Multilayered cell sheet of neural crest stem cells and method of preparing the same

A method of manufacturing a multilayered cell sheet of neural crest stem cells (NCSCs) without stacking a cell sheet, in the order recited, consisting of:

• (1) isolating and culturing NCSCs from peripheral nerves (PN);
• (2) embedding the cultured NCSCs in a mixed hydrogel of collagen and fibrinogen consisting of collagen at a final concentration in a range of about 0.1% to about 1% and fibrinogen at a final concentration in a range of about 0.1% to about 1%,
• wherein step (2) comprises mixing the cultured NCSCs having a PN-NCSCs density in a range of about 1×106/ml to about 1×108/ml with the mixed hydrogel of collagen and fibrinogen in a solution phase and converting the solution phase to a gel phase so that the NCSCs are uniformly distributed in a three-dimensional manner in the mixed hydrogel of collagen and fibrinogen to have the multilayered cell sheet without stacking the cell sheet;
• (3) casting the mixed hydrogel of collagen and fibrinogen comprising the NCSCs embedded therein on a physical support so that the NCSCs are cultured under attached hydrogel culture conditions in which a physical support is applied,
• wherein the physical support is a mold having a circular, rectangular, or square shape;
• and
• (4) removing the physical support and culturing the resulting mixed hydrogel of collagen and fibrinogen under free floating hydrogel culture conditions, wherein a structure of the mixed hydrogel is maintained without the physical support, wherein the free floating hydrogel culture conditions of step (4) induces cell-mediated hydrogel compaction so that water and culture media in the hydrogel is extruded,
• wherein the culturing under the attached hydrogel culture conditions of step (3) comprises accumulating factors in the multilayered cell sheet, the factors including:
• 1) Extracellular matrix (ECM) comprising fibronectin, laminin, and collagen type IV,
• 2) angiogenic factors comprising at least one angiopoietin (ANGPT) comprising ANGPT-1, ANGPT-2, ANGPT-3, and ANGPT-4, a vascular endothelial growth factor (VEGF), or a platelet-derived growth factor (PDGF),
• 3) anti-inflammatory factors comprising interleukin (IL) comprising IL-6 or IL-10, or a transforming growth factor (TGF) comprising TGF-β,
• 4) neurotrophic factors comprising:
• at least one neurotrophin (NT) selected from the group consisting of a nerve growth factor (NGF), a brain-derived growth factor (BDNF), NT-3, and NT-4/5;
• at least one glial cell line-derived neurotrophic factor (GDNF) selected from the group consisting of GDNF and artemin (ARTN);
• at least one ephrin (EFN) selected from the group consisting of EFN A1, EFN A2, EFN A4, EFN A5, EFN B1, EFN B2, and EFN B3;
• at least one ciliary neurotrophic factor (CNTF) selected from the group consisting of CNTF, a leukemia inhibitory factor (LIF), and IL-6;
• a glial maturation factor (GMF);
• or
• neuregulin (NRG) or an insulin-like growth factor (IGF)-1, and
• 5) neuroprotective factors comprising at least one fibroblast growth factor (FGF) selected from the group consisting of FGF-7, FGF-9, FGF-16, FGF-19, FGF-12, FGF-5, FGF-6, and FGF-14, which are produced and secreted from the NSCSs, in the mixed hydrogel of collagen and fibrinogen.