Engineered leather and methods of manufacture thereof
一种皮革、工程的技术,应用在工程的皮革和其制造领域,能够解决缺乏天然皮革质量、耐久性和声望、没有开发出科学上合理天然皮革等问题
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Embodiment 1
[0173] Embodiment 1 - preparation of support substrate
[0174] To prepare a 2% agarose solution, 2 g of ultrapure low melting point (LMP) agarose was dissolved in 100 mL of ultrapure water / buffer (1:1, v / v). The buffer is optionally PBS (Dulbecco's Phosphate Buffered Saline lx) or HBSS (Hanks Balanced Salt Solution lx). The agarose solution was placed in a beaker with warm water (over 80°C) and placed on a hot plate until the agarose was completely dissolved. As long as the temperature exceeds 36°C, the agarose solution remains liquid. Below 36°C, a phase transition occurs, viscosity increases and eventually the agarose forms a gel.
[0175] To prepare the agarose support substrate, 10 mL of liquid 2% agarose (temperature >40° C.) was placed in a 10 cm diameter petri dish and spread evenly to form a consistent layer. Agarose was allowed to form a gel in a refrigerator at 4 °C.
Embodiment 2
[0176] Example 2 - Culture of bovine keratinocytes, fibroblasts and epithelial cells
[0177] Fresh isolated bovine keratinocytes, fibroblasts and epithelial cells were grown in low glucose DMEM with 10% calf serum (Hyclone Laboratories, UT), 10% porcine serum (Invitrogen) , L-Vitamin C, Copper Sulfate, HEPES, L-Proline, L-Alanine, L-Glycine, and Penicillin G (all aforementioned supplements from Sigma, St.Louis, MO purchase). Cell lines were cultured on Petri dishes (Techno Plastic Products, St. Louis, MO) coated with less than 0.5% gelatin (porcine skin gelatin; Sigma) and maintained at 37°C in the presence of 5% CO. 2 in the humid air. Keratinocytes were cultured again to passage 7 before being used to form multicellular bodies.
Embodiment 3
[0178] Example 3 - Preparation of multicellular spheroids and cylinders
[0179] Cell cultures were washed twice with phosphate-buffered saline (PBS, Invitrogen), treated with 0.1% insulin (Invitrogen) for 10 minutes, and then centrifuged at 1500 RPM for 5 minutes. Cells were resuspended in 4 mL of cell-type specific media and then incubated at 37 °C with 5% CO 2 Incubate in a 10 Ml tissue culture flask of a 100 ml tissue culture flask and continue for 1 hour on a rotary shaker (New Brunswick Scientific, Edison, NJ) for adhesion recovery, and centrifuge at 3500 RPM. The resulting pellet was transferred to a 300 μm (Sutter Instrument, CA) or 500 μm (Drummond Scientific Company, Broomall, PA) diameter capillary micropipette at 37°C with 5% CO 2 Incubate for 15 minutes. For spherical multicellular bodies, extruded cylinders were cut into equal pieces, which were collected overnight on a rotary shaker. Depending on the diameter of the micropipette, this step provides conventi...
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