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.
