Method for obtaining homogeneous amniotic membrane homogenates

Abstract

The subject of the application is a method for obtaining homogeneous amniotic membrane homogenates, which consists in collecting the amniotic membrane using known methods, then decontaminating it and rinsing it in an antibiotic bath using known methods, collecting samples for microbiological and histopathological examination, placing the tissue in sterile containers with a PBS solution, and all activities are performed in a sterile manner under laminar flow beds with class A air supply in class B clean rooms, then the samples are cryopreserved by placing them at a temperature of -20°C for 24 hours, and then cooling them in a low-temperature freezer at a temperature of -80°C in a thermally insulating packaging surrounded by dry ice, and immediately after being removed from the low-temperature freezer, the sample is crushed in PBS without Ca and Mg and placed in dry ice and stored in glass containers or sterile disposable chambers in dry ice,and then the samples are placed in liquid nitrogen and homogenized in a batch mill with impact-shearing action and ground with dry ice for 2 to 15 minutes, placed in a glass vessel or sterile disposable chambers and stored at a temperature of -55°C until the dry ice sublimates, then weighed and centrifuged for 2 to 5 minutes, preferably 3 minutes in a ratio of 1:1 to 1:1.5, preferably 1:1 homogenate with dry ice, then the homogenate is placed in a laboratory centrifuge at a temperature of +4°C and centrifuged for 1 to 3 minutes, preferably 2 minutes at a speed of 3000 rpm to 8000 rpm, preferably 5000 rpm and a relative centrifugal force of 1000 g to 1800 g, preferably 1400 g, and then cooled at a temperature of -30 to -10, preferably -20°C for 15 to 48 hours, preferably 24 hours, then the homogenates are centrifuged by placing them in a low-temperature freezer at -20°C, adding 5 ml of cooled PBS solution without Ca and Mg,the mixture is vortexed for 30 seconds, then subjected to another centrifugation at +4°C for 2 minutes at a speed of 5000 rpm and a relative centrifugal force of 1400 g, then the sample is purified using sterile syringe filters with a pore size of 0.22 µm, after which the supernatant is transferred to sterile tubes, from which 1 ml of supernatant is then taken and the supernatant is subjected to ultracentrifugation at +4°C for 5 minutes at a speed of 14,000 rpm and a relative centrifugal force of 14,000 g, after which it is cooled in a laboratory freezer at -80°C.,

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