Medicine
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Lumican Extraction from Amniotic Membrane and Determination of its Storage Temperature
Chapters
Summary October 14th, 2022
The present protocol describes the extraction of lumican from the amniotic membrane (AM) and their storing conditions as AM extract (AME) at -20 °C, 4 °C, and room temperature (RT) for 6, 12, 20, and 32 days to quantify its proteins and lumican concentration.
Transcript
This method aims to demonstrate a straightforward technique for protein extraction, useful for the obtaintion of bountiful proteins and other factors. This technique could be used to extract other factors and proteins present in the amniotic membrane and also, to obtain proteins from other sources such as animals and vegetal tissues. Begin by thawing 100 milligrams of the amniotic membrane obtained from the amnion bank.
Once thawed, add 10 milliliters of the sterile balanced salt solution to a Petri dish and wash the membrane for two minutes. Then, remove the used balanced salt solution into a beaker and repeat the process until no trace of glycerol medium is visible in the Petri dish. Next, add 10 milliliters of dispase II to the membrane and incubate at 37 degrees Celsius and 5%carbon dioxide for 30 minutes.
At the end of the incubation, perform a mechanical de-epithelialization with a rubber policeman cell scraper. Then, visualize the membrane under an inverted microscope to confirm de-epithelialization. Next, wash the de-epithelialized amniotic membrane with the sterile balanced salt solution as demonstrated earlier, and place it in a two-milliliter microcentrifuge tube.
Then, immerse the tube containing the membrane in liquid nitrogen for 40 minutes. Post freezing with liquid nitrogen, manually grind the membrane for two to three minutes in a mortar, pre-chilled at minus 85 degrees Celsius, until a fine powder is obtained. Next, add 2.5 milliliters of protease inhibitor solution and solubilize the finely-powdered membrane.
Clean the mortar walls with the help of a scalpel knife and transfer the mixture to a five-milliliter tube. Vortex the tube for 30 seconds. Then, homogenize the tissue mixture by first centrifuging at 34 G for 20 minutes at four degrees Celsius, and immediately centrifuging it again at 3, 360 G for 20 minutes at four degrees Celsius.
At the end of centrifugation, collect the supernatant, which is the amniotic membrane extract, in a new tube. Then, aliquot 0.7 milliliters in a different two-milliliter microcentrifuge tube for various time points and temperatures. The total protein quantity and lumican concentration in the amniotic membrane extract were affected by time and storage conditions.
Basal protein concentration was similar among all the amniotic membrane extracts. However, a significantly high protein concentration was noted in the extract stored for 32 days at four degrees Celsius and 20 days at minus 20 degrees Celsius. Lumican in the amniotic membrane extract was significantly higher in the sample stored for a period of 12 days compared with the samples stored for 30, 20, and six days at four and minus 20 degrees Celsius.
These results suggested that the appropriate storage time and temperature to achieve the highest concentration of lumican is 12 days at minus 20 degrees Celsius. Exercise utmost care while grounding the frozen amniotic membrane. The grounding should be gentle as it could fall out from the mortar.
Further studies are needed to determine the role of amniotic membrane extract lumican in corneal epithelial cells and to determine the ideal dose of lumican for corneal re-epithelialization.
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