Three-Dimensional Culture of Vascularized Thermogenic Adipose Tissue from Microvascular Fragments

This protocol describes the generation of three-dimensional vascularized and functional thermogenic fat from microvascular fragments, or MVFs. Current strategies for engineering thermogenic adipose tissue are complex and don't fully depict its multicellular and functional properties. MVFs are a single source of cells that enable vascularization and adipose tissue formation.

Being a simple sole-source method to create biological imitations of beige fat, MVFs hold significant potential for promoting the understanding or development of treatments for obesity and metabolic disease. Place the appropriately shaved and euthanized animal treated with 70%ethanol in a supine position and begin isolation of the inguinal fat. Lift the skin below the penis with a pair of scissors and perform the incision, starting at the center and cutting laterally, forming a V shape, before looping to the animal's backside to access the entire fat deposit.

While cutting, ensure the separation of the skin from the fat by cutting the interconnected fascia tissue. Once the fascia is appropriately cut, ensure the inguinal fat from both sides, extending from the groin towards the back, is visible. Next, remove the fat from the two sides in separate conical tubes containing 10 milliliters of BSA in PBS, having a concentration of one milligram per milliliter.

To harvest the epididymal fat, cut through the abdominal skin, followed by carefully cutting through the thin layer surrounding the testicles. Gently pull the fat tissue using forceps and cut it out using scissors, while avoiding the dissection of any major visible blood vessels. Now, place the removed fat into a 50-milliliter conical tube containing 10 milliliters of one milligram per milliliter BSA in PBS.

To isolate the posterior subcutaneous fat, turn the rat prone and use large scissors to cut the thick skin of the back all the way up to the scalp, taking care not to cut too deep below the skin. Cut the fascia connecting the skin to the tissue. Differentiate the subcutaneous fat, located in the intrascapular region, from the brown fat located closer to the spine before isolating and placing the subcutaneous fat in a 50-milliliter conical tube containing 10 milliliters of one milligram per milliliter BSA in PBS.

While working inside a biohood, use forceps to place the excised fat into a standard 100-milliliter Petri dish containing 0.5 milliliters of one milligram per milliliter BSA in PBS. Remove any visible blood vessels, muscles or extraneous tissue from the fat. Using scissors, mince the fat for 10 minutes and check for lumps by adding some more BSA.

Continue mincing, if necessary, before transferring the minced fat suspension to a sterile 250-milliliter flask with a 10-milliliter pipette. Make the volume in the flask up to 20 milliliters by adding enough BSA in PBS. Now add BSA in PBS to collagenase, and homogenize the solution by shaking it gently before filtering it sterile through a 0.22-micron nylon net filter.

Immediately add the required amount of collagenase solution to the minced fat suspension, and digest the fat by shaking the flask in a circular motion in a water bath at 37 degrees Celsius for the appropriate amount of time. Transfer the digested fat into a 50 milliliter conical tube labeled digested fat or minced fat. Centrifuge the tube at 400 G for four minutes to spin down the microvascular fragments, or MVFs, into a red pellet.

Gently decant the supernatant into a 50-milliliter conical tube labeled waste without disturbing the pellet. Next, add 10 milliliters of one milligram per milliliter BSA in PBS to the tube containing the pellet, and mix the pellet suspension by gently pipetting it up and down twice without disrupting the fragments. Now, take the 500-micron screen pre-soaked in a sterile Petri dish containing five milliliters of one milligram per milliliter BSA in PBS and place it over the plastic screen holder kept on a new Petri dish.

Pipette 10 milliliters of suspension from the digested pellet tube onto the screen in concentric circles. Wash the filter with an additional five milliliters of one milligram per milliliter BSA in PBS and discard it, while retaining the filtrate inside the Petri dish. Then place the pre-soaked 37-micron screen above the plastic screen holder on a new Petri dish.

Using a fresh pipette, transfer the filtrate from the first filtration onto the screen in concentric circles. As described previously, wash the screen with BSA in PBS, and this time, discard the filtrate while retaining the 37-micron screen. Slide the 37-micron screen into a new Petri dish containing five milliliters of one milligram per milliliter BSA in PBS.

Dislodge the fragments by gently tapping the dish against a conical holder, without spilling any liquid. Rinse the filter with an additional five milliliters of the BSA in PBS. Next, transfer the liquid from the Petri dish into a sterile 50-milliliter conical tube.

Rinse the 37-micron screen several more times, adding the wash to the conical tube til the total volume collected is 15 to 20 milliliters. Discard the screen after the final rinse. Cut the end of a 20-microliter pipette tip using scissors.

Gently shake the tube containing the liquid before drawing two aliquots of 20 microliters using the cut pipette tip, and pipette each of the aliquots into a clean 35-millimeter Petri dish. Count the number of fragments in the sample in each Petri dish using a standard light microscope, and using the formula mentioned in the manuscript, obtain the total number of isolated microvascular fragments. Spin the remaining liquid in the 50-milliliter conical tube at 400 G for four minutes to collect the MVF.

After spinning, decant most of the supernatant from the conical tube and use a pipette to remove the small volume of liquid remaining on the rim of the tube. Next, add thrombin into the wells designated for casting gels. Cut the end of a 200-microliter pipette tip, and using it, gently resuspend the MVFs in fibrinogen to obtain the desired final density.

Pipette the suspension into the thrombin solution in the well and quickly homogenize the mixture by pipetting up and down. Once all the gels are cast, place the well plate in an incubator at 37 degrees Celsius and 5%carbon dioxide for about 15 minutes to allow for gel cross-linking. The lipids and the differentiated adipocytes were observed by confocal microscopy imaging of the hydrogels using BODIPY as the lipid stain.

Culturing of non-vascularized adipose tissue using white adipogenic or beige adipogenic media resulted in the formation of non-vascularized white or beige adipose tissue, respectively, from both lean as well as diabetic rodent-derived MVFs. Similarly, culturing of vascularized adipose tissue using white adipogenic or beige adipogenic media resulted in the formation of vascularized white or beige adipose tissue, respectively, from the rodent-derived MVFs. Non-vascularized white and beige adipose tissue obtained from human MVFs were also observed by confocal microscopy.

The ability of MVFs to differentiate into beige adipose tissue was genetically confirmed using RT-qPCR. Lean and diabetic Rodent MVFs exposed to direct white adipogenic or beige adipogenic media were evaluated for adipogenesis, thermogenesis and angiogenesis. The expression of uncoupling protein 1 as expected, was significantly higher in beige adipogenic tissue.

A similar trend was observed in rodent MVFs exposed to indirect white adipogenic or beige adipogenic media, as well as in human MVFs exposed to direct white adipogenic or beige adipogenic media. Lastly, from mitochondrial bioenergetics, it was evident that functionally, the beige adipose tissue had characteristically higher oxygen consumption rate, or OCR, level in all cases. The enzymatic digestion of the adipose tissue should be optimized to consistently reproduce MVFs of similar size and quality.

Post-digestion, MVFs should be handled gently, with special care to avoid further breaking up of the fragments. This technique helped to further the balance of vessel growth and adipocyte differentiation, proven to be dependent on the factors introduced.