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Custom architecturally defined 3D stem cell derived functional human neural networks for transformative progress in neuroscience and medicine

Periodic Reporting for period 3 - MESO_BRAIN (Custom architecturally defined 3D stem cell derived functional human neural networks fortransformative progress in neuroscience and medicine)

Reporting period: 2019-09-01 to 2020-02-29

MESO-BRAIN aims to grow human brain cells in the laboratory and to image for the first time brain cells communicating with each other. This work will allow scientists to study how the human brain works and could one day help combat the damage caused by Alzheimer's and other brain traumas. To do this, new three-dimensional structures are needed to grow cells in a similar way to how they grow in our own brains. New techniques which allow the cells to be studied without damage will allow the basic functions of the brain to be better understood, ultimately leading to new therapies for brain related diseases and conditions.
MESO-BRAIN has succeeded in using novel laser-based multiphoton printing technology to build 3D nanoscale biocompatible scaffolds with integrated conductive electrodes. These have been populated with human brain cells (neurons) and imaged to observe the process by which neurons send out signals that connect them to other neuronal cells. The 3D structures have successfully demonstrated the technology of building layers of cells with different types of cells in each layer and witnessing the connections that develop between them in a brain-like way. Further development of the 3D scaffolds is expected beyond the MESO-BRAIN project to micro-structure the surface of the scaffolds so that cells can be positioned within a given layer to control function and realise an even more brain-like model to support drug development activities.

The conductive electrodes can support stimulation of the cells. In this way, measurements and imaging observations of the response of the cell network to a given stimulation pattern can support the evaluation of drug therapy by comparing patterns of response in the scaffolds.

MESO-BRAIN has produced four main areas of exploitation as follows:

3D Scaffolds are being exploited via spin-out company Laser nanoFab GmbH
• Further development of novel three-dimensional scaffolds with embedded electrodes
• Development of new complex scaffold designs, exploiting IP in two-photon polymerisation, 3D scaffold design and micro-structuring
• A range of scaffold designs have been developed for customers based expressed needs
• An internet link has been established to allow potential users to specify scaffold requirements for their research and development activities

Axol Bioscience lead the commercialisation of neuronal cell types developed in the project:
• Working with their existing customers and in partnership with Laser nanoFab
• Supplemented by fast maturation protocols to reduce development times

ICFO is leading the exploitation and commercialisation of the fast 3D imaging system developed in the project based on light-sheet techniques
• The Light-sheet microscope features a module for aberration correction and fast-volumetric imaging
• A prototype system will be transferred to an industrial partner for demonstration and evaluation

University of Barcelona is continuing to develop the NETCAL software developed in the project used to analyse the brain-like activity of the populated scaffolds
• Developing user-friendly tools, 2D and 3D in-vitro analysis models for neurological disorders

MESO-BRAIN partners have published a total of 16 papers to-date in peer-reviewed journals based on the research, analysis and findings of the project. All are open-access.
The tools and resources developed along the 3-year duration of the project advanced the present state of the art in nanofabrication of scaffolds, cell culturing, imaging and data analysis, and opened interesting prospects for further advances.

1) Progress in nanofabrication - the creation of miniature 3D platform with integrated electrodes for the in vitro modelling of human cortex is unimaginable without the application of the unique technology ‘two-photon polymerisation (2PP)’. In the course of the project, we have developed and demonstrated the possibilities that this technology offers to create such a 3D platform.

The 3D scaffolds developed represent an attractive tool for in vitro modelling of many types of tissues due to their unique features, which can be produced and specified by the application of 2PP technology. 2PP produced 3D scaffolds are reproducible and can any achieve different grades of complexity. Therefore, this type of 3D platform represents an appealing and reliable tool for in vitro tissue modelling, which should quickly find the wide acceptance in basic research and pharmacological industry for drug therapy testing.

2) Progress in cell culturing and tailored biological circuits
Protocols for cell differentiation and maturation were optimized. Co-cultures of excitatory cortical neurons and astrocytes and co-cultures of excitatory, inhibitory neurons and astrocytes were grown. Electrical recordings indicated that neurons could fire trains of action potentials in response to step inputs. Calcium imaging and multi-electrode array (MEA) recordings revealed neurons exhibiting independent activity patterns and also participating in synchronized events. All these studies indicate a functionality that suggests that the differentiated cells may indeed faithfully reproduce human neural activity. Additionally, the experiments provided a detailed characterization of the interaction between neurons and glia. This opens new avenues in medicine for understanding the role of glia in neurological disorders. In numerous neurodegenerative diseases, , it is known that altered neuron-glia interactions accelerates the degradation of the entire neuronal circuit. Thus, the protocols and expertise developed in combination with patient derived iPSC cell lines, can be used to develop accurate in vitro models of the neuron-glia interactions in disease and advance towards the development of treatments.

3) Progress in imaging and industrial partnership.
Imaging neuronal activity in a 3D environment is a challenging endeavour, with strong industrial interest, and at the frontline in photonics and microscopy. Traditionally, imaging techniques are used for retrieving structural or functional information contained in a plane (i.e. in 2D). Although accessing the third dimension is possible, the current high-resolution imaging techniques are cumbersome and slow, as they are based on point scanning methodologies. In addition, current measurement devices cannot cover large volumes. Technological developments have therefore been urged by the need to explore noninvasively the behaviour of complex and large 3D tissues, as these represent in a more accurate way the complicated conditions found in biological structures.

The partner ICFO within MESOBRAIN went beyond the state of the art by pushing the capabilities of light-sheet microscopy in terms of volumetric imaging speed, spatial volume covered, high resolution imaging and data treatment. In collaboration with the rest of the partners, by the end of the project it was possible to access a volume comprised of a rectangular area of 0.8 x 0.5 mm2 and a height of 0.4 mm with excellent image quality. This 0.16 mm3 volume was scanned as 6 planes/volume and an effective speed of 22 images/s in each plane. The achieved volumetric imaging speed is a factor of 2 X faster than other competitive imaging techniques (not commercially available) and allows for the precise monitoring of fast dynamics in 3D that occur in biological samples, including the functional traits of living neuronal networks.
Image of Neuronal brain cells