Our protocol demonstrates that the ECM may promote adipogenic differentiation in the absence of classic adipogenic mediators and is the first to demonstrate disease-specific regulation of cellular metabolism by the ECM. Our technique provides a tool for dissecting the roles of the ECM and adipocytes in adipose tissue metabolism and permit study of the role of the ECM in regulating adipose tissue homeostasis. Compared with standard 2D cell culture, our 3D model allows for more robust examination and dissection of cross-talk between adipocytes and adipose tissue ECM in the context of type 2 diabetes.
We recommend beginning with smaller samples to gauge how well it is cellularized. Keep in mind there is interpatient variability in how well the cells grow and how well the tissue decellularizes. It is important to observe the tissue before and after each step to ensure the tissue is becoming decellularized and to understand potential differences between samples.
After obtaining visceral adipose tissue or VAT from surgery, prepare it for storage by adding 5-10 grams of the tissue to 15-25 milliliters of freezing buffer solution in a 50 milliliter conical tube. Completely immerse the sample and store it at minus 80 degrees Celsius until decellularization. For preadipocyte isolation, place two grams of a fresh sample of intact VAT in 20 milliliters of collagenase type II solution, then insert sterile scissors into the tube and thoroughly mince the tissue.
Once minced to a fine slurry, incubate it in the collagenase solution at 37 degrees Celsius on an orbital shaker for 60 minutes. After the incubation, filter the resultant digested through a 100 micrometer nylon mesh into a fresh 50 milliliter conical tube. The digested should be a yellow-orange liquid with moderate viscosity and a small amount of residual strands of undigested tissue.
Centrifuge the sample at 270 times g for 10 minutes, remove the supernatant and resuspend the cell pellet in two milliliters of 1X RBC lysing solution with a pipette. Incubate the solution for one minute at 25 degrees Celsius, then add 10 milliliters of 15%FBS DMEM F12 and centrifuge it at 270 times g for 10 minutes. To prepare the ECM, freeze/thaw the previously frozen VAT sample to 37 degrees Celsius by incubating it in a pre-heated water bath for 20 minutes with periodic gentle manual agitation.
Once thawed, transfer the tissue back to minus 80 degrees Celsius for 20 minutes and repeat the freeze/thaw process three times ending with the thawing. Use sterile forceps to transfer the VAT sample to a fresh 50 milliliter conical tube containing 15-25 milliliters of enzymatic solution 1 making sure that the sample is fully immersed. Then incubate it overnight at 37 degrees Celsius while shaking at 130 RPM.
On the next day, wash the sample three times with 15-25 milliliters of rinsing buffer solution, pouring off the solution after each wash. Transfer the sample to a fresh 50 milliliter tube with 15-25 milliliters of enzymatic solution 2 and incubate it overnight. Repeat the washes with the rinsing buffer solution and transfer the sample to a fresh tube containing 15-25 milliliters of polar solvent extraction solution.
Incubate the sample overnight while shaking. After incubation with the polar solvent solution, it is important to examine the sample for lipid removal. If most of the lipid is not removed, it may be necessary to repeat this step.
Shorter incubation time may be used. After the incubation, transfer the sample to a fresh tube containing 15-25 milliliters of rinsing buffer solution and wash the sample three times on the shaker. Then wash the sample three times with 70%ethanol solution, pouring off the ethanol after each wash.
Wash the sample once with storage solution, then use sterile forceps to transfer to a fresh tube containing 15-25 milliliters of storage solution making sure to fully immerse it. The sample can then be stored at four degrees Celsius for up to one month. The isopropanol and ethanol are flammable and must be at room temperature and disposed of in flammable waste.
Any human waste must be disposed of separately from the usual biohazard waste. Transfer the stored ECM fragments to individual wells of a 24-well plate with sterile forceps adding as many fragments as required for the planned downstream assays. Wash them three times with 500 microliters of 70%ethanol on an orbital shaker, then rehydrate them by washing three times with PBS.
Use sterile scissors to cut the ECM into 100 milligram fragments. Place each fragment into one well of a 24-well plate and incubate the plate at 25 degrees Celsius for 15 minutes to allow excess PBS to extrude from the fragments. Then carefully remove any leftover PBS from the wells with a pipette.
Seed each fragment with 20 microliters of preadipocyte cell suspension by pipetting the cells directly into the ECM. Place the pipette tip in the ECM and gently expel it into the center of the matrix making sure that the cell suspension does not overflow and end up at the bottom of the well. If the cell suspension overflows from the ECM, remove the pipette tip from that location and insert it elsewhere in the ECM.
After cell seeding, incubate the ECM for 40 minutes at 37 degrees Celsius. Fill each well with 500 microliters of growth media to cover the seeded ECM fragments. Culture the cells at 37 degrees Celsius and 5%carbon dioxide for 72 hours.
After 72 hours, carefully aspirate the growth media without disturbing the ECM fragment. Add 500 microliters of differentiation media and culture the cells for 14 days changing the media every two to three days. Check for differentiation using light microscopy.
Cells will accumulate lipids, turn brown-yellow in color and become more spherical as they differentiate. Preparation of adipose tissue ECM, seeding with preadipocytes, and in vitro differentiation into mature adipocytes was monitored with scanning electron microscopy which revealed decellularization of ECM and subsequent recapitulation of lipid containing adipocytes upon reseeding and differentiation. Collagen 1 specific immunohistochemistry of decellularized ECM showed maintenance of collagen micro architecture while Oil Red-O staining of live and fixed 3D ECM-adipocyte constructs showed lipid containing adipocytes.
The adipocytes differentiated in ECM were screened for adipogenic genes using quantitative real-time PCR which demonstrated that these genes are upregulated in the differentiated cells relative to undifferentiated preadipocytes cultured in ECM. The full difference in transcript levels is shown. Four combinations of ECM and adipocytes from diabetic and nondiabetic subjects were subjected to metabolic phenotyping.
Impaired glucose uptake in lipolytic function was discovered in constructs from diabetic tissues. Importantly, it was found that nondiabetic ECM rescues insulin-stimulated glucose uptake and lipolytic capacity in diabetic adipocytes. Contamination of the isolated preadipocytes, the decellularized tissue, and the reseeded ECM is not uncommon.
Sterility must be diligently maintained throughout the protocol.
