Development of synthetic vasculature, 4D optical oxygen mapping and their implementation in human 3D liver tissue models

3D in-vitro organ models for applications in toxicology, pharmacology and disease modelling that closely recapitulate in vivo microenvironments have come into increasing focus since both microenvironmental cues and cell-cell signalling have been shown to improve maintenance of mature primary phenotypes and improving maturation of induced pluripotent stem cell derived cells, thus improving the accuracy of in vitro to in vivo translation of pharmacological assays and disease models.
While a wide variety of complex 3D organ models have been presented, efficient synthetic vascularization of 3D tissues with in vivo like cell densities, 3D oxygen mapping methods for optimizing cellular microenvironments and 3D liver organ models with dense interconnected tissues, cultured at acinus-like oxygen microenvironments are still missing.
Here we present the development of a phosphorescent lifetime microsensor probe based 4D oxygen mapping method capable of visualizing 3D oxygen distributions in dense tissues in both static and dynamic microenvironments. We present the development of diffusion-open, 3D projection lithography printed synthetic vasculature scaffolds that enable meso-scale tissue cultures at in-vivo like cell densities in controlled oxygen microenvironments. Further we present the combination of the synthetic vasculature scaffolds and 3D oxygen mapping to create 3D liver organ models with interconnected tissues cultured at in vivo like cell densities and liver acinus like oxygen microenvironments.
For the physiologically most relevant oxygen tensions between 0% and 10% and approximate cell concentrations of 50 mio. cells mL^-1, 3D resolved localized oxygen readings up to 500 μm into tissues with an error below 1%  O₂ could be acquired. The synthetic vasculature scaffolds allowed the culture of interconnected tissues at in-vivo like cell densities without embedding hydrogel that enable unhindered cell-cell signalling. With maximum cell to vasculature distances of 190 μm, tissue oxygenation gradients could be flexibly adjusted in steepness, starting at 13% O₂ as maximum tissue oxygenation level.
iPSC-based liver organ models significantly improved maturation of hepatocyte-like cells compared to spheroid suspension cultures, with downregulation of α-fetoprotein and strong upregulation of CYP3A4 and CYP1A2 expression. Developed primary liver MPS vastly improved maintenance of key liver functions over a 14 day culture period compared to 2D adhesion cultures and markedly over 3D spheroid cultures.
In addition, the developed methods were successfully applied to a variety of other culture formats and organ models to validate oxygenation and establish in-vivo like microenvironments.