Research in cell biology greatly relies on cell-based in vitro assays and models that facilitate the investigation and understanding of specific biological events and processes under different conditions. The quality of such experimental models and particularly the level at which they represent cell behavior in the native tissue, is of critical importance for our understanding of cell interactions within tissues and organs. Conventionally, in vitro models are based on experimental manipulation of mammalian cells, grown as monolayers on flat, two-dimensional (2D) substrates. Despite the amazing progress and discoveries achieved with flat biology models, our ability to translate biological insights has been limited, since the 2D environment does not reflect the physiological behavior of cells in real tissues. Advances in 3D cell biology and engineering have led to the development of a new generation of cell culture formats that can better recapitulate the in vivo microenvironment, allowing us to examine cells and their interactions in a more biomimetic context. Modern biomedical research has at its disposal novel technological approaches that promote development of more sophisticated and robust tissue engineering in vitro models, including scaffold- or hydrogel-based formats, organotypic cultures, and organs-on-chips. Even though such systems are necessarily simplified to capture a particular range of physiology, their ability to model specific processes of human biology is greatly valued for their potential to close the gap between conventional animal studies and human (patho-) physiology. Here, we review recent advances in 3D biomimetic cultures, focusing on the technological bricks available to develop more physiologically relevant in vitro models of human tissues. By highlighting applications and examples of several physiological and disease models, we identify the limitations and challenges which the field needs to address in order to more effectively incorporate synthetic biomimetic culture platforms into biomedical research.

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Advances in Engineering Human Tissue Models.

Neural progenitor cells generated from human induced pluripotent stem cells (hiPSCs) are the forefront of ″brain-on-chip″ investigations Viable and functional hiPSC-derived neuronal networks are shaping powerful in vitro models for evaluating the normal and abnormal formation of cortical circuits, understanding the underlying disease mechanisms, and investigating the response to drugs They therefore represent a desirable instrument for both the scientific community and the pharmacological industry However, culture conditions required for the full functional maturation of individual neurons and networks are still unidentified It has been recognized that three-dimensional (3D) culture conditions can better emulate in vivo neuronal tissue development compared to 2D cultures and thus provide a more desirable in vitro approach In this paper, we present the design and implementation of a 3D scaffold platform that supports and promotes intricate neuronal network development 3D scaffolds were produced through direct laser writing by two-photon polymerization (2PP), a high-resolution 3D laser microstructuring technology, using the biocompatible and nondegradable photoreactive resin Dental LT Clear (DClear) Neurons developed and interconnected on a 3D environment shaped by vertically stacked scaffold layers The developed networks could support different cell types Starting at the day 50 of 3D culture, neuronal progenitor cells could develop into cortical projection neurons (CNPs) of all six layers, different types of inhibitory neurons, and glia Additionally and in contrast to 2D conditions, 3D scaffolds supported the long-term culturing of neuronal networks over the course of 120 days Network health and functionality were probed through calcium imaging, which revealed a strong spontaneous neuronal activity that combined individual and collective events Taken together, our results highlight advanced microstructured 3D scaffolds as a reliable platform for the 3D in vitro modeling of neuronal functions

In Vitro Development of Human iPSC-Derived Functional Neuronal Networks on Laser-Fabricated 3D Scaffolds.

Many neurodegenerative diseases (NDs) such as Alzheimer’s disease, Parkinson’s disease, frontotemporal dementia, amyotrophic lateral sclerosis and Huntington’s disease, are characterized by the progressive accumulation of abnormal proteinaceous assemblies in specific cell types and regions of the brain, leading to cellular dysfunction and brain damage. Although animal- and in vitro-based studies of NDs have provided the field with an extensive understanding of some of the mechanisms underlying these diseases, findings from these studies have not yielded substantial progress in identifying treatment options for patient populations. This necessitates the development of complementary model systems that are better suited to recapitulate human-specific features of ND pathogenesis. Three-dimensional (3D) culture systems, such as cerebral organoids generated from human induced pluripotent stem cells, hold significant potential to model NDs in a complex, tissue-like environment. In this review, we discuss the advantages of 3D culture systems and 3D modeling of NDs, especially AD and FTD. We also provide an overview of the challenges and limitations of the current 3D culture systems. Finally, we propose a few potential future directions in applying state-of-the-art technologies in 3D culture systems to understand the mechanisms of NDs and to accelerate drug discovery.

/pdf/modeling-neurodegenerative-diseases-with-cerebral-organoids-y7v2m9k2zw.pdf

Modeling neurodegenerative diseases with cerebral organoids and other three-dimensional culture systems: focus on Alzheimer’s disease

Functional Characterization of Three-Dimensional Cortical Cultures for In Vitro Modeling of Brain Networks.

https://backend.orbit.dtu.dk/ws/files/245220732/1_s2.0_S0197018621000899_main.pdf

3D biomaterial models of human brain disease.

The prevalence of dementia and other neurodegenerative diseases continues to rise as age demographics in the population shift, inspiring the development of long-term tissue culture systems with which to study chronic brain disease. Here, it is investigated whether a 3D bioengineered neural tissue model derived from human induced pluripotent stem cells (hiPSCs) can remain stable and functional for multiple years in culture. Silk-based scaffolds are seeded with neurons and glial cells derived from hiPSCs supplied by human donors who are either healthy or have been diagnosed with Alzheimer's disease. Cell retention and markers of stress remain stable for over 2 years. Diseased samples display decreased spontaneous electrical activity and a subset displays sporadic-like indicators of increased pathological β-amyloid and tau markers characteristic of Alzheimer's disease with concomitant increases in oxidative stress. It can be concluded that the long-term stability of the platform is suited to study chronic brain disease including neurodegeneration.

A Long-Living Bioengineered Neural Tissue Platform to Study Neurodegeneration.

Injury progression associated with cerebral laceration is insidious. Following the initial trauma, brain tissues become hyperexcitable, begetting further damage that compounds the initial impact over time. Clinicians have adopted several strategies to mitigate the effects of secondary brain injury; however, higher throughput screening tools with modular flexibility are needed to expedite mechanistic studies and drug discovery that will contribute to the enhanced protection, repair, and even the regeneration of neural tissues. Here we present a novel bioengineered cortical brain model of traumatic brain injury (TBI) that displays characteristics of primary and secondary injury, including an outwardly radiating cell death phenotype and increased glutamate release with excitotoxic features. DNA content and tissue function were normalized by high-concentration, chronic administrations of gabapentinoids. Additional experiments suggested that the treatment effects were likely neuroprotective rather than regenerative, as evidenced by the drug-mediated decreases in cell excitability and an absence of drug-induced proliferation. We conclude that the present model of traumatic brain injury demonstrates validity and can serve as a customizable experimental platform to assess the individual contribution of cell types on TBI progression, as well as to screen anti-excitotoxic and pro-regenerative compounds.

https://www.mdpi.com/2218-273X/10/8/1196/pdf

A 3D Tissue Model of Traumatic Brain Injury with Excitotoxicity That Is Inhibited by Chronic Exposure to Gabapentinoids.

Learning and synaptic plasticity in 3D bioengineered neural tissues

The brain's extracellular matrix (ECM) is a dynamic protein-based scaffold within which neural networks can form, self-maintain, and re-model. When the brain incurs injuries, microscopic tissue tears and active ECM re-modelling give way to abnormal brain structure and function including the presence of ectopic cells. Post-mortem and neuroimaging data suggest that the brains of jet pilots and astronauts, who are exposed to rotational forces, accelerations, and microgravity, display brain anomalies which could be indicative of a mechanodisruptive pathology. Here we present a model of non-impact-based brain injury induced by matrix deformation following mechanical shaking. Using a bioengineered 3D neural tissue platform, we designed a repetitive shaking paradigm to simulate subtle rotational acceleration. Our results indicate shaking induced ectopic cell clustering that could be inhibited by physically restraining tissue movement. Imaging revealed that the collagen substrate surrounding cells was deformed following shaking. Applied to neonatal rat brains, shaking induced deformation of extracellular spaces within the cerebral cortices and reduced the number of cell bodies at higher accelerations. We hypothesize that ECM deformation may represent a more significant role in brain injury progression than previously assumed and that the present model system contributes to its understanding as a phenomenon.

William Rusk

Papers

A Long-Living Bioengineered Neural Tissue Platform to Study Neurodegeneration.

A 3D Tissue Model of Traumatic Brain Injury with Excitotoxicity That Is Inhibited by Chronic Exposure to Gabapentinoids.

Learning and synaptic plasticity in 3D bioengineered neural tissues

Matrix Deformation with Ectopic Cells Induced by Rotational Motion in Bioengineered Neural Tissues