This protocol provides a simple strategy for generating cell cultural models of white and brown adipocytes that faithfully recapitulate in vitro, the physiology of the adipose tissue that we have in vivo. This protocol enables simultaneous isolation of both white and brown preadipocytes from newborn mice and the cells that we isolate using this method assess high proliferative capacity and differentiation potential. Cells isolated using this approach reflect the complexity of adipose tissue better than other culture models.
And it can be used to more accurately study adipocyte function in obesity and diabetes. We focus primarily on understanding adipose tissue dysfunction in obesity. However, cells prepared with this method can also be used to study basic questions about cell in developmental biology.
The key to a successful experiment is determining how to identify the white adipose depots as these depots, which are small, but very rich and proliferating preadipocytes can be difficult to distinguish in pups. Visual demonstration illustrates how simple this protocol is and allows the provision of tips for locating and dissecting the white and brown adipose depots. Seeing the depot isolation is very helpful.
To collect subcutaneous white adipose tissue, cut the skin around the abdomen of a euthanized newborn pup, and gently pull the skin below the legs. Without collecting any skin tissue, carefully harvest the fat depot, which will appear as a clear or white, thin elongated tissue attached to the inside of the skin or on top of the quadriceps. Rinse the harvested depot in PBS and place the adipose tissue in a 1.5 milliliter tube containing 250 microliters of PBS and 200 microliters of 2X isolation buffer on ice.
To collect interscapular brown adipose tissue, pull the skin from the shoulder blades over the head and lift the deep red brown adipose tissue located between the shoulder blades. Carefully make incisions all around the adipose to detach it from the body and check the tissue for consistency and color. After rinsing, place the tissue in a second 1.5 milliliter tube containing 250 microliters of PBS and 200 microliters of 2X isolation buffer on ice.
When all of the depots have been collected, use scissors to gently mince each depot four to six times directly inside the tubes and add 50 microliters of 10X collagenase in 2X isolation buffer to each tube. Then invert the tubes quickly to mix and place the tissues in a temperature controlled mixer for 30 minutes at 37 degrees Celsius and 1400 revolutions per minute. At the end of the digestion, place the tubes back on ice and filter the digestive tissues through 100 micron cell strainers into new 50 milliliter tubes.
To maximize the cell yield, rinse each tube with one milliliter of isolation medium and filter this wash through the strainer. Dilute the brown and white adipose tissue solutions to two milliliters of tissue suspension per well per depot in fresh isolation medium. Then plate two milliliters of white adipose tissue suspension to each of four to six wells and two milliliters of brown adipose tissue suspension to each of two to three wells of a six well plate through one 100 micron filter per well.
When all of the cells have been plated, place the plate in the cell culture incubator for 60 to 90 minutes. At the end of the incubation, wash each well with two milliliters of serum-free medium and gently agitate the plate to detach any blood cells from the bottom of the wells. After three washes, add two milliliters of fresh isolation medium to the wells and return the plate to the cell culture incubator.
When the cells reach 80 to 90%confluency, coat new destination plates with sterile 0.1%gelatin in distilled water at 37 degrees Celsius for 10 minutes. While the plates are incubating, wash the cells with PBS before treating with trypsin for three minutes in the cell culture incubator. When the cells have detached, pipette the cell suspension several times to maximize the cell recovery and transfer the cells to a new tube.
When all of the cells have been collected, wash each well with one milliliter of isolation medium and pull the washes with the cell suspension. Collect the cells by centrifugation and resuspend the pellet in three to five milliliters of isolation medium for counting. Dilute the cells to the appropriate concentration for plating in fresh isolation medium and wash the gelatin coated plates with PBS to remove any excess gelatin.
Then seed the cells onto the gelatin coated plates and return the cells to the cell culture incubator. When the cells reach confluency, replace the isolation medium with differentiation medium. If differentiating brown adipose tissue preadipocytes, also add one nanomolar triiodothyronine.
After 48 hours, refresh the medium with the appropriate volume of maintenance medium per culture. At this time, the accumulation of small liquid droplets within the cells should become visible under bright field microscopy. Even after filtering, some cellular debris and blood cells remain within the cell suspension.
Gentle washes one hour after plating will remove non-relevant cells as the preadipocytes attach rapidly to the bottom of the well. Although both white and brown preadipocytes isolated from newborn pups have a high proliferative capacity, the yield of white preadipocytes is generally twice that of brown preadipocytes on a per depot basis. Therefore, if synchronized cultures are desired, this starting density of the white preadipocytes must be calculated accordingly.
At the end of differentiation, the cells will appear to be loaded with lipid droplets and will express classical markers of white and brown adipocytes, respectively. In this comparative analysis of oxygen consumption in a mitochondrial stress test of primary white and brown adipocytes under basal conditions, as well as in response to known stimulators of mitochondrial function, this specific bioenergetic capabilities of each cell type can be observed. Remember that we are isolating live cells and that we want to culture them for several days.
So be sure to maintain sterile conditions during the isolation to avoid contamination, which can compromise the subsequent culture. This method generates fully mature adipocytes that can be used for different applications, including functional assays, genetic or chemical screens, or omics profiling to study cell-autonomous mechanisms that impact adipocyte function.
