One challenge in the field of adipose biology has been to maintain mature adipocyte in culture. We have developed a new method that allows the study of long term adipocyte throughout big changes. Our adipocyte culture technique allows the study of cell interactions and will hopefully be useful for the development of new treatments for diabetes and obesity.
It is important to follow the protocol carefully as there are some critical steps that greatly impact the gene quality and the viability of the mature adipocytes. For example, before plating make sure that all of the free lipid and wash buffer has been removed otherwise the lipocytes could rip off when the inserts are inverted. After receiving the human subcutaneous adipose tissue sample, place the tissue in a sterile biological safety cabinet in a 15 centimeter Petri dish.
A small volume of medium 199 to the dish. Use tweezers to grasp large fibrotic vessels within the tissue and use the backside of the tip of a closed pair of scissors to gently scrap the adipose along the vessel to release the adipocytes. When all of the cells have been collected discard the large pieces of fibrotic tissue and weigh the trimmed fat.
To further release the cells from the fat tissue, transfer 10 grams of adipose tissue into a new 15 centimeter Petri dish and use curved scissors to carefully mince the fat until it becomes a smooth homogenous mixture with no large pieces of adipose. When all of the fat has been processed, use a spoon to transfer 10 milliliters of minced tissue into individual 50 milliliter tubes and add 30 milliliters of digestion buffer to each tube. Then place the tubes in a shaking incubator at 37 degrees Celsius and 150 rotations per minute for 30 to 35 minutes.
The digestion is complete when the adipose solution is homogenous, apricot colored, and absent of large pieces. At the end of the digestion, decant the digested fat solution into a 250 micrometer mesh filter inside a funnel set into sterile one liter flask. When the entire volume of adipocyte suspension has been eluded, gentle squeeze the mesh filter to increase the yield of adipocytes and wash the filter with 50 to 100 milliliters of wash buffer before squeezing the filter again.
Transfer the isolated adipocyte solution into a separation funnel and add wash buffer until the funnel is almost completely filled. Gently invert the funnel a few times to mix the adipocyte suspension with the buffer and let the suspension stand for two to three minutes until there is a distinct separation of the layers. When the layers have separated, open the nozzle on the funnel and slowly elude the bottom solution into a sterile flask.
When all of the collagenase has been removed, collect the purified mature adipocyte solution in 50 milliliter conical tubes and lightly pack the cells by centrifugation. Then, use a 10 milliliter syringe equipped with an 18 gauge needle to remove the remaining wash buffer below the adipocyte suspension and use a pipette to remove the free lipid layer floating above the mature adipocytes. For seeding of the mature adipocytes, place the permeable membrane insert component upside down on a sterile surface and gently invert the tubes of packed adipocytes a few times to ensure an even distribution of cells.
Using a wide bore pipette tip, add 30 microliters of packed mature adipocytes onto each membrane. Invert the insert panel in one smooth motion so that the seeded adipocytes are on the bottom of the inserts. Place the panel of inserts into a 24 well plate containing 500 to 1, 000 microliters of complete 37 degree Celsius medium per well.
Then, cover the plate and carefully place the plate into a tissue culture incubator. Every seven days of culture, replace the medium via the cutout hole in each insert. After one week of culture, mature adipocyte aggregates isolated from subcutaneous adipose tissue maintain the characteristic unilocular lipid droplet morphology observed only in mature adipocytes.
Treatment with the PPAR-GAMMA agonists pioglitazone and rosiglitazone results in an increased expression in PPAR-GAMMA responsive genes, whereas treatment with the glucocorticoid receptor agonist dexamethasone has no effect on these genes. Similarly, the glucocorticoid receptor agonist robustly drives the gene expression of glucocorticoid receptor target genes, while the PPAR-GAMMA agonists have no significant effects on these genes. In addition, seven day treatment with the PPAR-GAMMA agonists robustly induces the gene expression of the brown fat specific gene, UCP1.
Our new adipocyte in vitro model can be used for the functional studies in mature adipocytes, including glucose uptake, lipogenesis, and lipolysis. With this new adipocyte culture technique we've been able to show that human mature white adipocytes can transdifferentiate into brown-like adipocytes.
