STACKS - GUTVIBRATIONS is based on a 3D printed platform called STACKS established at the Technical University of Denmark. STACKS are simple to use, easy to fabricate and are a readily scalable and stackable system. It is a multilayer system consisting of 3D printed multiwell plates with each plate mimicking a different organ. A STACKS plate is very similar to Transwell® inserts. Unlike Transwell® inserts, STACKS have an opening onto which an extra cellular matrix (ECM) like layer can be cast, which provides the natural cellular microenvironment as found in vivo. The well plates can be stacked on top of each other creating a multi-organ system.

GUTVIBRATIONS will use Biosilk, a biomaterial made from recombinant spider silk protein. Biosilk is a biocompatible, biodegradable, and non-immunogenic biomaterial that can be functionalised with specific ECM proteins such as laminins and collagen. The spider silk protein is produced by recombinant expression in a bacterial system that makes it highly robust and reproducible. This is a significant improvement on traditionally used ECM layers such as Matrigel and collagen that are animal-derived and show lot-to-lot variability. Biosilk is also highly versatile and can be remodeled in different forms depending on the physiological segment that needs to be mimicked. In GUTVIBRATIONS, Biosilk will be used in two different forms - Biosilk nanomembrane (representing the basement membrane) and Biosilk (representing the underlying ECM). Both forms of Biosilk are provided by Biolamina AB and KTH Royal Institute of Technology.

Together with the STACKS and Biosilk, gut and brain organoids will form the basis for the next-generation gut-brain axis organ-on-chip (Ooc). Gut organoids are derived from intestine-specific stem cells. Stem cells self -organise into 3D structures to form organoids. Brain organoids are brain-like 3D cultures derived from pluripotent stem cells and contain brain-specific cell types. The gut and brain organoids are provided by the Amsterdam University Medical Centers, UniQure and KU Leuven.

STEMCELL Technologies (SC) will provide validated media for the formation and culture of human brain organoids. In addition to the different cell types found in the gut and brain organoids, these organs are supported by several other cell types with specific functions in vivo. For the brain layer, it is vital to include a Blood Brain Barrier (BBB) supported by brain associated cells such as pericytes, astrocytes, and microglia. Media for generating these different cell types from iPSCs will also be provided by SC. In GUTVIBRATIONS, specialised media will be developed by SC for each co-culture system. Each layer of the gut-brain axis organ-on-chip will be supplied with a different media, enabling straightforward integration.

The brain-gut axis organ-on-chip provides a model to investigate important questions regarding the pathogenesis of viral diseases. The viruses that will be investigated within GUTVIBRATIONS are Enterovirus (EV), Human immunodeficiency virus (HIV) and Human norovirus (HuNoV) which all initiate infection in the gut and can result in neurological symptoms later on.

The brain-gut axis organ-on-chip provides a system that can accurately simulate diseases caused by viruses, allowing for drugs to be tested against these specific diseases. Within GUTVIBRATIONS, the potential treatment of two different viral infections will be studied.

To make the STACKS platform suitable for industrial applications, the STACKS will be scaled up to a 96-well format for high throughput screening. In addition, GUTVIBRATIONS will ensure that the 96-well format is compatible with pipetting and will adapt the plate format to a polystyrene base for high throughput manufacturing.