Acellular matrix with natural level of glycosaminoglycan and preparation and application thereof
A technology of acellular matrix and glycosaminoglycan, which is applied in the field of acellular matrix and its preparation, can solve the problems of biological function change, small application range, influence on cell growth, etc., achieves good biocompatibility, and improves decellularization. Efficiency, good product reproducibility
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Embodiment 1
[0051] 1. The following processes are all aseptic operations, and the reagents used are all sterilized
[0052] I. Preparation of the carrier of the decellularized amniotic membrane cross-linked heparin of the present embodiment:
[0053] (1) Decellularization of natural biological tissues;
[0054] At room temperature, under routine aseptic operation, fresh porcine amniotic membrane (25cm 2 ) was scrubbed repeatedly with sterile normal saline to remove blood clots and serous fluid until clean, placed in a sterile square dish, and used carbonate buffer (0.05 μg / mL penicillin G and 100 μg / mL streptomycin sulfate) mol / L, pH=9.0~9.6) after immersion, place on a shaker and shake and wash for 30 minutes, repeat 4 times. Next, the amnion tissue was treated with 0.25% trypsin, 1% Triton X-100, 0.5% SDS, 0.2% glutaraldehyde and neutral protease, respectively. Specifically: ① Soak the cleaned amnion in 0.25% trypsin for 1 hour and shake it for 1 hour, wash it with PBS buffer (0.01mo...
Embodiment 2
[0071] Example 2 Preparation of acellular corneal grafted keratan sulfate carrier
[0072] 1. The following processes are all aseptic operations, and the reagents used are all sterilized
[0073] I, preparation of the carrier of the decellularized cornea grafted keratan sulfate of the present embodiment:
[0074] (1) Decellularization of natural biological tissues;
[0075]Under normal aseptic operation at room temperature, scrub the fresh rabbit cornea repeatedly with sterile saline to remove blood clots and serous fluid until it is clean, place it in a sterile square plate, and use antibiotics (100 U / mL penicillin G and 100 μg / mL sulfuric acid) Streptomycin) carbonate buffer solution (0.05mol / L, pH=9.0-9.6) soaked 5 times, 10 minutes each time. Put the cornea into sterile pure water and soak in a water bath at 37°C for 60 minutes. After soaking, put it into 10mL sterile phospholipase solution (prepared with 0.05mol / L carbonate buffer, pH=8~9; phospholipase A1=200U / mL, pho...
Embodiment 3
[0094] Example 3 Preparation of the carrier of decellularized pericardium adding hyaluronic acid
[0095] 1. The following processes are all aseptic operations, and the reagents used are all sterilized
[0096] I. Preparation of the carrier of the decellularized pericardium cross-linked hyaluronic acid of the present embodiment:
[0097] (1) Decellularization of natural biological tissues;
[0098] At room temperature, under routine aseptic operation, the fresh porcine pericardium (25cm 2 ) repeatedly scrubbed with sterile saline to remove blood clots and serous fluid until they were clean, placed them in a sterile square dish, and used carbonate buffer (0.05 μg / mL penicillin G, 100 μg / mL streptomycin sulfate) mol / L, pH=9.0~9.6) Soak 5 times, 10 minutes each time. The porcine pericardium was repeatedly frozen and thawed (frozen at -80°C, thawed at 37°C) three times, each time for 1 hour. Place 10 mL of 0.05% (w / v) trypsin-EDTA solution in a 37°C water bath for shaking (rot...
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