Formation of three-dimensional tubular endothelial cell networks under defined serum-free cell culture conditions in human collagen hydrogels

Birgit Andrée,Sarah Strauß,Houda Ichanti,Alexander Heisterkamp,Axel Haverich,Stefan Kalies,Andres Hilfiker,Peter-Maria Vogt

doi:10.1038/s41598-019-41985-6

Birgit Andrée, Sarah Strauß + Show 6 more

Open Access

https://doi.org/10.1038/s41598-019-41985-6

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Abstract

Implementation of tubular endothelial cell networks is a prerequisite for 3D tissue engineering of constructs with clinically relevant size as nourishment of cells is challenged by the diffusion limit. In vitro generation of 3D networks is often achieved under conditions using serum containing cell culture medium and/or animal derived matrices. Here, 3D endothelial cell networks were generated by using human umbilical vein endothelial cells (HUVECs) in combination with human adipose tissue derived stromal cells (hASCs) employing human collagen I as hydrogel and decellularized porcine small intestinal submucosa as starter matrix. Matrigel/rat tail collagen I hydrogel was used as control. Resulting constructs were cultivated either in serum-free medium or in endothelial growth medium-2 serving as control. Endothelial cell networks were quantified, tested for lumen formation, and interaction of HUVECs and hASCs. Tube diameter was slightly larger in constructs containing human collagen I compared to Matrigel/rat tail collagen I constructs under serum-free conditions. All other network parameters were mostly similar. Thereby, the feasibility of generating 3D endothelial cell networks under serum-free culture conditions in human collagen I as hydrogel was demonstrated. In summary, the presented achievements pave the way for the generation of clinical applicable constructs.

Highlights

In natural tissues, the nutrition of cells is facilitated by a dense capillary network which is generated during development
Formation of endothelial cells (ECs) networks visible by GFP expression of human umbilical vein endothelial cells (HUVECs) was followed over the course of the days (Fig. 1)
Constructs cultivated in serum-free medium (SFM) exhibited EC network formation from day 2–3 onwards resulting in a stable, filigree network of GFP-HUVECs

Summary

Introduction

The nutrition of cells is facilitated by a dense capillary network which is generated during development. Successful EC network formation was described in many approaches, but most studies employed animal derived components like Matrigel or cultivation of the constructs was conducted in medium containing fetal bovine serum (FBS). For envisioned clinical application of such constructs well defined materials as well as serum-free cultivation will be indispensable. As previously demonstrated by us, the combination of ECs and hASCs in a hydrogel construct containing Matrigel and rCOL leads to EC network formation regardless of the source of ECs11. This model is hampered by the usage of animal derived matrices as well as the cultivation in FBS containing medium. The substitution of Matrigel and rCOL by hCOL from fibroblasts in combination with cultivation under serum-free conditions was investigated

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