This breakthrough method allows us, for the first time, to keep fat alive and healthy ex vivo for several consecutive weeks for the study of adipose biology. The main advantage of this technique is that the tissue remains stable over eight weeks in the culture while recapitulating the adipose microenvironment including the surrounding native adipose stromal support cells. Begin by seeding ASC at an approximately 80%confluency in tissue culture plates.
For each well of SWAT desired, seed one conventional tissue culture well and one well of corresponding size on a PNIPAAm coated tissue culture plastic plate. Maintain the ASC in complete culture medium at 37 degrees Celsius and 5%CO2 for six to eight days, changing the medium every two days. Twenty-four hours before the SWAT seeding, wrap the rim of the plunger disk of each plunger for the SWAT seeding apparatus at least two times with lab tape to prevent leakage of the gelatin solution.
In an ethanol-disinfected biosafety cabinet, spray down a 15 milliliter conical tube rack containing empty, uncapped 15 milliliter conical tubes, as well as each tape wrapped plunger with 70%ethanol, placing each plunger into an empty tube as it is disinfected. Spray approximately 6.3 gram metal washers and place them on the rack along with a pair of hooked forceps. Then close the cabinet sash and turn on the UV light to facilitate overnight drying and sterilization of the seeding apparatus materials.
When the cells reach 100%confluency and demonstrate a striated pattern, add 0.75 grams of gelatin powder to 10 milliliters of HBSS. And balance the pH of the solution with 100 microliters of sodium hydroxide. Add the gelatin to a 75-degree Celsius water bath and shake the tube vigorously every five minutes until the powder dissolves into solution.
Strain the homogenous gelatin solution through a syringe filter and apply the gelatin to the plungers. The taped edges of the plungers will serve as a mold while the gelatin solidifies. When the gelatin has solidified unwrap the tape and use the hooked forceps to remove the gelatin from the outer edge of the plunger, taking care that the remaining gelatin in the center of the plunger is completely level, to maximize the contact with the ASC sheet.
Once the gelatin has been removed from all of the plungers, gently apply each plunger to a PNIPAAm coated ASC plate using the metal washers to weigh down the plungers. After a 1.5-hour incubation at room temperature, transfer the plates to an ice-water bath for another 1.5 hours to complete the dissociation of the cell sheet from the PNIPAAm coated plate surface. Then, clean the bottom of the PNIPAAm coated plates to remove any non-sterile water.
Upon receipt of the human adipose tissue, add cold cell culture medium to one 1.5 milliliter microcentrifuge tube per culture to be seeded. And wash large segments of the adipose tissue three times in sterile PBS, removing as much PBS as possible between washes. After the last wash, use forceps and a sterile razor to coarsely mince the tissue, removing as much vasculature and fascia as possible until the solution takes on thick liquid consistency with as few visible individual segments of white adipose tissue as possible.
Once a proper tissue consistency has been reached, use a modified P1000 micro-pipette tip to transfer the appropriate volume of minced tissue to each previously prepared 1.5 milliliter tube and briefly mix the minced tissue with the medium within the tubes. Next, replace the medium in each ASC culture with one volume of white adipose tissue solution, and gently transfer the gelatin plungers to the white adipose tissue mixture on the base ASC plates. Examine the monolayers in the PNIPAAm coated plates under a microscope to confirm cellular detachment and move the plates to the surface of a 37 to 40 degree Celsius heat block in the biosafety cabinet.
Then add two to three milliliters of warmed culture medium to incubate the cells and to facilitate gelatin melting. After 30 minutes gently remove the plungers from the plate surface, cover the cultures with the appropriate plate lids and place the plates in the cell culture incubator. When the gelatin has completely liquified, aspirate and replace the cell culture medium with the appropriate volume of phenol red free M199 medium supplemented with insulin, dexamethasone, and antibiotics.
Over 7.5 weeks of culture, the SWAT clusters remain secured in place on the monolayer with only slight morphological changes observed in individual adipocytes. Clear Nile red staining of unilocular adipocyte clusters at two weeks of culture demonstrates that the cells are indeed adipocytes with intact membranes, rather than artifact, cysts, or dead adipocytes. Propidium iodide staining of 53-day-old SWAT and Oil Red O staining of 51-day-old SWAT cultures confirm that the adipocytes maintain viability even at advanced time points.
Immunocytochemistry demonstrates the appropriate expected protein expression and localization of mature adipocyte markers within the SWAT and quantitative PCR indicates the expression of key adipocyte genes at the transcriptional level. The evaluation of basil endocrine function indicates basil leptin secretion from the SWAT cultures as detected by ELISA, and glycerol release is also able to be induced in response to chemical stimulation. Xenotransplantation of SWAT-cultured adipocytes reveals clearly visible and readily recovered fat deposits 10 days after transplant that stain for human but not mouse adipocyte markers.
Once mastered, this technique can be completed in about five hours if it is performed properly. It's important to remember to process the primary tissue quickly to minimize cell death. Following this procedure, other methods like real time quantitative PCR, immunostaining, or functional assays can be performed to answer additional questions about how adipocytes respond to various drugs or toxins.
After watching this video you should have a good understanding of how to perform SWAT culture as a stable and reliable platform for your adipose research. Don't forget that working with primary human tissue can be hazardous, and that precautions such as wearing the proper PPE and handling the tissue under biosafety cabinets should always be taken while performing this procedure.
