Characterization of cell spheroids as promising tool as 3D printable building blocks for vascular network formation

Fabian Stein, Vasileios W.D. Trikalitis, Denise M. Feitosa-Afonsso, Jeroen Rouwkema

    Research output: Contribution to conferencePosterAcademic

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    The human vasculature is a highly structured and organized system to supply each cell in the body with oxygen and nutrients and remove metabolic waste. Blood vessels mainly consist of three different cell types. The inner layer (Tunica Intima) of endothelial cells which form a monolayer which are responsible for new blood vessel formation, allow blood flow and also act as a semi-selective barrier to the surrounding tissue followed by the middle layers (Tunica media) of mural cells (smooth muscle cells and pericytes) which are responsible for the vessel maturation and stabilisation. The outer layer (Tunica adventitia) consists mostly of fibrous connective tissue and myofibroblast which can act as progenitor cells. One promising tool to recreate a vascular network in artificial tissue constructs is the use of 3D printable spheroids. 3D printing in general offers the opportunity to have spatial control about release of different materials depending on its properties. In this project we are aiming for using cell spheroids as a 3D printable material. Spheroids display a scaffold free multicellular tissue construct which is characterized by three dimensional cellular self-assembly and by strong cell-cell and cell-ECM interactions1. Furthermore spheroids represent more physiological and functional characteristics compared to 2D cell culture 3,4. By using spheroids consisting of cells which reflect the physiological cell composition and having the ability to fuse with each other to larger scale multicellular constructs, they show a promising potential for usage as printable building blocks

    Cultivation and characterisation of cell spheroids consisting of different cell types regarding their time related compaction rate and fusion ability as prediction for use as 3D printable building blocks. Additional analysis of cell survival and cell proliferation at different states of the spheroid characterization.

    Materials and Methods
    We use manufactured SU8/Si wafer as a positive mold to create patterned elastomeric stamps of poly (dimethylsiloxane) (PDMS) for microwell array platforms. A 4 % agarose solution was cast onto the PDMS stamp for non-adherent cell spheroid culture to cultivate spheroids made of 100 % smooth muscle cells (SMC), 100 % mesenchymal stem cells (MSC) and a co-culture of 90 % SMC and 10 % human umbilical vein endothelial cells (HUVEC). Cell spheroids, consisting each of approximately 267 cells, were cultivated for 9 days to analyse the spheroid formation and spheroid compaction over time using a custom developed MATLAB based program for automate data analysis. Spheroids at day 9 were stained for Live/Dead and Ki67. Afterwards ,the spheroids were transferred to another agarose made microwell array platform to analyse the fusion between different spheroids of the same cell types (MSC-Spheroid : MSC-Spheroid, SMC-Spheroid : SMC-Spheroid, SMC-HUVEC-Spheroid : SMC-HUVEC-Spheroid) and spheroids of different cell types (SMC-Spheroid : MSC-Spheroid, SMC-HUVEC-Spheroid : MSC-Spheroid) for 48 hours by using different cell trackers with subsequent staining for Live/Dead and Ki67.

    Figure 1. Schematic overview of the current workflow to analyse spheroid cultivation, compaction and fusion as a final use as 3D printable building blocks

    Results and Conclusions
    Depending on the cellular composition of the spheroids, different compaction times could be observed, resulting in a final size range of 70-120µm despite the same cell number in each spheroid type. After the final compaction it could be shown, that the spheroids didn’t developed a necrotic core or specific proliferation zones. Due to the differences in final compaction size, heterologous spheroid fusion showed a different dynamic compared to homolog spheroid fusion.

    1. Whitesides GM, Grzybowski B. Self-assembly at all scales. Science 2002; 295: 2418–213
    2. Fukuda J, Nakazawa K. Orderly Arrangement of Hepatocyte Spheroids on a Microfabricated Chip. Tissue Eng 2005; 11: 1254–1262.
    3. Bhang SH, Cho S-W, La W-G, Lee T-J, Yang HS, Sun A-Y et al. Angiogenesis in ischemic tissue produced by spheroid grafting of human adipose-derived stromal cells. Biomaterials 2011; 32: 2734–2747.

    This work is supported by an ERC Consolidator Grant under grant agreement no 724469.
    Original languageEnglish
    Publication statusPublished - 30 Nov 2018
    Event27th NBTE Annual Meeting 2018 - Congrescentrum De Werelt, Lunteren, Netherlands
    Duration: 29 Nov 201830 Nov 2018
    Conference number: 27


    Conference27th NBTE Annual Meeting 2018
    Internet address


    • Vascular tissue engineering
    • Tissue Engineering
    • 3D Printing


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