Method for manufacturing body substitutes by additive deposition
A substitute and addition technology, applied in the field of body tissue substitutes, the manufacture of body substitutes, and the substitutes of skin tissue, which can solve the problems of no solution, no natural epidermal layered structure, etc.
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Embodiment 1
[0117] Example 1 : Cell culture and collection
[0118] This example illustrates a method for expanding and harvesting cells (fibroblasts and keratinocytes) which can then be used to manufacture a skin substitute according to the invention.
[0119] Skin keratinocytes and fibroblasts isolated from human foreskin.
[0120] Keratinocytes were cultured on human fibroblasts irradiated using techniques well known to those skilled in the art using a medium known as "Green's medium" containing DMEM and Ham's F12 (in a 3:1 ratio), Add adenine (24.3 μg / mL) and human epidermal growth factor (10 ng / mL), hydrocortisone (0.4 μg / mL), insulin ( 5μg / mL), 2×10 -9 M's triiodo-L-thyronine (5 μg / mL), 10 -10 M isoproterenol, penicillin (100 U / mL), streptomycin (100 μg / mL) and 10% fetal bovine serum. Keratinocytes collected during passages 2, 3 and 4 were used.
[0121] 5% CO at 37°C 2 Fibroblasts were cultured in an appropriate medium containing DMEM, 20% newborn calf serum and antibiotic...
Embodiment 2
[0122] Example 2 : deposited by additive techniques (method according to the invention)
[0123] A first aqueous solution of gelatin was prepared by dissolving gelatin powder at 20% m / v in 0.9% m / v NaCl solution. A second aqueous solution of alginate was prepared by dissolving alginate powder (very low viscosity) at 4% m / v in 0.9% m / v NaCl solution. A third aqueous solution of 8% m / v fibrinogen was prepared, to which was added the fibroblast suspension (obtained in Example 1) at a cell concentration of 2 million cells / mL.
[0124] These three solutions are then mixed to obtain a mixture (called "bioink") comprising 50% by volume of the first solution (gelatin), 25% by volume of the second solution (alginate) and 25% by volume of the second solution (alginate). Trisolution (fibroblasts recovered in fibrinogen).
[0125] Prepare an aqueous polymerization solution containing 3% m / v calcium and thrombin at a final concentration of 20 U / mL.
[0126] The bioink has a viscosity ...
Embodiment 3
[0128] Example 3 : maturation of skin substitute precursors (method according to the invention)
[0129] The skin substitute precursors were cultured for 12 days in fibroblast medium containing 1 mM ascorbic acid 2-phosphate; the skin substitute precursors were nourished daily. Twelve days later, the keratinocytes were 2 2.5×10 5 A concentration of 300 cells is coated on the surface of the skin substitute precursor.
[0130] The skin substitute precursor was cultured for the first seven-day culture period by immersion in Green's medium with ascorbic acid 2-phosphate and antibiotics at a concentration of 1 mM, as described above; the skin substitute precursor was nourished daily .
[0131] The skin substitute precursors were then cultured for a second 21-day culture period by maintaining them at the liquid surface in differentiation medium containing DMEM with hydrocortisone (0.4 μg / mL), insulin (5 μg / mL), ascorbic acid 2-phosphate and antibiotics; differentiation mediu...
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