To begin, thaw the passage, one human white subcutaneous preadipocytes, and add the cells into a T75 flask containing 12 to 15 milliliters of warm preadipocyte growth media-2. After the cells attain 90%confluency, wash them with PBS and incubate them with one milliliter of 0.25%Trypsin-EDTA solution for two minutes. Using a light microscope, confirm the cell detachment and add 23 milliliters of preadipocyte growth media-2 to the cells.
Dispense one milliliter of cell suspension into each well of a 24 well co-culture companion plate and incubate the plate to attain cell confluency. Then replace the media with one milliliter of preadipocyte differentiation medium and incubate at 37 degrees celsius with 5%carbon dioxide for 10 days. On day six of differentiation, seed five times 10 to the power of four human SAT MVECs in 500 microliters of complete MVEC growth media on a transwell insert.
Place the insert in a well containing 500 microliters of complete MVEC growth media and incubate at 37 degrees Celsius with 5%carbon dioxide. On day 10, when the adipocytes are fully differentiated, remove half the media from each well and transfer the transwell inserts containing confluence SAT MVEVs to each well. Incubate the plate for 24 hours to obtain a co-culture.
As the human subcutaneous white preadipocytes become more differentiated, the development of lipid vacuoles can be noticed.
