Neurologic disorders affecting human brain have often proved difficult to recapitulate in animal models. The efforts to develop a 3D culture model of neurons and glia have contributed a more realistic microenvironment, enabling cell diversity, migration, homogeneous distribution of nutrients and oxygen. Consequently, cell morphology and cell-cell interactions through gradients of endogenous growth factors and chemokines are maintained.

These models have been largely contributing to neuroscience research and regenerative medicine, providing results with a greater accuracy than conventional 2D method. Cerebral organoids, bioengineered tissues, brain-on-a-chip and 3D bioprinting are some of the three-dimensional examples that have emerged to revolutionize the drugs discoveries and to study a variety of neurological diseases.

As mentioned before on the blog Mini Brains!, cerebral organoids were developed in 2013, by Madeline Lancaster. Growing human brain cells as miniature three-dimensional organ is a real breakthrough! The cytoarchitecture of these self-organizing brain tissues presents a variety of brain regions with appropriate cortical cell layers, recapitulating some hallmarks of embryonic brain development and can model human neurogenesis (differentiation and maturation), neuronal migration and network formation.

When mini brains are generated from induced pluripotent stem cells (iPSCs) - obtained from patients with brain disorders-, they can acquire the same compendium of cell types and genetic predisposition to develop the disease. For example, in microcephaly studies, cerebral organoids are designed with smaller neuroepithelial tissues reminiscent of the reduced brain size seen in patients, while those designed for Alzheimer studies develop spontaneously amyloid plaques and abnormal tau accumulation over time (figure 1).

Initially, due to the absence of some type cells, researchers were skeptical whether organoids could develop a functional neural network. A researcher in University of California, San Diego, plated mini-brains on culture dishes containing multi-electrode arrays and showed that neurons in cortical organoids were capable of firing, indicating that neural networks were present. In addition, they used a virtual branch of artificial intelligence, called machine learning, to predict the organoid developmental timeline, applying a cross-validation between electrophysiological neural activity of the in vitro model and premature babies aged 24 to 38 weeks electroencephalogram (EEG). Data collected from the cortical organoids on multi-electrode array showed that network development resembles with high fidelity after 25 weeks some neonates EEG features (Figure 2).

Although there are some limitations in this approach regarding lack of vascularization and immune system, the results have been showing the ability to study not only brain development and several neurological diseases, but also neurophysiology. With future improvements, brain organoids hold a great promise for neuroscience and regenerative medicine.

Tissue engineering in regenerative medicine focuses on restoring cells, tissues and organs, and produces scaffolds to delivery drugs/proteins or for implants. In the research field, on the other hand, biomaterials and cells produce a more realistic in vivo environment, which can be further submitted for experiments, such as drug screening, cell migration, neurodevelopment and molecular studies.

Biomaterials must have biocompatibility and cause a positive effect on cell growth, differentiation, migration and degradability in vivo after implantation. They can be made of natural polymers (e.g., collagen, gelatin, fibrin, chitosan, cellulose), synthetic [e.g., poly (lactic-co-glycolic acid) – (PLGA); poly-ethylene glycol – (PEG)], or hybrid (e.g., PLGA/collagen, PEG-liposomes).

Zamproni et al (2017) combined two synthetic polymers, PLGA and PEG to produce nanoparticles charged with a chemoattractant and pro-neurogenic protein (SDF1 – Stromal derived Factor-1) in brain-injured mice. This molecule is responsible to stimulate neural stem cells migration from neurogenic niches towards brain injury and, thus, restore tissue loss. A significant increase of migrating cells was observed when nanoparticles were injected and SDF1 was released, showing that this model can contribute to ameliorate brain injuries (figure 3).

 

               Brain-on-a-chip is a technology that enables long-term culture of multiple cell types (such as progenitor and neural stem cells) in a controlled and monitored environment with high precision. Many approaches to produce a brain-like tissue fail to recapitulate the neurovascular unit and neural circuitry, which are essential to the homeostasis, cell communication and nutrients diffusion. Brain-on-a-chip uses micro-channels with different heights and widths that inter-connect the micro-environments, simulating the most complex organ in the human body – the brain.

               Researchers from John Hopkins Hospital, USA, used a multi-layered device with neurons and astrocytes to build a model resembling the brain parenchyma. The biocompatibility of the device allowed neuronal maturation and axons could spread through the channels, interconnecting with astrocytes. This neuronal-glial environment was also interfaced with a layer of human brain microvascular endothelial cells, mimicking the blood brain barrier. This platform is, therefore, not only attractive for the ability of reproducing brain tissues, but it also resembles the integrated compartments of central nervous system and allows a variety of neurotoxicity and drug delivery studies.

Replications of 3D structures similar to what we see in vivo are difficult using conventional bioengineering methods. The 3D bioprinting technology has recently emerged bringing more complexity for tissue engineering with a higher accuracy, organization and a broad range of medical and research applications.

The approach uses a bioink, which is a hydrogel carrier vehicle formed by natural, synthetic or hybrid polymers. Through three different methods (inkjet, extrusion or laser-assisted printing), the bioink previously mixed with cells is deposited via a print-head to produce a biomimetic construct. Multiple bioinks and cells can be used in the same construct to give the tissue layers, compartments and, thus, more complexity. After deposition, the hydrogels are crosslinked by using physical (e.g., temperature) or chemical methods (e.g., calcium chloride) to stabilize the constructs (figure 5). The constructs can be maintained in the incubator and submitted for molecular and cellular experiments.

The tissue is designed on computer software with adequate structure to homogenously receive nutrients, stimulate cellular proliferation and differentiation and allow cells to migrate in any direction through the porous flexible network. Entire or partial organs can be submitted for drug screening, molecular and cellular experiments. Implants are also produced and engrafted in damaged tissues to promote recovery.

3D bioprinting technology is potentially revolutionizing the field of neural tissue engineering by increasing its throughput, reproducibility and getting us through a new era of a more personalized medicine.

Lovejoy, Christopher Edward James, et al. "3D CEREBRAL ORGANOIDS AS IN VITRO MODELS FOR ALZHEIMER’S DISEASE." Alzheimer's & Dementia: The Journal of the Alzheimer's Association 13.7 (2017): P327.

Trujillo, Cleber A., et al. "Complex oscillatory waves emerging from cortical organoids model early human brain network development." Cell Stem Cell 25.4 (2019): 558-569.

Zamproni, Laura N., et al. "Injection of SDF-1 loaded nanoparticles following traumatic brain injury stimulates neural stem cell recruitment." International journal of pharmaceutics 519.1-2 (2017): 323-331.

Kilic, Onur, et al. "Brain-on-a-chip model enables analysis of human neuronal differentiation and chemotaxis." Lab on a Chip 16.21 (2016): 4152-4162.