Studies on neural cell behavior in decellularized extracellular matrix-based three-dimensional culture systems

Abstract

Over the past few decades, tissue engineering and regenerative medicine (TERM) technologies have experienced vast expansion, allowing for the creation of proper structural and biochemical environment for cells that closely mimics those found in living organisms. Conventional two-dimensional (2D) surfaces, such as micro-well plates, flasks, and plastic substrates, have limitations in providing microenvironments that are compatible with in vivo conditions due to their inherent differences in properties. As a result, the concept of three-dimensional (3D) cell culture has gained popularity as it allows for the creation of an in vivo-like environment in vitro. This is particularly relevant for studies involving in vitro construction of environment that emulate native tissues for neural cells, as more proximate resemblance to the intricacies of native central nervous system (CNS) can lead to a more accessible understanding of neural cell behaviors at the cellular and molecular levels. As one of the biggest targets of the TERM technologies is to repair the nerve system, thereby researchers were strived to find an ultimate key to cure neurological disorders, such as stroke, traumatic brain injury, Alzheimer’s disease, and Parkinson’s disease. However, due to the extra-sensitive response to the environment and poor regenerative ability of neural cells, limited access to in vivo brain tissue, and a lack of simulation systems that can be replicated for in vitro studies, progress in understanding the central nervous system (CNS) and illuminating mechanisms of these neurological disorders has been impeded. In this aspect, we designed strategies for the construction of novel 3D cell culture system based on brain-derived, decellularized extracellular matrices. To provide a more accurate imitation of tissue-specific environment to cells, using ECMs of CNS tissue itself has recently emerged as a reliable candidate as a plausible alternative. This method involves decellularization of cells from desired tissue, and using the remnants for the provision of a tissue-like environment, called decellularized ECMs (dECMs). The dECM can not only be utilized as a scaffold for implantation but also as an injectable, in situ self-assembling hydrogel for regenerative medicine. In this thesis, we utilized brain-derived dECMs (bdECMs) as a form of hydrogels to take advances of biologically relevant properties, such as tissue-like stiffness, tunable viscoelasticity, and ease of diffusion of oxygen, nutrients, and waste products. First, we proposed the decellularization of brain tissue derived from Sus scrofa domesticus as model bdECM for further construction of a 3D neural cell culture system. The bdECMs were decellularized and characterized by various methods, including proteomic analysis. Decellularization processes that were adapted to form bdECMs enabled the retention of the unique chemical compositions of the original tissue while minimizing cytotoxicity. Next, we induced 3D bdECM hydrogel scaffolds as astrocyte culture platform. Astrocytes, cultured in 3D bdECM scaffolds, exhibited in-vivo-like characteristics, such as stellate morphology, profoundly distinct from the flat, polygonal structures of astrocytes on the 2D culture platform. The transcriptomic analysis confirmatively showed significant differences in gene expression of astrocytes between 3D bdECM and 2D cultures. Lastly, we incorporated 3D bdECM hydrogel scaffolds neuronal and neuron-astrocyte co-culture platform. Microfluidic device was induced for the fabrication of bdECM hydrogel in micro-scale, results to the neuron-seeded microstructure that can aggregate and form macrostructure. Furthermore, neurons cultured on astrocyte-encapsulated bdECM hydrogels presented advanced neuronal development and neural circuit formation, implying the neurosupportive properties of astrocytes in bdECMs. Together, we developed bdECM-based 3D neural cell culture platforms that can be incorporated with both neurons and astrocytes in various dimension and formation. Although the methods were only suggesting the possibilities of the system as a model, we expect that these strategies can well-suited throughout neural tissue engineering fields, from biomaterial-based 3D neural cell culture platform to actual therapeutics.