Adipose-derived stem cell isolation and proliferation produces a large number of stem cells that can be used for several downstream applications and experimental techniques. A major intended use for differentiated adipocytes in this protocol is metabolic assays such as insulin stimulated glucose uptake, lipogenesis, and stimulated lipolysis. To begin, create a sterile working environment by disinfecting the biosafety hood and all the tools.
Pipette approximately 10 milliliters of five PBS buffer into four 100 millimeter cell culture dishes. Transfer a 50 gram adipose sample into one of the dishes and wash it four times by sequentially transferring it across the other dishes containing five PBS. Then transfer the washed adipose tissue into a clean 100 millimeter culture dish and thoroughly mince it using scissors or sterile forceps.
Transfer a one to three centimeter section of the minced tissue to a 50 milliliter conical tube containing 13 milliliters of collagenase buffer. Rinse the remaining tissue from the culture dish with two milliliters of collagenase buffer to ensure a total volume of 15 milliliters. Thoroughly mix the sample using a 25 milliliter serological pipette and incubate the tube at 37 degrees Celsius on a rocker for 30 to 60 minutes.
In order to neutralize the enzyme activity, add 10 milliliters of ADSC growth medium into the tube after incubation and mix it well with a pipette to separate any tissue aggregates. Transfer the liquid portion to a new sterile 50 milliliter conical tube, leaving the solid tissue. Wash the tissue three times using seven milliliters of two PBS and transfer the liquid to the new tube.
Next, centrifuge the tube at 500 times G for five minutes and carefully remove as much supernatant as possible without disturbing the pellet. Resuspend the pellet in one milliliter of 1X red blood cell or RBC lysis buffer and incubate it at room temperature for 10 minutes. Then wash the cells twice by adding five milliliters of ADSC growth medium to the tube and centrifuging to remove the supernatant.
After the last wash, resuspend the pellet in two milliliters of ADSC growth medium and filter it through a 70 micron cell strainer into a sterile 50 milliliter conical tube. Rinse the strainer with an additional two milliliters of the medium and transfer four milliliters of the suspension into a sterile 100 milliliter culture dish. Rinse the conical tube twice with three milliliters of medium to collect a total volume of 10 milliliters in the culture dish.
Observe the collected cells under an inverted microscope at 10X magnification to check for floating cells. Incubate the cells for 24 hours in a carbon dioxide incubator at 37 degrees Celsius, 5%carbon dioxide and 100%relative humidity. To ensure sterility of the culture media, include a plate with media alone.
After 24 hours, view the cells under the inverted microscope to check for cell adherence. Remove and replace the medium with warm medium every 48 hours until cells are 80 to 90%confluent. For proliferation, remove the growth medium from the confluent ADSC cells and wash them twice with two milliliters of sterile room temperature PBS.
Aspirate the PBS and add two milliliters of 0.25%trypsin EDTA into each culture plate, covering the entire surface area of the plate. Incubate the plate for seven minutes at 37 degrees Celsius and 5%carbon dioxide, then mechanically dislodge the trypsinized cells by forceful pipetting. Add two milliliters of ADSC growth medium and gently mix the cells.
Transfer the cells into a 50 milliliter conical tube, rinsing the dish with an additional two milliliters of medium to ensure maximal cell recovery from the plate. Next, centrifuge the tube at 500 times G for five minutes at room temperature and decant the supernatant to obtain the cell pellet. Resuspend the cell pellet in five to six milliliters of ADSC growth medium per million cells.
Count the cells using a hemocytometer and plate them as described in the text manuscript. Once the cells reach 80 to 90%confluency, aspirate the growth medium and rinse the cells with sterile room temperature PBS, then add 10 milliliters of ADSC differentiation medium. Replace the medium every three days for approximately 14 to 21 days.
Observe the cells for the presence of lipid droplets under the inverted microscope at 40X magnification. Mature adipocytes are generally observed after 14 days. On the day of plating, the ADSCs were non-adherent and floated in culture.
The cells became 80%confluent after 72 hours and were ready for adipocyte differentiation. After 14 days of ADSC differentiation, mature adipocytes exhibited strong adipogenic characteristics which were observed under 40X magnification. On day 14, cells were stained and fixed with Oil Red O and BODIPY for lipid droplet visualization.
Differentiated adipocytes could be observed under 20X magnification. When attempting this protocol, it is important to remember that a sterile working environment is critical for healthy isolation, adequate expansion, and efficient differentiation of adipose-derived stem cells. Following this procedure, these cells can be used for several metabolic assays such as glucose uptake, lipogenesis, and lipolysis which is a major focus of our research.
This technique allows for the investigation of how chronic alcohol and SIV impact adipocyte metabolic capacity.
