3D bioprinting is ideally positioned to promote the (vascular) complexity of in vitro engineered tissues. However, the resolution of extrusion-based printing, the most widespread bioprinting technique, is too limited to create microstructural features such as capillaries. Therefore, we have previously developed printable vascularized spheroids (Ø≈100-130 µm) by seeding human umbilical vein endothelial cells (ECs), human foreskin fibroblasts and adipose-tissue derived mesenchymal stem cells (MSCs) onto agarose microwell chips. These spheroids remain stable and viable over at least 10 days and the ECs organize into a capillary-like network1.
The spheroids can be printed in a methacrylamide-modified gelatin (GelMA) hydrogel and extensively grow out in the gel. Sprouts of individual spheroids inosculate and the spheroids fuse, but only when they are placed in close proximity2. Unfortunately, the encapsulation density is currently too low to obtain extensive fusion of all spheroids into one tissue. Therefore, our current efforts focus on the optimization of the printing protocol and the maturation of the constructs post-printing.
We aim to advance the spheroid outgrowth and fusion by lowering the hydrogel concentration and enhancing the encapsulation density. We print 6-layered grid constructs with a 0°/90° lay-down pattern or macrovascular cylindrical constructs with an inner diameter of 1 mm.
Since hypoxia is a known stimulator of angiogenesis, we are also assessing the impact of low-oxygen culture on non-printed and printed spheroids.
Spheroids are analyzed via light, fluorescence and confocal microscopy. Immunohistochemistry/qRT-PCR is applied to detect growth factors as vascular endothelial growth factor and vascular elements as VE-cadherin, collagen type IV, laminin and EC markers as CD31.
In addition, we are innovating our protocol using human induced pluripotent stem cells (hiPSCs). We are currently differentiating hiPSCs, transfected with the endothelial transcription factor ETV2, to ECs3. These cells will be combined with hiPSC-derived MSCs to create hiPSC-derived vascularized spheroids.
We were able to reduce the GelMA concentration from 10 to 8 w/v% while maintaining a stable construct integrity. This reduced concentration allowed us to double our original encapsulation density of 22 920 spheroids/ml gel. The spheroid outgrowth and fusion was strongly increased compared to our previous results, but it was not complete yet so further optimizations are required.
Preliminary data indicate that hypoxia slightly increases the vascularization in non-encapsulated spheroids. Data on the influence on printed spheroids will be gathered in the coming months.
Lastly, we could successfully differentiate hiPSCs to ECs and we will combine them with hiPSC-derived MSCs in a spheroid culture in the following weeks.
Spheroids form versatile building blocks for the 3D bioprinting of vascularized tissues. Lowering the hydrogel concentration, enhancing the encapsulation density and the implementation of hypoxia show to promote spheroid vascularization and fusion. Future experiments will further optimize and synergize these strategies to engineer one whole microvascularized tissue and answer the need for novel vascularization approaches in tissue engineering.
1. De Moor, L. et al., Biofabrication 10 (2018).
2. De Moor, L. et al., Biofabrication 13 (2021).
3. De Smedt, J. et al., Cell Death Dis. 12, 84 (2021)."