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4-Deep Brain Reconstruction

Periodic Reporting for period 1 - 4-DBR (4-Deep Brain Reconstruction)

Reporting period: 2022-09-01 to 2023-08-31

Neurodegenerative diseases (NEDs) are a heterogeneous group of disorders that occur when neurons in the brain or peripheral nervous system either progressively lose their function and structure or die. There is no curative treatment for these diseases, with available treatments only slowing down progression or reducing patient symptomatology. Unfortunately, neurodegenerative diseases affect around 180 million of Europeans, with 1 of 3 of us expected to be diagnosed with one of these disorders during our lifetime. Consequently, the social impact and economic healthcare burden is enormous, with an annual cost of 798 billion €.

A key bottleneck in developing curative treatments for these disorders, is that once symptoms appear the patient’s brain region is already affected. Given the complexity of neuronal cells, it is difficult for standard treatments to reverse adult brain degeneration. Hence, advanced therapies have been referred as an alternative treatment as healthy brain cells can be implanted in the degenerated region. Nevertheless, trials with these therapies in neurodegenerative patients have not proven successful, as cells are not able to appropriately implant or remain active in the given region.

The 4-DBR project aims to develop a 4D-minibrain that will integrate into the specifically allocated area of the brain, and these cells will only mature once it has been transplanted. Moreover, the high quality of our scaffold will ensure the stability of the cells and the patient-based or organic origin of the material used will minimize tissue rejection. The 4-DBR consortium will join expertise to generate a new implantable 4D-bioprinted reconstructed brain that will contain (I) the corresponding 3D architecture; (II) a vascular system, which will provide the functional cells with nutrients and chemical cues; and (III) a customised optogenetics system to stimulate brain cells to differentiate and record neuronal activity after implant.

In addition, the resulting 4D-minibrain will also be useful for drug development in neurodegenerative disorders. Using functionalised human tissue to screen medication candidates would speed up development and provide key tools to facilitate personalised medicine, while saving money and reducing the number of animals used for research. This is particularly important in neurodegenerative diseases, where existing animal models have proven to be inadequate in predicting human response due to major differences in brain functionality and composition.

The main breakthrough envisaged of the 4-DBR project are:

- Printable committed hNPCs
- New decellularization methods for brain specific areas
- 3D imaging decellularized blood capillaries.
- Novel 4D bioprinting technologies to obtain a tridimensional blood-brain-barrier (BBB)
- Cerebral stimulation system based on a 3D printed opto-chip
The main objective of 4-DBR is to generate a new implantable 4D-bioprinted RB that contains a vascular system, specific human neurons that will be stimulated by a customized optogenetic system to bypass the bottlenecks in cell-based therapies for NEDs.

Performed work:

- Developed and optimised successful neuronal differentiation protocol.

- Freeze, thaw and expand the differentiated cells at several time points.

- Test differentiation protocols for (I) astrocytes; (II) endothelial cells; and (III) pericytes.

- Test different media with co-culture to find out which one works best.

Ongoing work:

- Set the parameters and characteristics for the bioprinted scaffold.

- Optimize the protocol for extracellular matrix (ECM) decellularization.

- Set bioprinter conditions for NPCs and other cell types.

- Improve bioink composition for bioprinting consistency.

- Developing opto-chip.

- Testing optogenetic stimulation.
Due to the lack of effective treatments for neurodegenerative patients, the 4-DBR impact will generate a huge advance in the study and treatment of the disorders. 4-DBR consortium wants to offer a new approach for clinical trials and thus, reduce the suffering of patients. Consequently, this will alleviate the health and economic burden by stopping costly treatments that only treat symptoms.

Moreover, the new alternative 4D minibrain could be also used as a platform for in vitro drug screening and therefore, save money and reduce the number of animals used for research.

The main achievements of the 4-DBR consortium beyond the state-of-the-art are:

- Differentiation of endothelial cells from hPSCs

- Selection of specific hNPCs using cell Surface markers

- Vascular networks for tissue engineering

- 3D printed hollow optoelectrical waveguides

- Functional human blood-brain barrier by in vitro differentiation of pluripotent stem cells