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Fine-tuning of a three-dimensional microcarrier-based angiogenesis assay for the analysis of endothelial-mesenchymal cell co-cultures in fibrin and collagen gels

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Abstract

A prerequisite for successful tissue engineering is the existence of a functional microvascular network. We hypothesized that such networks can be created and quantified in an in vitro setting by co-culturing endothelial cells (ECs) with tissue-specific ‘bystander cells’ in 3-D gel matrices. To test this hypothesis we adapted a previously described in vitro microcarrier-based angiogenesis assay (V. Nehls and D. Drenckhahn, 1995, Microvasc Res 50: 311–322). On optimizing this assay, we noted that the initial EC-microcarrier coverage depended on EC type and seeding technique employed to coat the microcarrier beads with the ECs. A confluent EC monolayer on the microcarrier surfaces formed only when bovine aortic endothelial cells (BAECs) were admixed to the beads under gentle agitation on an orbital shaker. After embedding BAEC-covered microcarrier beads into a sandwich-like arrangement of collagen or fibrin gels, we assessed cellular outgrowth at different serum concentrations in terms of migration distance and sprout formation. Quantifiable sprout formation was highest at 1% fetal bovine serum (FBS) in collagen matrices and at 0.1% FBS in fibrin matrices. At higher serum concentration, excess cell migration and formation of clusters prevented quantitative analysis of sprouting. Following the fine-tuning of this angiogenesis assay, we co-cultured BAECs with adipose tissue-derived fibroblasts (FBs) and vascular smooth muscle cells (SMCs). While FBs were able to increase the average migration distance of BAECs in both matrices, SMCs enhanced BAEC migration in fibrin, but not in collagen gels. By contrast, the number of newly formed sprouts in fibrin gels was increased by both cell types. We conclude that in this model bystander cells enhance EC network formation in a matrix-dependent manner. Additionally, these results stress the importance of carefully selecting␣the experimental parameters of a given in vitro angiogenesis model.

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Abbreviations

3-D:

three-dimensional

AMD:

average migration distance

BAEC:

bovine aortic endothelial cell

bFGF:

basic fibroblast growth factor (also FGF2)

DMEM:

Dulbecco’s modified Eagle’s medium

EC:

endothelial cell

ECM:

extracellular matrix

ECGS:

endothelial cell growth supplement

FB:

bovine adipose fibroblast cell

FBS:

fetal bovine serum

HAEC:

human aortic endothelial cell

HMEC-1:

transformed human microvascular endothelial cell

HMVEC:

primary human microvascular endothelial cell

PBS:

Dulbecco’s phosphate buffered salt solution

SMC:

porcine pulmonary artery smooth muscle cell

rhEGF:

recombinant human epidermal growth factor

VE-cadherin:

vascular endothelial cadherin

VEGF:

vascular endothelial growth factor

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Acknowledgements

This work was supported in part by grants from the Nanotechnology Institute of Southeastern Pennsylvania (NTI), the Calhoun Funds, and the National Aeronautics and Space Administration (NASA, NAG-1436, NNJ04HC81G-01, and NCC9–130).

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  1. Franziska Dietrich

    Present address: Interdisciplinary Center for Clinical Research (IZKF), University of Leipzig, Leipzig, 04103, Germany

Authors and Affiliations

  1. School of Biomedical Engineering, Science, and Health Systems, Drexel University, 3141 Chestnut Street, Philadelphia, PA, 19104, USA

    Franziska Dietrich & Peter I. Lelkes

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  1. Franziska Dietrich
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  2. Peter I. Lelkes
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Correspondence to Peter I. Lelkes.

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Dietrich, F., Lelkes, P.I. Fine-tuning of a three-dimensional microcarrier-based angiogenesis assay for the analysis of endothelial-mesenchymal cell co-cultures in fibrin and collagen gels. Angiogenesis 9, 111–125 (2006). https://doi.org/10.1007/s10456-006-9037-x

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  • DOI: https://doi.org/10.1007/s10456-006-9037-x

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