In vitro bioengineered model of cortical brain tissue

Abstract

A bioengineered model of three-dimensional (3D) brain-like tissue was developed using silk-collagen protein scaffolds seeded with primary cortical neurons. The scaffold design provides compartmentalized control for spatial separation of neuronal cell bodies and neural projections, resembling the layered structure of the brain (cerebral cortex). Neurons seeded in a donut-shaped porous silk sponge grow robust neuronal projections within a collagen-filled central region, generating 3D neural networks with structural and functional connectivity. The silk scaffold preserves the mechanical stability of the engineered tissues, allowing for ease of handling, long-term culture in vitro, anchoring of the central collagen gel to avoid shrinkage, and neural network maturation. This protocol describes the preparation and manipulation of silk-collagen constructs, including the isolation and seeding of primary rat cortical neurons. This 3D technique is useful for mechanical injury studies, as a drug screening tool and could serve as a foundation for brain-related disease models. The protocol of construct assembly takes 2 days and the resulting tissues can be maintained in culture for several weeks.

Automated quantification of three-dimensional organization of fiber-like structures in biological tissues

“…Recently, we engineered functional 3D brain-like cortical tissues [29,48], and we sought to assess the potential use of 3D directional variance as a quantitative metric of axonal organization alterations in response to injury within these cortical tissues. A cortical unit module consists of neuron-rich grey matter-like regions and axon-only white matter-like regions (Fig.…”

“…Salt leached silk scaffolds are prepared and used for 3D neuronal culture as previously established [29]. Briefly, silk scaffolds are made by combining 30 ml of 6% silk solution with 60 g of 500–600 μm NaCl particles for a period of 48 hours at room temperature to allow the crosslinking of silk solution.…”

“…Immunostaining of the fixed samples is done using a previously described protocol [29]. Primary antibody against β-III tubulin (rabbit, Sigma), a neuron-specific marker is diluted by 1:500 in a blocking solution (4% goat serum, 0.2% triton-X100, 0.05% bovine serum albumin).…”

Three‐Dimensional Models of the Human Brain Development and Diseases

“…[12] For example, Tang-Schomer et al developed a 3D brain-like cortical tissue using silk-collagen scaffolds and primary cortical neurons, resembled the layered structure of the cerebral cortex with capability to study traumatic brain injury. [9,191] A 3D in vitro human neural cell culture model of AD using human neural progenitor cells embedded in Matrigel showed the two pathological hallmarks of AD: extracellular aggregation of A β and accumulation of intercellular hyperphosphorylated tau proteins. [12,192] Excessive accumulation of A β leads to the aggregation of hyperphosphorylated tau, [58,193] however, no animal model could verify this hypothesis prior to this study.…”

Hydrogels and bioreactors for cartilage research and functional tissue engineering

“…Such a 3D culture approach for ovarian cancer represents an organotypic model that can be further expanded with other tissue-specific cell types that are present in the tumour microenvironment, and it enables the high-throughput screening of novel therapeutics [297]. Other approaches include the combined silk-collagen scaffolds developed by the Kaplan group [298] to build a brain-like tissue, in which the biphasic architecture enables the spatially controlled growth of a neuronal network, thus mimicking the 3D morphology of the brain cortex. Other scaffold technology platforms enable researchers to monitor oxygen levels and determine hypoxia in a 3D cell culture setup [299], as well as to investigate mechanisms that are altered during bone metastasis in patients with prostate cancer [300].…”