Bridging from bench to bedside

In vivo evaluation of sound-patterned endothelial cells

Highlights

  • High local cell density condensation by sound leads to enhanced vascular structures

  • Patterns were retained and successfully implanted

  • Vascular structures were apparent after two weeks of implantation

Within the scope of personalised healthcare in the field of regenerative medicine, patient-derived cells are key players. Their successful application is, however, often hampered by low cell numbers at the expense of donor-site morbidity and lengthy in vitro expansion.


Novel biofabrication methods requiring lower initial cell numbers are therefore timely to address this unmet clinical challenge. In vitro, local cell density enhancement by use of sound induced morphogenesis (SIM) at low frequency of <100 Hz was shown to induce increased microvasculature formation at lower cell concentration than conventional methods.1 Based on these promising results, we initiated an in vivo study to investigate anastomosis of subcutaneously implanted cell-hydrogel biografts.


Endothelial cells and mesenchymal stem cells were patterned in two different hydrogels. These biografts were prepared at a cell density of 2⋅106 cells per mL (compared to standard cell densities of 10 to 20⋅106 cells per mL in conventional approaches) and implanted for 2 weeks at the back of nude mice. Based on preliminary data, we provide evidence that SIM is an excellent method to prepare dozens of samples in a few hours at high reproducibility.

The method proved stable, reliable, and safe with respect to sterility in this pre-clinical setting.


 

Figure 1

A’) Cell patterns obtained in a PEG-based hydrogel and B’) in a dextran-based hydrogel. A’’) and B’’) At higher magnification, increased cell-cell contacts at higher cell density (within the pattern) are apparent.


 

Figure 2

A) Cell patterns were retained throughout a 3-day in vitro culture and visible by the naked eye. B) The specially designed mimiX labware can easily be removed from the petri dish to release the biograft. C) Hydrogel properties were adapted to match viscosities that allow for SIM and result in a solid biograft after gelation. The biograft can be lifted and implanted with spatula. D) Four biografts were subcutaneously implanted for 2 weeks. Subsequently, mice were euthanised and biografts explanted for histological and microscopical analysis. SIM and mimiX labware are perfectly integrated in this complex workflow.


 

Figure 4

After two weeks, biografts were explanted and imaged with a confocal laser scanning microscope (CLSM). A) Image of the full construct and B) image at higher magnification.

Implanted endothelial cells express green fluorescent protein (GFP) and can therefore be visualised after explantation. Based on CLSM images, implanted endothelial cells remained viable over two weeks and assembled into microvascular structures. Future experiments will investigate whether they connected with the host vasculature.


 

Experimental Conditions

Biomaterial: PEG-based and dextran-based hydrogels.

Cell Type: Endothelial Cells, Human Mesenchymal Stem Cells

Labware: mimiX Labware S10A


References

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