# Construction of an In Vitro Blood–Brain Barrier Micro-Organoid Model Using Decellularized Squid Mantle Scaffold Film

## TL;DR

Researchers created a new in vitro model of the blood-brain barrier using a squid mantle scaffold to better study neurological diseases and drug permeability.

## Contribution

A novel BBB micro-organoid model using decellularized squid mantle scaffold films for a more authentic NVU microenvironment.

## Key findings

- The DSMS film supported the growth of endothelial and astrocyte cells, forming a BBB-like structure.
- The model showed functional barrier properties with high TEER and low permeability to macromolecules.
- LRP1 expression was used to assess model integrity and response to thrombotic conditions.

## Abstract

Although blood–brain barrier (BBB) models are of great value in investigating neurological diseases, the structural complexity and intricate function based on cell–cell interactions of the BBB bring various limitations to the applications of existing models. In this study, a novel BBB micro-organoid model was established by culturing neurovascular unit (NVU) cells on a decellularized squid mantle scaffold (DSMS) film to reconstitute a more authentic and reliable NVU microenvironment for in vitro research. The DSMS applied was obtained from squid mantle scaffolds via decellularization, followed by defatting, and showed good biocompatibility with no cytotoxicity. The DSMS film was finally prepared by lyophilization. The lyophilized film exhibited a void ratio and pore size suitable for the adhesion and growth of endothelial cells (hCMEC/D3) and astrocytes (hACs), which led to the formation of a BBB-like spatial structure. The BBB micro-organoid model exhibited functional barrier properties, including an effective transendothelial electrical resistance (TEER) of approximately 230 Ω/cm2, restricted permeability to macromolecules—with apparent permeability coefficients (Papp) of 6.3 × 10−7 cm/s for 10 kDa and 2.7 × 10−7 cm/s for 70 kDa FITC–dextran—and expression of tight junctional complex (TJC) proteins such as vascular endothelial cadherin (VE-cad) and Zonula Occludens-1 (ZO-1). Furthermore, low-density lipoprotein receptor-related protein 1 (LRP1), a key receptor stably expressed in these two NVU cell types, was utilized as a critical indicator to assess the integrity of the BBB micro-organ model and its responsiveness to pathophysiological stimuli, particularly under thrombotic conditions. This study not only validates the feasibility of constructing a functionally competent BBB micro-organ model using DSMS films integrated with NVU cells but also provides a promising in vitro platform for subsequent studies on the BBB-related pathological mechanisms and the evaluation of drug permeability across the BBB.