Methodology

The research team developed a custom hydrogel based on polyethylene glycol diacrylate (PEGDA), which is modified with pentaerythritol triacrylate (PETA) and Triton-X 100 to balance polymer density and porosity. This formulation allows for the creation of non-swelling, cell-permeable hydrogel structures, crucial for maintaining the fidelity of the printed microvessels.

Using high-resolution 2-photon stereo-lithography, the team printed microfluidic grids directly onto a hard plastic base. These grids feature capillaries as small as 10 µm in diameter, enabling the perfusion of large, multi-millimeter scale tissue constructs. The microvessels are embedded in a supportive hydrogel matrix, facilitating nutrient and waste exchange throughout the tissue.

Results

Perfusion of Neural and Liver Constructs:

The researchers demonstrated the viability of their method by perfusing neural and liver tissue constructs. Single-cell RNA sequencing (scRNAseq) and immunohistochemistry revealed accelerated neural differentiation and reduced hypoxia in the perfused constructs compared to non-perfused ones. This suggests that perfusion significantly improves the functional maturation and viability of large tissue constructs.

Cell Viability and Proliferation:

Brightfield microscopy and flow cytometry showed a substantial increase in cell proliferation in perfused constructs, with a 5-fold increase in total cell numbers compared to non-perfused constructs. Immunohistochemistry for apoptosis markers (cleaved Caspase 3) and hypoxia markers (HIF1α) further confirmed reduced cell death and hypoxia in perfused tissues.

Gene Expression Analysis:

Differential gene expression analysis indicated significant transcriptional differences between perfused and non-perfused tissues. Perfused constructs exhibited upregulation of genes related to cell proliferation and neural differentiation, while non-perfused constructs showed higher expression of hypoxia and stress-related genes. Gene Ontology (GO) enrichment analysis supported these findings, highlighting processes like cell division and neural precursor proliferation in perfused tissues.

Conclusion

This study showcases a groundbreaking approach to creating vascularized tissue constructs using synthetic 3D microfluidics. The ability to generate dense, perfusable microvessels at capillary scales opens new avenues for tissue engineering, enabling the development of large, functional tissue models. These advances could have significant implications for drug discovery, disease modeling, and regenerative medicine.

References

The original article by Sergei Grebenyuk, Abdel Rahman Abdel Fattah, Manoj Kumar, Burak Toprakhisar, Gregorius Rustandi, Anja Vananroye, Idris Salmon, Catherine Verfaillie, Mark Grillo, and Adrian Ranga can be found in Nature Communications here.