Isolation and Culturing of Primary Murine Adipocytes from Lean and Obese Mice

Our research focuses on investigating the function of mature adipocytes from different fat depots and dietary conditions. We are specifically interested in understanding how mature adipocytes communicate with surrounding cells and how this crosstalk can influence disease progression and be altered in obesity. Mature adipocytes are large and delicate cells, which makes commonly used cell isolation methods such as FACS ineffective.

Moreover, high levels of free lipid often found in adipose tissue can be toxic to mature adipocytes complicating their maintenance. In contrast to commonly used methods which rely on the in vitro differentiation of preadipocytes, this protocol allows us to study ex vivo isolated adipocytes, which are functionally and transcriptionally like in vivo adipocytes. This is particularly important as adipocytes vary greatly between fat depots and in different diet conditions.

This protocol efficiently isolates, mirroring mature adipocytes and is easily scalable. Adjustments are included to optimize the isolation of adipocytes from mice of different sexes and with different dietary conditions. Once isolated, the adipocytes can be cultured, imaged or otherwise used as needed.

To begin, prepare a collagenase solution with DNase by adding 0.00017 grams of DNase per one milliliter of collagenase solution, and vortex the mixture to ensure complete mixing. Using a razor blade, mince the isolated mice adipose tissue until it appears homogeneous. Then transfer the mince tissue to a sterile 50-milliliter conical tube.

Add the prepared collagenase and DNase solution to the minced adipose tissue and vortex at maximum speed for seven to 10 seconds to thoroughly mix the components. Then place the 50 milliliter conical tubes horizontally in a shaker set to 220 RPM at 37 degrees Celsius for 10 minutes. After incubation, remove the tubes, vortex for five seconds and check the samples.

Now using a serological pipette, pass the digested tissue through a sieve into a new sterile 50-milliliter conical tube. Add FBS equal to the volume of tissue and collagenase solution through the sieve into the same conical tube to neutralize the collagenase. Spin the tubes at 300 G for three minutes at the temperature specified in the table shown here.

Then using a 200-microliter pipette, carefully discard the top layer of free lipids. Next, use a P1000 pipette with the tip cut off to transfer the mature adipocytes to a prepared 15-milliliter tube containing 10 milliliters of growth media. Gently invert the 15-milliliter tubes containing mature adipocytes three to five times until the white layer disperses throughout the media.

After resting the tubes for 10 minutes, spin them at the previously determined temperature to further separate the layers. After that, use a pipette to discard the top layer of free lipids. Now attach a 21 gauge needle to a five-milliliter syringe and carefully slide the needle down the edge of the tube until the bottom of the needle is below the mature adipocytes site layer.

Then slowly pull up the syringe to remove the culture media and any cellular debris. Gently add 10 milliliters of culture media to the tube. Using gel loading pipette tips Slide the tip down the edge of the tube to remove the remaining media from below the mature adipocyte layer.

Then centrifuge the cells at 100 G for one minute. Using a pipette remove the free lipids. Using a gel loading pipette tip remove the culture media below the adipocytes.

Remove the cell culture inserts from a tissue culture-treated 24-well plate and place them membrane side up on the surface of the hood. Fill alternating wells of the plate with warm culture media. Using a cut P200 pipette tip transfer 30 microliters of packed mature adipocytes onto the top of a Transwell insert.

Then carefully pick up the Transwell insert, invert it, and place it into a prefilled well in the 24-well plate. To remove the media from the wells hold the Transwell insert steady and aspirate with a P200 pipette through the small openings on the sides. After removing the media, while still holding the Transwell in place, slowly add one milliliter of fresh media along the sidewall of the well.