Co-culture of Endothelial and Mesenchymal Stem Cells on 3D Scaffolds

Objectives: For the repair of bone defects, tissue engineering approaches are necessary that combine cells capable of osteogenic activity and stimulation of angiogenesis together with an appropriate scaffolding material. The present in vitro study was aimed to optimize the culture conditions to grow mesenchymal stem cells (MSCs) and endothelial cells (ECs) on a 3D scaffold and to model heterotypic cell-cell interactions.

Methods: MSCs and ECs were co-cultured on tissue culture plates (2D) and in 3D polymer scaffolds made by copolymerization of the commonly used L-lactide (LLA) polymer and the completely amorphous polymer 1,5-dioxepan-2-one (DXO-CO-LLA) or with caprolactone (CL-CO-LLA) as monomers for 1, 3 and 5 days. At each time point, the morphology of the cells was studied by light, fluorescence and confocal microscopy. 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di- phenytetrazoliumromide (MTT) assay was used to evaluate the cellular proliferation. At day 5, total RNA was extracted, cDNA was prepared and the expression of alkaline phosphatase (ALP) was quantified by real time reverse transcriptase PCR (QRT-PCR).

Results: The morphological analysis showed that both MSCs and ECs could grow well within 5 days in a direct contact co-culture model. Furthermore, the cells had attached and spread well on the scaffold. MTT method showed good proliferation of the co-cultured cells at 1, 3, and 5 days. Furthermore, high expression of ALP was detected by the PCR.

Conclusion: This in vitro study demonstrated that MSCs and ECs grew well both on 2D and on 3D polymer scaffolds in a co-culture model indicating that co-culture of MSCs and ECs in tissue engineering is feasible. Expression of alkaline phosphatase is influenced by co-culturing of MSCs and ECs.

The study is supported by Helse Vest and the Research Council of Norway.