| 主权项 |
1. A method of preparing a tissue engineered nerve graft for repairing a peripheral nerve defect including a nerve conduit and an ECM, being prepared with a three-dimensional microgravity rotational culture to generate a support cells-containing nerve graft and then obtaining an autologous or allogeneic cell-derived ECM-modified nerve graft via decellularization, the method comprising the steps of:
preparing the nerve conduit by electrostatically spinning a biodegradable material selected from the group consisting of silk fibroin, chitosan and a combination thereof;
preparing the support cells-containing nerve graft, wherein a complete medium is added to a culture vessel, and then 2.5×107 cells selected from the group consisting of Schwann cells, skin-derived fibroblasts, skin stem cells, bone marrow mesenchymal stem cells, induced pluripotent stem cells and a combination thereof and the nerve conduit are added to the culture vessel;processing with a peristaltic pump to ensure a final cell density of 1×105/ml, after air is exhausted from the culture vessel;initiating a rotational microgravity circulation perfusion culture with a rotational bioreactor placed in a 37° C. CO2 incubator to ensure a full contact and adhesion of the cells onto the nerve conduit by setting a rotating speed of 10 r/min at the first 24 h, and then suspending the nerve conduit in a culture solution comprising Iscove's Modified Dulbecco's Media (IMDM) by adjusting the rotating speed after 24 h, further wherein following culture for 2 d, the medium is changed with a differentiation medium to promote ECM secretion, and allowed to culture for two more weeks before the nerve conduit, now coated with support cells is taken out; and constructing a decellularized tissue engineered nerve graft, wherein the support cell-containing tissue engineered nerve graft is subjected to decellularization, and is washed with phosphate buffered saline, placed in sterile deionized water at 37° C. for hypotonic treatment for 10 min, followed by cell lysis in a nonionic detergent for 10-15 min at 37° C., and adding of DNAse I at a concentration of 4 mg/ml to the nonionic detergent at 37° C. to remove DNA, and the acellular product is kept at −80° C. for use. |
