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

Summary

Here, we present a detailed protocol outlining the use of microvascular fragments isolated from rodent or human fat tissue as a straightforward approach to engineer functional, vascularized beige adipose tissue.

Abstract

Engineering thermogenic adipose tissue (e.g., beige or brown adipose tissues) has been investigated as a potential therapy for metabolic diseases or for the design of personalized microtissues for health screening and drug testing. Current strategies are often quite complex and fail to accurately fully depict the multicellular and functional properties of thermogenic adipose tissue. Microvascular fragments, small intact microvessels comprised of arteriole, venules, and capillaries isolated from adipose tissue, serve as a single autologous source of cells that enable vascularization and adipose tissue formation. This article describes methods for optimizing culture conditions to enable the generation of three-dimensional, vascularized, and functional thermogenic adipose tissues from microvascular fragments, including protocols for isolating microvascular fragments from adipose tissue and culture conditions. Additionally, best practices are discussed, as are techniques for characterizing the engineered tissues, and sample results from both rodent and human microvascular fragments are provided. This approach has the potential to be utilized for the understanding and development of treatments for obesity and metabolic disease.

Introduction

The goal of this protocol is to describe an approach for developing vascularized beige adipose tissue from a single, potentially autologous source, microvascular fragment (MVF). Brown and beige adipose tissues have been demonstrated to display beneficial properties related to metabolic regulation; however, the small volume of these adipose tissue depots in adults limits the potential impact on systemic metabolism, particularly in diseased conditions such as obesity or type 2 diabetes^{1,2,3,4,5,6,7}. There is significant interest in brown/beige fat as a therapeutic target for preventing the harmful metabolic effects linked with obesity and its comorbidities^8,9,10,11,12.

MVFs are vessel structures that can be directly isolated from adipose tissue, cultured, and maintained in a three-dimensional configuration for extended periods of time^13,14,15. Previous work from our group, and others, have begun to exploit the multicellular and multipotent capacity of MVFs, specifically as it relates to adipose tissue formation^16,17,18. As a buildup of this work, we recently demonstrated that MVFs derived from rodent models of healthy and type 2 diabetes¹⁹ and from human subjects (adults over 50 years of age)²⁰ contained cells capable of being induced to form thermogenic, or beige, adipose tissue.

Herein is an innovative approach from which a single source MVF is utilized, not only capable of creating beige adipose tissue but also its associated and critical vascular component²¹. The use of this technique could be of great value for studies looking for a straightforward tissue-engineered approach for thermogenic adipose tissue formation. Unlike other methods aspiring to engineer beige adipose tissue^{22,23,24,25,26,27,28}, the process described in this study does not require using multiple cell types or complex induction regimens. Vascularized beige and white fat models can be created with MVFs originating from rodent and human sources, demonstrating great translation potential. The end product of this protocol is an engineered beige thermogenic fat tissue with a structure and metabolic function comparable to brown adipose tissue. Overall, this protocol presents the idea that an easily accessible and possibly autologous source MVF may be a worthwhile therapeutic intervention and tool for studying metabolic disorders.

Protocol

This study was conducted in compliance with the Animal Welfare Act and the Implementing Animal Welfare Regulations in accordance with the principles of the Guide for the Care and Use of Laboratory Animals. All animal procedures were approved by the Institutional Animal Care and Use Committee at the University of Texas at San Antonio.

NOTE: For the steps described below, male Lewis Rats are utilized. Slight protocol adjustments must be made for a female, as well as mouse microvascular fragment (MVF) collection²⁹. For protocols using human MVFs (h-MVFs), the only steps required are the resuspension of h-MVFs following the manufacturer's protocol, growth media preparation (1.3), formation of fibrin hydrogels (5), and culturing (6). For an overview of the protocol, please see Figure 1.

Figure 1: Experimental outline. Breakdown of six key steps, prior to analysis, for the formation of thermogenic adipose tissue using MVF. Please click here to view a larger version of this figure.

1. Reagent preparation

NOTE: Reagents below correspond to one rat, weighed and made inside a biohood.

1. Prepare bovine serum albumin (BSA) in PBS.
   1. Prepare 10 mg/mL (1.0%) BSA in PBS to be diluted for wash steps (1 mg/mL, 0.1%) and digestion (4 mg/mL, 0.4%).
   2. Prepare different concentrations of BSA, as mentioned in step 1.1.1, following steps 1.1.3-1.1.5.
   3. 10 mg/mL BSA in PBS (1% BSA in PBS)
      1. Add 500 mg of BSA and 500 mL of PBS to a 50 mL conical tube and vortex the solution to dissolve the BSA.
      2. If excessive bubbles form, centrifuge the solution at 350 x g for 2 min. Filter sterilize the solution with a 0.22 µm nylon net filter.
   4. 1 mg/mL BSA in PBS (0.1% BSA in PBS)
      1. Dilute 15 mL of 10 mg/mL BSA in PBS 1:10 with PBS by adding 15 mL of 10 mg/mL BSA in PBS to 135 mL of PBS in a 500 mL sterile bottle and gently shake the bottle to ensure a homogenous mixture.
   5. 4 mg/mL BSA in PBS (0.4% BSA in PBS)
      1. Dilute 35 mL of 10 mg/mL BSA in PBS 1:2.5 with PBS by adding 35 mL of 10 mg/mL BSA in PBS + 57.5 mL of PBS in a 100 mL sterile bottle and gently shake bottle to ensure a homogenous mixture.
2. Prepare collagenase in BSA for the digestion of minced fat pads.
   1. Prepare 6 mg/mL collagenase.
      1. In a 50 mL conical tube, weigh 72 mg of collagenase (label "for Epi").
      2. In three 50 mL conical tubes, weigh 144 mg of collagenase (label "for Ing 1", "for Ing 2", and "for SubQ") each.
      3. Store the weighed out collagenase at 4 ˚C until use.
         NOTE: Do not add BSA/PBS until right before digestion.
      4. Add 12 mL of 4 mg/mL BSA in PBS to the tube containing 72 mg of collagenase.
      5. Add 24 mL of 4 mg/mL BSA in PBS to the tubes containing 144 mg of collagenase.
      6. Shake the tubes to ensure a homogenous solution and filter sterilize the solution with a 0.22 µm nylon net filter.
3. Prepare growth media supplemented with Aminocaproic Acid (ACA) to feed/differentiate the isolated MVF.
   1. Growth media (GM): Supplement DMEM with 20% FBS, 1% penicillin-streptomycin (pen strep), 0.2% mycoplasma prophylactic, and 1 mg/mL ACA.
   2. Prepare white adipogenic media (WAM).
      1. WAM induction: Supplement DMEM/F12 with 20% FBS, 1% pen strep, 0.2% mycoplasma prophylactic, 10 µg/mL insulin, 10 µm of forskolin, 1 µm of dexamethasone, and 1 mg/mL ACA.
         NOTE: For h-MVFs, in addition, add 125 µM of indomethacin.
      2. WAM maintenance: Supplement DMEM/F12 with 20% FBS, 1% pen strep, 0.2% mycoplasma prophylactic, 5 µg/mL insulin, and 1 mg/mL ACA.
         NOTE: For h-MVFs, change the insulin concentration to 10 µg/mL.
   3. Prepare beige adipogenic media (BAM).
      1. BAM induction: Supplement DMEM/F12 with 20% FBS, 1% pen strep, 0.2% mycoplasma prophylactic, 10 µg/mL insulin, 10 µm of forskolin, 1 µm of dexamethasone, 1 µm of rosiglitazone, 20 nM 3,3′,5-Triiodo-L-thyronine (T3), and 1 mg/mL ACA.
         NOTE: For h-MVFs, change the concentration of T3 to 120 nM.
      2. BAM maintenance: Supplement DMEM/F12 with 20% FBS, 1% pen strep, 0.2% mycoplasma prophylactic, 5 µg/ml insulin, 10 µm of forskolin, 1 µm of rosiglitazone, 20 nM T3, and 1 mg/mL ACA.
         NOTE: For h-MVFs, change the insulin concentration to 10 µg/mL and the T3 concentration to 120 nM.
4. Prepare thrombin (only need to be made if the stock aliquoted solution is not available) to make the clotting agent to be used in fibrin gels.
   1. 10 U/mL thrombin in ddH₂O:
      1. Resuspend thrombin powder using 1-5 mL of ddH₂O in the vial from the manufacturer and transfer the resuspension to a 250 mL beaker.
      2. Bring up the solution to 100 mL and pipet the solution up and down numerous times to ensure a homogenous mixture. Aliquot the solution into 15 mL conical tubes (~10 mL/tube) and store the aliquots in a -20 ˚C freezer.
         NOTE: To use, thaw the thrombin at room temperature (RT).

2. Tool/materials preparation

NOTE: All instrumentation should be autoclaved/sterilized prior to use.

1. Epididymal/inguinal/subcutaneous fat isolation (surgery to be done in a defined surgical area)
   1. Ensure the availability of disposable underpads, two scissors, one hemostat (optional), two forceps, and four 50 mL conical tubes with 10-15 mL of 1 mg/mL BSA (0.1%) in PBS.
2. Microvascular fragment isolation
   1. For mincing (to be done in a biohood), ensure the availability of three uncoated sterile 100 mm Petri dishes, one pair of forceps, and one pair of curved scissors.
   2. For digestion and isolation (to be done in a biohood), ensure the availability of one pair of scissors, one pair of forceps, eight uncoated sterile 100 mm Petri dishes, three uncoated sterile 35 mm Petri dishes, collagenase weighed and at 4 ˚C, four 250 mL flasks, one plastic holder with a hole at the center, four 500 µM screens (cut into 3 in rounded squares), four 37 µM screens (cut into 3 in rounded squares), and 11 50 mL conical tubes.
3. Fibrin resuspension
   1. For fibrin hydrogels (to be done in a biohood), ensure that fibrinogen, thrombin, and growth media (made during reagent preparation) are available.

3. Fat isolation protocol

1. Preliminary steps
   1. Fill a beaker with ethanol to wash and sanitize the surgical instruments before using them. Next, prepare the table for handling the rat and have a vacuum ready to aspirate the fur generated during the shaving step.
   2. Prepare a bucket with ice where the previously prepared 50 mL conical tubes, each containing 10 mL of 1 mg/mL BSA, will be placed. Label the tubes accordingly.
      NOTE: It might be necessary to have two separate conical tubes for the inguinal fat, since two sides need to be extracted and the amount of fat gathered from this region tends to be the largest compared to epididymal and posterior subcutaneous.
   3. Start with a euthanized rat in the defined surgical area with the required surgical tools. Generally, CO₂ is used to euthanize the rats following IACUC protocols.
   4. Using hair clippers, shave the rat around the area of interest. Specifically, shave between the groins and half of the abdomen for epididymal and inguinal fat isolation (use the smaller shaver for the fur in this region). Shave the entire back to get the posterior subcutaneous fat located between the scapulas (it is best to use a larger shaver as the fur is thicker on the backside of the rat).
   5. Aseptically prepare the rat by spraying it down with 70% ethanol. Generally, it is recommended to clean the area that is going to be cut twice.
2. Isolation of different fat depots
   1. Inguinal (subcutaneous)
      1. In the supine position, lift the skin below the penis with a pair of scissors. Begin the incision with scissors, starting in the center and cutting laterally, forming a "V" shape and looping to the rat's backside to access the entire fat depot. Remember to cut superficially so that the fat below is intact. During the cutting step, ensure the separation of the skin from the fat by cutting the interconnected fascia tissue (Figure 2A).
      2. Once the fascia is appropriately cut, ensure that the two sides of the inguinal fat are visible (the inguinal fat extends from the groin area toward the back). Next, remove the fat from the two sides in two separate conical tubes containing 10 mL of 1 mg/mL BSA (Figure 2B).
   2. Epididymal (visceral)
      1. Harvest the epididymal fat by cutting through the abdominal skin and carefully through the thin layer surrounding the testicles (Figure 2C).
      2. With forceps, gently pull the fat tissue out and cut it out using scissors. Avoid dissecting any major visible blood vessels (should the testis and epididymis be harvested, remove them during the cleaning step in the biohood).
      3. Carefully place the removed fat into a 50 mL conical tube with 10 mL of 1 mg/mL BSA in PBS.
         NOTE: The removal of the epididymal fat should be done after the inguinal fat has been removed. The epididymal fat is generally smaller in volume and located below the inguinal fat, below the abdominal skin surrounding the testis and epididymis.
   3. Posterior subcutaneous
      1. Turn the rat prone (dorsal side up) and, using large scissors, cut the skin of the back (the skin in this region is thick) all the way up to the scalp, taking care not to cut too deep, just below the skin (Figure 2D).
      2. Cut the fascia connecting the skin to the tissue; the fat is located in the interscapular region. Make a note to differentiate/separate the subcutaneous fat from the brown fat. The brown fat is closer to the spine (Figure 2E).
      3. Isolate and place the fat in the corresponding 50 mL conical tube(s) with 10 mL of 1 mg/mL BSA in PBS.

Figure 2: Isolation of different adipose tissue depots. (A) Initial incisions needed for excision of the inguinal adipose tissue. (B) Location of the inguinal fat depot. (C) Location of the epididymal fat depot, noting incision of the outer skin needed for access. (D) Additional incisions needed once the mice are placed prone to access additional fat. (E) Location of the posterior subcutaneous fat depot. Please click here to view a larger version of this figure.

4. Microvascular fragment isolation protocol

1. Place 50 mL conical tube(s) containing excised fat from the rat into biohood.
2. Using forceps, place the fat in a standard 100 mm Petri dish (with ~0.5 mL of 1 mg/mL BSA in PBS to keep the tissue hydrated).
3. Clean and remove any visible blood vessels and muscle/extraneous tissue from fat.
4. Mince the fat with scissors for ~10 min (mince enough so that it can be transferred with a 10 mL pipet).
5. Check for lumps by adding ~0.5 mL of 1 mg/mL BSA in PBS; continue mincing if necessary.
6. Transfer the minced fat to a sterile 250 mL flask with a 10 mL pipet.
   NOTE: Note the volume using a pipet.
7. Add enough BSA (1 mg/mL) so the final volume is 20 mL.
8. Add 4 mg/mL BSA in PBS to collagenase (collagenase final concentration: 6 mg/mL) (i. e., 12 mL for the 72 mg of collagenase or 24 mL for the 144 mg of collagenase).
   NOTE: Do not add until the mincing is complete as it is time sensitive.
9. Gently shake the conical tube to ensure a homogenous solution and filter sterilize the solution with a 0.22 µm nylon net filter.
10. For the epididymal fat, digest for ~8-10 min; for the inguinal and posterior subcutaneous fat, digest for ~15-20 min in a 37 °C water bath, shaking the flask in a circular motion throughout (stop at the moment the fat gets to where there are only a few clumps remaining).
11. Transfer the digested fat into a 50 mL conical tube (there should be ~30 mL/tube), and label the tube as "digested fat".
12. Spin the tube at 400 x g for 4 min; post spinning, the pellet must be red (Figure 3A).
13. During the spin, place the sterile 37 µM screen and 500 µM screens in a sterile Petri dish with 5 mL of 1 mg/mL BSA in PBS to presoak before use.
14. After the spin, decant the supernatant into a 50 mL conical tube labeled as "waste". Perform the decantation gently to remove the superficial fat and to not disturb the pellet made of MVFs.
15. Add 10 mL of 1 mg/mL BSA in PBS to the tube containing the pellet ("digested fat"). Triturate (pipet up and down) the pellet 2x.
16. Avoid being too rough on the pellet in order not to disrupt the fragments.
17. Place the 500 µM screen in a new Petri dish above the plastic screen holder (Figure 3B).
18. Pipet 10 mL from the "digested pellet" tube over a 500 µM screen using concentric circles (Figure 3C).
19. Wash the filter with an additional 5 mL of 1 mg/mL BSA in PBS. The desired cells will filter through into the Petri dish; therefore, discard the 500 µM screen but save the filtered liquid inside the Petri dish.
20. Place the 37 µM screen in a new Petri dish above the plastic screen holder.
21. Change out the pipet to eliminate any clumps before using the 37 µM screen.
22. Pipet the liquid obtained from the first filtration over the 37 µM screen using concentric circles.
23. Wash the filter with an additional 5 mL of 1 mg/mL BSA in PBS. The desired cells will remain in the filter, therefore discard the filtered liquid, but save the 37 µM screen.
24. Slide the 37 µM screen into a new Petri dish containing 5 mL of 1 mg/mL BSA in PBS.
25. Shake the dish by tapping it against a conical holder to dislodge the fragments. Do not shake too vigorously, as the liquid/cells may spill out of the Petri dish.
26. Rinse the filter with an additional 5 mL of 1 mg/mL BSA in PBS. The desired cells will remain in liquid solution in the Petri dish. Save the 37 µM screen and the dislodged fragment containing liquid inside the Petri dish for the following steps.
27. Transfer the BSA + fragment containing liquid to a sterile 50 mL conical tube.
28. Repeat rinsing of the 37 µM screen several more times (each time with ~5 mL of 1 mg/mL BSA in PBS) and add to the conical tube. Repeat until the total volume collected is ~15-20 mL. Ultimately, the desired cells will be collected from the liquid solution in the Petri dish and placed in a conical tube; at this point, discard the 37 µM screen after the final rinse but save the dislodged fragments containing liquid inside the 50 mL conical tube.
29. Clip the end off of a 200 µL pipet tip using scissors. Gently shake the 50 mL tube, remove two samples of 20 µL, and put them in a clean 35 mm Petri dish.
30. Count the number of fragments in each sample in the Petri dish to obtain the total number of MVFs isolated.
    Total Fragments =
31. Spin the remaining dislodged fragment containing liquid in a 50 mL conical tube at 400 x g for 4 min to collect the MVF.

Figure 3: Isolation of MVFs. (A) Post digestion of the adipose tissue, depiction of the separation of MVFs containing pellet and supernatant following a spin-down. (B) Layout of supplies for filtration and entrapment of MVFs. (C) Illustration of the concentric circle method for filtration/washing steps. Please click here to view a larger version of this figure.

5. Formation of fibrin hydrogels

1. Example calculations:
   NOTE: Below are the calculations for MVFs seeded at ~15,000-20,000 MVF/mL and the fibrinogen:thrombin gel ratio at 2:5, with fibrinogen used at a 20 mg/mL concentration.
   1. For making five 250 µL gels, a total volume of 1,250 µL is required. Always account for pipetting errors, therefore, make enough for 1.5 mL of gels.
      1. Calculate the volume of fibrinogen required as follows:
         , X₁ = 428.57 µL of fibrinogen
      2. Calculate the volume of thrombin required as follows:
         , X₂ = 1,071.43 µL of thrombin
      3. For the required volume of 428.57 µL, make 500 µL of fibrinogen as follows:
         20 mg/mL * 0.5 mL = 10 mg of fibrinogen. Resuspend this in 500 µL of DMEM
      4. To obtain each gel, calculate the volume of fibrinogen and thrombin as follows:
         1. Fibrinogen:
            , X₁ = 71.43 µL of fibrinogen (in DMEM) + MVF
         2. Thrombin:
            , X₂ = 178.57 µL of thrombin
2. MVF fibrin gel casting
   1. Decant most of the liquid in the spun-down fragment in the 50 mL conical tube. Use a pipette to remove the small volume of liquid that catches on the rim of the conical tube.
   2. Add thrombin into wells where gels will be made (Figure 4A).
   3. Clip the end of a 200 µL pipet tip and resuspend the MVFs gently using fibrinogen to obtain a final density of ~15,000-20,000 MVF/mL once in the gel.
   4. Clip the end of a 200 µL pipet tip and pipet MVF+Fibrinogen into the thrombin solution gently. Quickly pipet up and down to ensure a homogenous mixture. Repeat until all gels are made (Figure 4B).
   5. Place the well plate(s) in an incubator (37 °C, 5% CO₂) for ~15 min to allow for gel crosslinking (Figure 4C).
   6. Add 100-150 µL of growth media to each well.

Figure 4: Formation of MVF fibrin gels. (A) A 5/7-part thrombin mixture gets pipetted into the corresponding well. (B) Next, with a clipped pipet tip (to not disturb MVFs), a 2/7-part fibrinogen+MVF mixture is pipetted into the well and gently mixed. (C) Lastly, all completed gels are placed into an incubator at 37 °C, allowing hydrogel to solidify fully before the media is placed on top. Please click here to view a larger version of this figure.

6. Culturing conditions of MVFs

1. For culturing non-vascularized white and beige adipose tissue (+ GM Control), use the timelines¹⁹ shown in Figure 5.
2. For culturing of vascularized white and beige adipose tissue (+ GM Control), use the timelines¹⁹ shown in Figure 6.
3. Hydrogels should be maintained in an incubator (37 °C, 5% CO₂) for the duration of culture for the study, with the media being changed every other day. For fixation and handling of samples for analysis, refer to previously published works^16,19,20.

Figure 5: Timing for non-vascularized adipose tissue formation. This figure has been modified from Acosta et al.¹⁹. Please click here to view a larger version of this figure.

Figure 6: Timing for vascularized adipose tissue formation. This figure has been modified from Acosta et al.¹⁹. Please click here to view a larger version of this figure.

Results

There are a few key phenotypic morphological characteristics of beige/brown adipose tissue: it is multilocular/contains small lipid droplets, possesses a large number of mitochondria (the reason for its characteristically "brownish" appearance in vivo), correspondingly has a high oxygen consumption rate/mitochondrial bioenergetics, is highly vascularized, has increased lipolysis/insulin-stimulated glucose uptake, and, most notoriously, expresses high levels of uncoupling protein 1 (UCP1), a mitochondrial...

Discussion

The field of brown/beige adipose tissue engineering is largely immature^{22,23,24,25,26,27,28}, with the bulk of adipose models being developed for white adipose tissue^8,22,31. Engineered brown/beige m...

Materials

Name	Company	Catalog Number	Comments
Aminocaproic Acid	Sigma Aldrich	A2504-100G	Added in DMEM at the concentration of 1 mg/mL
Blunt-Tipped Scissors	Fisher scientific	12-000-172	Sterilize in autoclave
Bovin Serum Albumin (BSA)	Millipore	126575-10GM	Diluted in PBS to 4 mg/mL and 1 mg/mL
Collagenase Type 1	Fisher scientific	NC9633623	Diluted to 6 mg/mL in BSA 4 mg/mL, Digestion of minced fat
Dexamethasone	Thermo Scientific	AC230302500	Diluted in ethanol at a 2 mg/ml stock concentration
Disposable underpads	Fisher scientific	23-666-062	For fluid absorption during surgery
Dissecting Scissors	Fisher scientific	08-951-5	Sterilize in autoclave
Dulbecco′s Modified Eagle′s Medium (DMEM)	Fisher scientific	11885092
Dulbecco′s Modified Eagle′s Medium/Nutrient Mixture F-12 Ham (DMEM/F12)	Sigma Aldrich	D8062
Fetal Bovine Serum	Fisher scientific	16140089	Added in DMEM to 20% v/v.
Fibrinogen	Sigma Aldrich	F8630-25G	Solubilized in DMEM at the concentration of 20 mg/mL, Protein found in blood plasma and main component of hydrogel
Flask, 250 mL	Fisher scientific	FB500250	Allows for digestion of fat using a large surface area
Forceps	Fisher scientific	50-264-21	Sterilize in autoclave, For handling of tissue and filters
Forskolin	Sigma Aldrich	F6886	Diluted in ethanol at a 10 mM stock concentration
Human MVF	Advanced Solutions Life Scienes, LLC	https://www.advancedsolutions.com/microvessels	Human MVFs (hMVFs) isolated from three different patients (52-, 54-, and 56-year old females) were used in the current study.
Indomethacine	Sigma Aldrich	I7378	Diluted in ethanol at a 12.5 mM stock concentration
Insulin from porcine pancreas	Sigma Aldrich	I5523	Diluted in 0.01 N HCl at a 5 mg/ml stock concentration
MycoZap	Fisher scientific	NC9023832	Added in DMEM to 0.2% w/v, Mycoplasma Prophylactic
Pennycilin/Streptomycin (10,000 U/mL)	Fisher scientific	15140122	Added in DMEM to 1% v/v.
Petri dishes, polystyrene (100 mm x 15 mm).	Fisher scientific	351029	3 for removal of blood vessels and mincing, 8 (lid) for presoaking of screens & 8 (dish) for use when filtering with 500 or 37 µM screens
Petri dishes, polystyrene (35 mm x 10 mm).	Fisher scientific	50-202-036	For counting fragments
Phosphate Buffer Saline (PBS)	Fisher scientific	14-190-250	Diluted to 1x with sterile deionized water.
Rat Clippers (Andwin Mini Arco Pet Trimmer)	Fisher scientific	NC0854141
Rosiglitazone	Fisher scientific	R0106200MG	Diluted in DMSO at a 10 mM stock concentration
Scissors	Fine Science Tools	14059-11	1 for initial incision, 1 for epididymal incision, 1 for tip clipping
Screen  37 µM	Carolina Biological Supply Company	652222R	Cut into 3" rounded squares and sterilized in ethylene oxide, Fragment entrapment and removal of very small fragments/single cells and debris
Screen 500 µM	Carolina Biological Supply Company	652222F	Cut into 3" rounded squares and sterilized in ethylene oxide, Removes larger fragments/debris
Serrated Hemostat	Fisher scientific	12-000-171	Sterilize in autoclave, For clamping of skin before incision
Steriflip Filter 0.22 μm	Millipore	SE1M179M6
Thrombin	Fisher scientific	6051601KU	Diluted in deionzed water to 10 U/mL, Used as a clotting agent turning fibrinogen to fibrin
Thyroid hormone (T3)	Sigma Aldrich	T2877	Diluted in 1N NaOH at a 0.02 mM stock concentration
Zucker diabetic fatty (ZDF) rats - obese (FA/FA) or lean (FA/+) male	Charles River	https://www.criver.com/products-services/find-model/zdf-rat-lean-fa?region=3611 https://www.criver.com/products-services/find-model/zdf-rat-obese?region=3611	Obtained from Charles River (Wilmington, MA). Rats were acquired at 4 weeks of age and fed Purina 5008 until euthanasia (15-19 weeks of age). Glucose levels (blood from the lateral saphenous vein) were greater than 300 mg/dL in all FA/FA rats used in the study. All animals were housed in a temperature-controlled environment with a 12-h light-dark cycle and fed ad libitum.