In Vitro Vascularization Controlled by Local Cell Density Enhancement

Sound-induced endothelial cell condensation leads to the formation of stable vascular structures


  • Endothelial cells can be patterned in different hydrogels, resulting in distinct sprouting

  • Local cell density matters and can be enhanced by sound at lower initial cell number

  • In reproducible systems, vascular structures can be observed over time

Engineering large scale in vitro tissues is often hampered by the lack of vascularisation, resulting in nutrient deprived and necrotic cores. In clinical scenarios, vascularisation is fundamental in wound healing after injury or surgery, and of paramount importance for proper integration of e.g. bone grafts. Our recent work showed that local cell density and close proximity of endothelial cells is crucial for the formation of perfusable, vascular structures(1). Specifically ,it was shown that endothelial cells can be assembled by low frequency sound, leading to increased branching at lower initial cell seeding density compared to a standard microfluidic approach. Our research is thus driven by the hypothesis that local cell density enhancement of endothelial cells recreates the physiological cell density and thus favours in vitro vasculogenesis. Here, we are exploiting the different possibilities within the mimix labware and biomaterial portfolio to generate vascular structures(2, 3). Ultimately, our robust and highly reproducible system will provide a basis for invitro evaluation of angiogenic/antiangiogenic drugs and will be implemented in tissue engineering approaches.


Figure 1

A 1:1 ratio of human mesenchymal stem cells (hMSC)and green fluorescent protein expressing human umbilical vein endothelial cells (GFP-HUVEC) was patterned in KYTO in labware of different shape or size A) Light microscopy pictures during patterning, B) fluorescent images of GFP-HUVEC (hMSC were not visualised). All scale bars 5 mm.


Figure 2

Sound induces local cell density enhancement: Local cell density has a significant influence on the formation of vascular structures. Left: at high local cell density, right: at low local cell density.


Figure 3

In the created in vitro model system, GFP-HUVEC assembly and sprouting can be observed over time. The chosen labware and applied sound frequency enable one to e.g. fine-tune the distance between concentric rings and their number. In square frames, a different pattern is created. This flexibility and versatility can be leveraged to study endothelial cell migration in response to the distance, curvature or size of the pattern. Controlled cell patterns and concomitant sprouting are particularly interesting to assess pro- or antiangiogenic drugs. The chosen hydrogel, KYTO, is based on Dextran and bears MMP-cleavable sites and RGD peptides. It therefore provides sites for cell adhesion and allows for potential cell migration and endothelial spreading which was observed over time. At the same time, the main structure is retained, with a maternal vascular structure in the form of two concentric rings from where endothelial sprots arise (inset).


Experimental Conditions

Biomaterial: ZAVA, KYTO

Cell Type: Endothelial Cells, Human Mesenchymal Stem Cells

Labware: mimiX Labware S10A and S10B, rings for small petri dishes


(1)Petta et al.,Sound-induced morphogenesis of multicellular systems for rapid orchestration of vascular networks, Biofabrication 2020.

(2)Results presented at the Swiss Symposium of Point of Care Diagnostics, 2021

Results presented at TERMIS World Congress 2021