and Meinhart that has convincingly demonstrated the feasibility and the effi cacy of such a procedure (Deutsch et al , 2009).…”