Method for in-vitro construction of tissue engineered nerves
A tissue engineering and neural technology, applied in the field of biomedical engineering, can solve different problems and achieve the effect of large surface area
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Example 1. Isolation, culture, purification and identification of precursor cells derived from rat skin
Take out the SPF SD rats 1-3 days after birth. After disinfection with 75% alcohol, they are decapitated and put to death under aseptic conditions. The back skin tissue is cut, and the subcutaneous tissue is removed mechanically until the skin is more transparent. Rinse with PBS 3 times. Cut skin tissue to 1mm 2 Digest the small tissue pieces with 1mg / ml type XI collagenase at 37℃, pipette repeatedly every 15min, digest for 30-60min, add 20ml DMEM / F12(3:1) medium to clean the tissue, centrifuge at 1200rpm for 10min and discard Then add 1ml of fresh DMEM / F12 medium and repeatedly blow the tissue with a 1ml pipette tip to separate the cells from the tissue. This process is repeated 3-4 times, and 1ml of medium can be added each time. Finally, centrifuge at 1200 rpm for 10 min, discard the supernatant, resuspend the cells in DMEM / F12 (3:1) medium containing FGF2 (40ng / ml), EGF (...
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Example 2. Induced differentiation, expansion and identification of rat skin-derived precursor cells into Schwann cells.
The obtained P2-P4 generation rat skin-derived precursor cells were digested with 1 mg / ml type XI collagenase at 37°C for 10-20 minutes, and gently pipetted repeatedly to mechanically separate the cell pellets. The single cell suspension was divided into 5×10 4 / ml density is planted on 75cm pre-coated with Laminin (0.02mg / ml) and Poly-D-lysine (0.2mg / ml) 2 Cultivate in a petri dish, use DMEM / F12 (3:1) medium containing FGF2 (40ng / ml), EGF (20ng / ml), B27 (2%) and FBS (5%) for 3 days, and after 3 days The medium was replaced with Forskolin (5μM), Heregulin-1 (50ng / ml), N 2 (2%) and FBS (2%) in DMEM / F12 (3:1) culture medium for 14-21 days to obtain Schwann cell colonies. The Schwann cell colonies are picked, expanded, and frozen for later use. See the results of immunofluorescence detection of Schwann cells induced by skin-derived precursor cells figure 2 .
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Example 3. Preparation of nerve catheter.
With reference to the patent application number CN201110324474.8, the chitosan nerve catheter is prepared by the method described in "Tissue Engineering Nerve Graft and Its Use", and the polylactic acid-glycolic acid copolymer fiber prepared by electrospinning is passed through Put it into the chitosan nerve duct (200 pieces per mm inner diameter), and coat it with a mixture of laminin (0.02mg / ml) and polylysine (0.2mg / ml) overnight for later use. The stent material is sterilized by Co60 irradiation or ethylene oxide.
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