Development of brain decellularized extracellular matrix bio-ink for 3D cell-printing of neural stem cell-laden construct to treat a damaged brain

Abstract

A damaged adult brain has a limited regenerative potential because of the little portion of neural stem cells. Hence, the transplantation of exogeneous neural stem cells to the defect area is promising for healing the damaged brain. However, the low cell delivery efficiency of the conventional methods, such as intravenous and intrathecal method, has resulted in the poor engraftment of the implanted cells and the low cell survival rate. Therefore, the advanced stem cell delivery method is needed to improve the stem cell delivery efficiency. In this regard, implantation of neural stem cell-seeded scaffold has shown benefits in increasing the cell survival and proliferation by providing a mechanical support. In the field of engineering scaffold, tissue decellularization is an emerging technology to provide a highly favorable microenvironment for the differentiation of stem cells into a tissue-specific lineage by preserving the complex extracellular matrix (ECM) composition of native tissue. In addition, the enzymatically solubilized decellularized ECM, called dECM bio-ink, has shown its potential as a building material for producing an implantable 3D cell-laden construct throughout a layer-by-layer cell-printing process. Since 3D cell-printing can produce an arbitrary shape fit for a defect area, the host-implant integration might be enhanced by minimizing the gap between the implant and the defect. Therefore, here, we developed a brain decellularized ECM (BdECM) bio-ink for the application to 3D cell-printing of implantable neural stem cell-laden construct. First of all, we isolated a brain from a farm pig and treated with several steps of physical and chemical methods to remove all cellular component in the tissue for decellularization, which is confirmed with residual DNA fraction (~5%). The decellularized porcine brain was solubilized to produce BdECM bio-ink and 20 mg/ml concentration showed a comparable softness with a native brain (~100 Pa) in rheology study and an excellent shape fidelity during 3D printing process. In addition, the neural stem cells encapsulated in the BdECM bio-ink showed high cell viability (~98%) and the similar proliferation rate to those in collagen type I hydrogel, a widely used in cell culture. These results suggest that our BdECM bio-ink has low cytotoxicity and a potential as a stem cell delivery material. Taken together, the BdECM bio-ink is expected to be processed with neural stem cells by 3D cell-printing to provide fit shape and size for injured brain. In the future, the BdECM bio-ink will be implanted to a corticectomized rat in order to see the efficacy as a neural stem cell delivery system. We also expect the favorable effect of BdECM bio-ink on neural differentiation with provision of native-tissue like environment.