Study of angiogenesis in brain-on-a-chip format – BrAngioChip

We exploited two different pathways to create 3D neural tissues. First, we succeeded to spontaneously form 3D network of neurospheroids differentiated from human induced pluripotent stem cells (hiPSCs) supported by gelatin nanofiber meshes on a substrate with honeycomb micro-compartments. We also have succeeded to differentiate hiPSCs into brain organoids starting from spherical aggregates of hiPSCs. The electrophysiological activities of those 3D neural tissues were confirmed by the measurements using homemade micro-needle electrode arrays in collaboration with Wolfrum laboratory at Technical University of Munich. A brain microvascular endothelial cell layer was added to the neurospheroid network, showing a clear interaction between neurons, glia, and endothelial cells. We then challenged in situ formation of brain organoids on an engineered substrate for vascularization of brain organoids. By developing bi-facial gelatin nanofiber mesh, we have successfully formed brain organoids at the early stage and demonstrated the co-culture with brain microvascular endothelial cells.

We have developed two different micro-/nano-engineered substrates, one for in situ formation of brain organoids, the other for spontaneous growth of an interconnected neurospheroid network. Both multi-cellular structures were obtained from human induced pluripotent stem cells, and exploited for co-culture with brain microvascular endothelium. Further, the electrophysiological activities of 3D neural tissues, i.e., the above-mentioned neurospheroid network and conventional brain organoids, were measured by a homemade 3D micro-needle electrode array developed in collaboration with a laboratory at Technical University of Munich in Germany.

We have reached several important milestones, but the following final objectives have not yet been achieved.
- establish a complete system for dynamically observing cerebral organoid vascularization
- to study the vascularization of cerebral organoids in the presence of brain tumor spheroids
- create a microfluidic drug screening platform.
Even taking into account the difficulties posed by Covid-19 pandemic, these objectives were probably too ambitious. Nevertheless, thanks to the support of the ANR JCJC, we were able to achieve good results as described above, which were published in high impact journals (ACS Applied Materials & Interfaces, 2022 and 2023). Fortunately, the progress made in this project enabled us to obtain new fundings (Prématuration PSL/Qlife, Institut Carnot IPGG) and a PhD grant (ED 388) to continue the project immediately after the end of the ANR JCJC funding. We will thus try to achieve the remaining objectives in coming years.

We have published two peer-reviewed articles in a high-impact journal, ACS Applied Materials & Interfaces, one on the formation of 3D neurospheroid network derived from hiPSCs and neurovascular unit mimicry within a micro-substrate, the other on the development of a 3D micro-needle electrode array and its application for the 3D neurospheroid network and human brain organoids. The project was presented in 2 international conferences (invited talks) and 4 French conferences (2 invited talks, 2 posters), as well as in 2 Japanese conferences for Japanese researchers in Paris and in Europe, respectively (invited talks).

We propose to develop unique platforms combining human iPSC-derived brain organoids and microfluidic organ-on-chip technologies to study angiogenesis in the context of brain vasculature and brain tumor evolution. We will focus our research on 3 main targets: i) spontaneous angiogenesis in a brain organoid, ii) tumor-induced angiogenesis in the presence/absence of brain organoids, and iii) development of drug testing platforms. Using our on-chip 3D cell co-culture technology, we will be able to position brain organoids and tumor spheroids at the vicinity of a reconstituted microvessel in extracellular matrix, and characterize angiogenesis mechanisms under different conditions. In parallel, we will develop a new microfluidic device combining a biomimetic nanofiber membrane, organoids, endothelial cells, and a microelectrode array to analyze the effects of drugs on neuronal activity in a non-invasive manner.