Method for fabricating activated artificial skin tissue in bilayer by using bioreactor
A bioreactor and artificial skin technology, applied in biochemical equipment and methods, microorganisms, tissue culture, etc., to achieve uniform distribution, delay degradation speed, promote cell proliferation and functional expression
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Embodiment 1
[0067] Take the foreskin of the child and cut it into 1cm 2 The left and right skin blocks were washed 3 times with PBS containing 200 U / mL gentamycin, 2 times with 75% alcohol, and 2 times with PBS. The skin tissue was added to 200 U / mL neutral protease (Sigma) and digested at 4°C for 18 hours to separate the dermis and epidermis. Keratinocytes are obtained from epidermal tissue and fibroblasts are obtained from dermal tissue.
[0068]Cut the epidermis into small pieces, act with 6 mL of 0.25% trypsin (Sigma) / 0.06% EDTA (the volume of trypsin / EDTA digestion solution is about 3 times the volume of the digested tissue) for 8 minutes, and add an equal amount of calf serum and trypsin activity.
[0069] Filter through a 150-mesh sieve to remove epidermal fragments and obtain keratinocyte suspension. Centrifuge at 2000r / min for 5min, discard the supernatant, wash twice with PBS, add keratinocyte serum-free medium (Gibco) containing 200U / mL gentamycin, and the cell inoculation v...
Embodiment 2
[0072] The fascia and lipids were removed from the tendon to make tendon powder, which was stored at -20°C for later use. Soak the tendon end in 0.1mol / L HAc solution to prepare a collagen-HAc solution with a final concentration of 8.0mg / mL. Add chitosan solution, heparin sodium solution, and chondroitin sulfate solution to the collagen solution so that the final concentrations are 10% (g / g), 7% (g / g) and 3% (g / g) of the dry weight of collagen, respectively. g), frozen at -20°C overnight, and vacuum freeze-dried for 24 hours to obtain a collagen sponge. Collagen sponges were chemically cross-linked with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (Sigma), rinsed thoroughly with distilled water, freeze-dried, and sterilized with ethylene oxide to make usable Composite collagen sea surface matrix material for preparing double-layer active skin tissue.
Embodiment 3
[0074] The same method as in Example 2 is adopted, but in the composite collagen sponge, the amino polysaccharide substance is hyaluronic acid, and the final concentration is 20% (g / g) of the dry weight of the collagen.
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