From inserts to chips: microfluidic culture and 3D astrocyte co-culture drive functional and transcriptomic changes in hiPSC-derived endothelial cells.

Background: The blood-brain barrier (BBB) exhibits a hurdle for drug delivery and development. In addition, the dysfunction of the BBB has been seen in several neurodegenerative diseases, although the mechanisms remain poorly understood. Thus, improved models are needed for the purposes of disease modelling and drug development. To overcome the constraints of conventional in vitro models, there has been a growing use of human induced pluripotent stem cells (hiPSCs) and organ-on-chip systems. However, the detailed characterization of these models is still mainly missing. We aimed to investigate how different culture platforms alter the functionality and, consequently, the transcriptomic phenotype of hiPSC-derived endothelial cells (ECs).

Methods: ECs were cultured on a microfluidic BBB chip platform (AKITA plate) or a standard cell culture insert model. Furthermore, we used hiPSC-derived astrocytes in the AKITA plate format to examine their effect on ECs. Astrocytes were cultured under either 2D or 3D conditions. The impact of pore size and culture system was studied using permeability assays and protein expression. Finally, we used single-cell RNA sequencing to analyze transcriptional changes in ECs cultured on insert or AKITA plate, both with and without astrocytes.

Results: First, we tested the impact of different membrane pore sizes in AKITA plate on EC morphology and barrier formation. We demonstrated that the AKITA plate supports confluent monolayer formation, even with higher pore sizes. Secondly, ECs cultured on AKITA plate showed improved barrier function and reduced migration in comparison to ECs cultured on inserts, supported by permeability experiments and transcriptomics. The single-cell RNA sequencing revealed the activation of cholesterol metabolism-related pathways in ECs cultured on an AKITA plate under flow conditions. At last, we discovered that astrocytes require 3D culture to sustain the EC monolayer. Moreover, astrocytes promote a slight shift in transcription levels by upregulating genes associated with EC-astrocyte interactions.

Conclusions: Complex cell culture systems are becoming accessible; still, additional research into their properties is needed. Our data highlights the importance of the cell environment and its impact on the cellular function and gene expression profiles. Understanding these changes can improve future models and facilitate the development of more physiologically relevant platforms.