Development of brain decellularized extracellular matrix bioink to recapitulate native microenvironment for 3D neural cell culture

Abstract

In the field of neurodegenerative disease research, animal models, such as mice and rats, have enhanced the understanding of basic mechanism and neural disease progress [1]. However, the animal models have critical limitations that the animals are restrained to mimic pathophysiology of the human neural disease due to genetic difference and usually require a lot of cost and labor. Researchers in bioengineering have taken note of the culture of human-derived neural cells in vitro. Last a few decades, 2D plastic dish were used for culturing cells; however, neural cells on 2D environment has shown different characteristics to that of the 3D native environment. In this regards, researchers have focused on the neural cell culture in 3D hydrogel (e.g. Matrigel, type I collagen) to mimic the native environmental features [2]. Recently, decellularization of the neural tissue is emerging for recapitulating the native neural tissue environment [3]. Particularly, this strategy has a potential to preserve the component and composition of native extracellular matrix (ECM) which is comprised of sophisticated combination of growth factors, proteins, glycoproteins. In addition, reconstitution of decellularized extracellular matrix (dECM) can provide tissue-specific biophysical and biochemical cues to the cells. In this study, we developed a brain decellularized extracellular matrix (BdECM) and BdECM bioink for the human neural cell culture. Briefly, brain was isolated from a farm pig and we treated the tissue to multiple solutions with the physical agitation for decellularization process. After removing all cellular component from the porcine brain, the tissue was lyophilized and pulverized into the powder. The decellularization process was validated by quantifying residual DNA fraction (less than 5 w/w%) and GAG assay (approximately 102 w/w%). BdECM powder was solubilized in the weak acid to prepare BdECM bioink. 2 w/v % (20 mg/ml) BdECM bioink showed comparable biocompatibility to the porcine type 1 collagen hydrogel. We employed the human neural stem cells (HB1. F3) to evaluate its beneficial effects on proliferation and differentiation of neural stem cells. The human neural stem cells were encapsulated in 2 w/v % of BdECM bioink and 1 w/v % of type 1 collagen hydrogel that has similar stiffness to that of the 2 w/v % BdECM bioink. The neural stem cells in BdECM bioink showed significantly enhanced morphology of differentiated neuron, which were not expressed in the group of type I collagen hydrogel. The encapsulated neural stem cells in BdECM bioink expressed Tuj1 marker after 7 days culture and MAP2 marker after 14 days higher than that of type I collagen hydrogel. In this regard, BdECM bioink could be applied for 3D culture platform of various neural cell culture, such as neural progenitor cells, microglia, and astrocytes. We also expect BdECM bioink can provide a favorable effect for the neural tissue regeneration.