Intra-Laboratory Pre-Validation of a Human Cell Based in vitro Angiogenesis Assay for Testing Angiogenesis Modulators

Sarkanen, Jertta-Riina; Mannerström, Marika; Vuorenpää, Hanna; Uotila, Jukka; Ylikomi, Timo; Heinonen, Tuula

doi:10.3389/fphar.2010.00147

Cited by 29 publications

(49 citation statements)

References 70 publications

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“…This might complicate the use of commercial media in toxicological studies as medium components may interact with test compounds. In addition to commonly used VEGF and FGF-2 defined by us earlier (Sarkanen et al, 2011), we found that the network formation can be further improved by addition of AA, HE and HY. Ascorbic acid (vitamin C) is an essential nutrient for human endothelial cells necessary for their effective migration and for the synthesis of collagen type IV, an important component of basement membrane (Telang et al, 2007).…”

Section: Discussionmentioning

confidence: 54%

“…Isolation and culture of human umbilical vein endothelial cells HUVEC were isolated from human umbilical cord veins using 0.05% collagenase I as described previously (Sarkanen et al, 2011). The cells were cultured in EGM™-2 Endothelial Cell Growth Medium-2 (EGM-2, Lonza Group Ltd).…”

Section: Methodsmentioning

confidence: 99%

“…Due to an increasing amount of compounds affecting the vascular system, accurate vasculogenesis and angiogenesis models are needed for chemical safety testing and for drug development (Sarkanen et al, 2011;Bishop et al, 1999). Currently, preclinical animal models are dominantly used for angiogenesis testing although they are not considered optimal in efficacy or relevance to humans.…”

Section: Introduction #mentioning

confidence: 99%

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Human vascular model with defined stimulation medium – a characterization study

Huttala

2015

ALTEX

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SummaryThe formation of blood vessels is a vital process in embryonic development and in normal physiology. Current vascular modelling is mainly based on animal biology leading to species-to-species variation when extrapolating the results to humans. Although there are a few human cell based vascular models available, these assays are insufficiently characterized in terms of culture conditions and developmental stage of vascular structures. Therefore, well characterized vascular models with human relevance are needed for basic research, embryotoxicity testing, development of therapeutic strategies and for tissue engineering. We have previously shown that the in vitro vascular model based on co-culture of human adipose stromal cells (hASC) and human umbilical vein endothelial cells (HUVEC) is able to induce an extensive vascular-like network with high reproducibility. In this work we developed a defined serum-free vascular stimulation medium (VSM) and performed further characterization in terms of cell identity, maturation and structure to obtain a thoroughly characterized in vitro vascular model to replace or reduce corresponding animal experiments. The results showed that the novel vascular stimulation medium induced an intact and evenly distributed vascular-like network with morphology of mature vessels. Electron microscopic analysis assured the three-dimensional microstructure of the network containing lumen. Additionally, elevated expression levels of the main human angiogenesis-related genes were detected. In conclusion, with the newly defined medium the vascular model can be utilized as a characterized test system for chemical testing as well as in creating vascularized tissue models.

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Section: Discussionmentioning

confidence: 54%

Section: Methodsmentioning

confidence: 99%

Section: Introduction #mentioning

confidence: 99%

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Human vascular model with defined stimulation medium – a characterization study

Huttala

2015

ALTEX

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“…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].…”

Section: Introductionmentioning

confidence: 99%

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

Sarkanen

Vuorenpää²,

Huttala³

et al. 2012

Cells Tissues Organs

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The current limitation in designing three-dimensional tissue models is the lack of adequate vascularization with mature and stable vessels. Adipose tissue is known to secrete several angiogenic factors, and human adipose stromal cells (hASC) are known to promote vessel growth, maturation and stabilization. In this study, hASC were induced to angiogenesis with growth factor-enriched medium either in monoculture or in coculture with human umbilical vein endothelial cells (HUVEC) and analyzed for vascular, pericytic and smooth muscle cell markers. hASC and HUVEC cocultures showed an accelerated proliferation rate and the cells self-assembled, independent of the cell passage number, into multilayered three-dimensional tubular networks. The networks of hASC and HUVEC expressed endothelial markers, a complete basement membrane and vessel-supporting cells with contractile properties. A hASC and green fluorescence protein-HUVEC-infection model revealed that cocultures consisted of a mosaic of von Willebrand factor-positive cells derived from both cell populations – hASC and HUVEC. hASC monoculture had passage- and donor-dependent ability to form tubular networks, with half of the cultures presenting tubule structures and basement membrane formation. Pericytic and smooth muscle cell markers were expressed in hASC monoculture even when tubules were absent. By combining the potential properties of hASC and features from the present angiogenesis assays, we generated a natural-like, xeno-free, prevascular-like network in vitro model with excellent reproducibility and minimal limitations in technical performance. This tubular network model is an excellent tool for studying cell interactions during vascular development, for chemical and drug testing and for developing natural-like, multilayered, vascularized, scaffold-free tissue models.

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“…A previous study showed that a linear dose-response relationship exists between the VEGF-A concentration and the tube formation potency in the co-culture assay in which concentrations of 10 ng/mL VEGF-A and 1 ng/mL FGF-2 are required for maximum response [29]. Our data demonstrated that the tube formation capacity of ASC-CM, even at passage 6, was equivalent or higher than that of medium containing 10 ng/mL VEGF-A as positive control (see Figure 4).…”

Section: Discussionmentioning

confidence: 99%

Influence of Donor Age and Passage Number on Angiogenic Activity in Human Adipose-Derived Stem Cell-Conditioned Media

Nakamura¹,

Kazama²,

Nagaoka³

et al. 2015

J Stem Cell Res Ther

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Introduction: Adipose-derived stem/stromal cells (ASCs) are considered a promising cell source for therapeutic angiogenesis because the cells can be prepared following a minimally invasive procedure and because they secrete a variety of angiogenic cytokines. In the present study, the influence of donor age and passage number on angiogenic activity of ASC-conditioned media (ASC-CM) was examined.Methods: Human ASCs (donor age, 5 months to 82 years; n = 10) were cultured, and ASC-CM were collected at passages 2, 4, and 6. The angiogenic activity of ASC-CM was evaluated with the tube formation assay using a system in which human umbilical vein endothelial cells (HUVECs) and fibroblasts were co-cultured. The concentrations of vascular endothelial growth factor-A (VEGF-A) and hepatocyte growth factor (HGF) in each ASC-CM were measured using an enzyme-linked immunosorbent assay. Results:A donor age over 60 years affected the proliferative capacity of ASCs at passage 4 and later. ASC-CM significantly enhanced HUVEC tube formation, and this response was not influenced by donor age. ASC-CM at passage 6 showed a lower tube formation capacity compared to ASC-CM at passage 4, although the capacity was still equivalent to the positive control (medium containing10 ng/mL VEGF-A). A donor age over 26 years affected VEGF-A but not HGF levels in ASC-CM, although we found no direct correlation between VEGF-A/HGF levels and the tube formation capacity. Conclusion:Our results demonstrate a passage number-dependent but a donor age-independent decline in the angiogenic activity of ASC-CM.

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Intra-Laboratory Pre-Validation of a Human Cell Based in vitro Angiogenesis Assay for Testing Angiogenesis Modulators

Cited by 29 publications

References 70 publications

Human vascular model with defined stimulation medium – a characterization study

Human vascular model with defined stimulation medium – a characterization study

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

Influence of Donor Age and Passage Number on Angiogenic Activity in Human Adipose-Derived Stem Cell-Conditioned Media

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