A quantitative ELISA‐based co‐culture angiogenesis and cell proliferation assay

Abstract

Since solid tumours and metastases depend on adequate blood supply, much research is focused on inhibition of angiogenesis. Unfortunately, most known angiogenesis inhibitors have serious side effects when used as therapeutic agents in man. It is therefore important to develop methods to identify well-tolerated and efficient angiogenesis inhibitors. As a method for identification of new angiogenesis inhibitors we have further developed the procedure described by Bishop et al. (Angiogenesis 1999;3:335-44) to a quantitative ELISA-based fibroblast and endothelial cell co-culture angiogenesis assay. In each well of a 96-microwell plate, human umbilical vein endothelial cells (HUVEC) are seeded onto normal human dermal fibroblasts (NHDF) and propagated in co-culture for 72 h with or without a potential angiogenesis inhibitor. The effect on total cell proliferation is evaluated by quantitative immunochemical measurement of DNA, and on endothelial tube formation by quantification of CD 31, von Willebrand factor, and collagen IV. After ELISA reading, the morphology of the tubular structures formed by HUVEC is visualised with BCIP/NBT, permitting a quantitative result and a qualitative evaluation of cell morphology from the same well. We have used the assay to demonstrate the effect of well-known angiogenesis inhibitors on HUVEC tube formation.

Levamisole Inhibits Angiogenesis in vitro and Tumor Growth in vivo

“…However, with the exception of Bevacizumab (Avastin Ò ) serious side effects or lack of significant prolongation of survival have so far ruled out their clinical use as routine treatments for cancer. In order to be able to efficiently identify new and effective inhibitors of angiogenesis, we have developed a screening assay based on endothelial cells and fibroblasts in co-culture [3]. The synthetic antihelmintic compound Levamisole has previously been used as adjuvant therapy in the treatment of colon cancer in combination with 5-fluorouracil [4,5].…”

Adipose Stromal Cell Tubule Network Model Provides a Versatile Tool for Vascular Research and Tissue Engineering

“…HUVEC, cultured as above (24 different cell lines derived from different donors, passages 1-6 used), were immediately carefully seeded on top of hASC at a density of 4,000 cells/cm 2 . The seeding densities of hASC and HUVEC were chosen according to previous studies by us and others [Donovan et al, 2001;Friis et al, 2003;Sarkanen et al, 2011]. The day after plating, the differentiation treatments were applied to hASC+HUVEC coculture.…”

“…In this prevascular-like network model, cells are induced with natural growth factors and allowed to self-assemble into tubular network and vascular supporting structures. We studied the hASC+HUVEC coculture model by combining some properties and analyses from previous studies [Friis et al, 2003;MerfeldClauss et al, 2010] and the fibroblast-HUVEC coculture angiogenesis assay previously validated in our laboratory [Sarkanen et al, 2011]. We induced the hASC+HUVEC coculture or hASC monoculture with naturally occurring angiogenic growth factors; epidermal growth factor, VEGF, bFGF and IGF-I [Mehta and Besner, 2007].…”

“…The failure of the endothelial cells to establish mature structures in vitro is often due to an absence of stabilizing mural cells [Merfeld-Clauss et al, 2010]. Coculture models that have been used in several studies [Bishop et al, 1999;Donovan et al, 2001;Friis et al, 2003Friis et al, , 2005MerfeldClauss et al, 2010;Montesano et al, 1993;Nicosia and Ottinetti, 1990] provide a useful method for studying angiogenesis in controlled conditions in vitro, with the advantage of being able to replicate some tissue-derived signaling [Rubina et al, 2009]. However, only a few studies [Merfeld-Clauss et al, 2010;Traktuev et al, 2008] [Miranville et al, 2004].…”

The Hematopoietic Chemokine CXCL12 Promotes Integration of Human Endothelial Colony Forming Cell–Derived Cells into Immature Vessel Networks

“…Ex vivo assays have been developed to mimic human vessel formation either in fibrin or collagen gels [28][29][30][31][32][33][34][35][36][37]. These have combined endothelial cells or ECFCs with mural cells (eg, lung fibroblasts, dermal fibroblasts, retinal pericytes, or mesenchymal stromal cells) and specialized endothelial growth medium containing cytokines [eg, VEGF and fibroblast growth factor-2 (FGF-2)].…”