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We describe a 3D human extracellular matrix-adipocyte in vitro culture system that permits dissection of the roles of the matrix and adipocytes in contributing to adipose tissue metabolic phenotype.
The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of cellular function. The ECM plays a particularly important role in adipose tissue function in obesity, and alterations in adipose tissue ECM deposition and composition are associated with metabolic disease in mice and humans. Tractable in vitro models that permit dissection of the roles of the ECM and cells in contributing to global tissue phenotype are sparse. We describe a novel 3D in vitro model of human ECM-adipocyte culture that permits study of the specific roles of the ECM and adipocytes in regulating adipose tissue metabolic phenotype. Human adipose tissue is decellularized to isolate ECM, which is subsequently repopulated with preadipocytes that are then differentiated within the ECM into mature adipocytes. This method creates ECM-adipocyte constructs that are metabolically active and retain characteristics of the tissues and patients from which they are derived. We have used this system to demonstrate disease-specific ECM-adipocyte crosstalk in human adipose tissue. This culture model provides a tool for dissecting the roles of the ECM and adipocytes in contributing to global adipose tissue metabolic phenotype and permits study of the role of the ECM in regulating adipose tissue homeostasis.
The extracellular matrix (ECM) not only provides a mechanical scaffold for tissues, but also engages in complex crosstalk with cells that reside within it, regulating diverse processes necessary for tissue homeostasis, including cell proliferation, differentiation, signaling, and metabolism1. While healthy ECM plays an essential role the maintenance of normal tissue function, dysfunctional ECM has been implicated in multiple diseases2.
Adipose tissue plays an important role in the pathogenesis of metabolic disease. Obesity is associated with excessive adipocyte hypertrophy and cellular hypoxia....
Adipose tissues are procured from human subjects undergoing elective bariatric surgery under institutional review board approval.
1. Preadipocyte isolation and culture reagent preparation
Preparation of adipose tissue ECM, seeding with preadipocytes, and in vitro differentiation into mature adipocytes result in clear sequential morphologic changes in tissue that permits visual assessment of progress throughout the protocol (Figure 1). Preadipocytes used to seed the ECM are isolated using collagenase digestion from separate VAT samples (Figure 2). Scanning electron microscopy of ECM-adipocyte constructs at each sta.......
The ECM-adipocyte culture model provides a valuable tool for dissecting the individual roles of ECM and cells in dictating ultimate tissue phenotype. The ECM isolation protocol is quite reproducible, but variability in the decellularization process may be observed. The Day 3 delipidation step is a critical point in the protocol. At the completion of the overnight extraction, delipidation of the matrix should be evidenced by the Polar Solvent Solution turning yellow, while the matrix should transition from a yellow-orange.......
We thank Danielle Berger, Marilyn Woodruff, Simone Correa, and Retha Geiss for assistance with study coordination. SEM was performed by University of Michigan Microscopy & Image Analysis Laboratory Biomedical Research Core Facility. This project was supported by NIH grants R01DK097449 (RWO), R01DK115190 (RWO, CNL), R01DK090262 (CNL), Veterans Affairs Merit Grant I01CX001811 (RWO), Pilot and Feasibility Grant from the Michigan Diabetes Research Center (NIH Grant P30-DK020572) (RWO), Veterans Administration VISN 10 SPARK Pilot Grant (RWO). Scanning electron microscopy performed by the University of Michigan Microscopy & Image Analysis Laboratory Biomedical Resea....
Name | Company | Catalog Number | Comments |
0.25% trypsin-EDTA | Gibco, ThermoFisher Scientific Inc., Waltham, MA, USA | Cat#25200056 | |
1.5 mL cryovial tube | Fisher Scientific, ThermoFisher Scientific Inc., Waltham, MA USA | Cat#02-682-557 | |
10% Neutral Buffered Formalin | VWR International LLC., Radnor, PA, USA | Cat#89370-094 | |
100 µm nylon mesh filter | Corning Inc., Corning, NY, USA | Cat#352360 | |
2-Deoxy-D-glucose | Sigma-Aldrich, Inc., St Louis, MO, USA | Cat#D8375 | |
2 nM 3,3’-5,Triiodo,L-thyronine sodium salt (T3) | Sigma-Aldrich, Inc. St Louis, MO, USA | Cat#T6397 | |
24-well tissue culture plates | VWR International LLC., Radnor, PA, USA | Cat#10861-700 | |
3-Isobutyl-1-methylxanthine (IBMX) | Sigma-Aldrich, Inc. St Louis, MO, USA | Cat#I5879 | |
96-well tissue culture plates | VWR International LLC., Radnor, PA, USA | Cat#10861-666 | |
Antibiotic-Antimycotic Solution (ABAM) | Gibco, ThermoFisher Scientific Inc., Waltham, MA, USA | Cat#15240062 | |
Biotin | Sigma-Aldrich, Inc. St Louis, MO, USA | Cat#B4639 | |
Bovine Serum Albumin (BSA) | Sigma-Aldrich, Inc., St Louis, MO, USA | Cat#A8806 | |
Buffer RLT | Qiagen, Hilden, Germany | Cat#79216 | |
Ciglitizone | Sigma-Aldrich, Inc. St Louis, MO, USA | Cat#C3974 | |
Deoxy-D-glucose, 2-[1,2-3H (N)]- | PerkinElmer Inc., Waltham, MA, USA | Cat#NET328A250UC | |
Deoxyribonuclease I from bovine pancreas, type II-S | Sigma-Aldrich, Inc. St Louis, MO, USA | Cat#D4513 | |
Dexamethasone | Sigma-Aldrich, Inc. St Louis, MO, USA | Cat#D4902 | |
Dimethyl Sulfoxide | Fisher Scientific, ThermoFisher Scientific Inc., Waltham, MA USA | Cat#BP231 | Flammable, caustic |
Disodium EDTA | Fisher Scientific, ThermoFisher Scientific Inc., Waltham, MA USA | Cat#BP118 | |
D-pantothenic acid hemicalcium salt | Sigma-Aldrich, Inc. St Louis, MO, USA | Cat#21210 | |
Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12 | Gibco, ThermoFisher Scientific Inc., Waltham, MA USA | Cat#11320033 | |
Ethanol | Decon Labs, Inc., King of Prussia, PA, USA | Cat#DSP-MD.43 | Flammable |
EVE Cell Counting Slides, NanoEnTek | VWR International LLC., Radnor, PA, USA | Cat#10027-446 | |
Fetal bovine serum (FBS) | Gibco, ThermoFisher Scientific Inc., Waltham, MA, USA | Cat#10437028 | |
Glutaraldehyde | Sigma-Aldrich, Inc., St Louis, MO, USA | Cat#G5882 | Caustic |
Hexamethyldisalizane | Sigma-Aldrich, Inc. St Louis, MO, USA | Cat#440191 | Flammable, caustic |
Human insulin solution | Sigma-Aldrich, Inc. St Louis, MO, USA | Cat#I9278 | |
Isopropanol | Fisher Scientific, ThermoFisher Scientific Inc., Waltham, MA USA | Cat#A415 | Flammable |
Isoproterenol | Sigma-Aldrich, Inc., St Louis, MO, USA | Cat#I5627 | Flammable |
KCl | Sigma-Aldrich, Inc. St Louis, MO, USA | Cat#S25484 | |
KH2PO4 | Sigma-Aldrich, Inc. St Louis, MO, USA | Cat#P5655 | |
Lipase from porcine pancreas, type VI-S | Sigma-Aldrich, Inc. St Louis, MO, USA | Cat#L0382 | |
MgSO4*7H2O | Sigma-Aldrich, Inc. St Louis, MO, USA | Cat#230391 | |
Na2HPO4 | Sigma-Aldrich, Inc. St Louis, MO, USA | Cat#S5136 | |
NaCl | Sigma-Aldrich, Inc. St Louis, MO, USA | Cat#S3014 | |
NaHCO3 | Fisher Scientific, ThermoFisher Scientific Inc., Waltham, MA USA | Cat#S233 | |
NH4Cl | Fisher Scientific, ThermoFisher Scientific Inc., Waltham, MA USA | Cat#A661 | |
Optimal cutting temperature (OCT) compound | Agar Scientific, Ltd., Stansted, Essex, UK | Cat# AGR1180 | |
Oil Red-O Solution (ORO) | Sigma-Aldrich, Inc., St Louis, MO, USA | Cat#O1391 | |
Oil Red-O Stain Kit | American Master Tech Scientific Inc., Lodi, CA, USA | Cat#KTORO-G | |
Osmium tetroxide | Sigma-Aldrich, Inc. St Louis, MO, USA | Cat#201030 | Caustic |
Phenylmethylsulfonyl fluoride (PMSF) | Sigma-Aldrich, Inc. St Louis, MO, USA | Cat#93482 | Caustic |
Phosphate Buffered Saline Solution (PBS) | Fisher Scientific, ThermoFisher Scientific Inc., Waltham, MA USA | Cat#SH3025601 | |
Ribonuclease A from bovine pancreas, type III-A | Sigma-Aldrich, Inc. St Louis, MO, USA | Cat#R5125 | |
RNAEasy Fibrous Tissue MiniKit | Qiagen, Hilden, Germany | Cat#74704 | |
Scintillation Fluid | Fisher Scientific, ThermoFisher Scientific Inc., Waltham, MA USA | Cat#SX18 | |
Scintillation Counter | |||
Scissors, forceps, sterile | |||
Sorensen's phosphate buffer | Thomas Scientific, Inc., Swedesboro, NJ | CAS #: 10049-21-5 | |
T-150 culture flask | VWR International LLC., Radnor, PA, USA | Cat#10062-864 | |
TaqMan Gene Expression Master Mix | ThermoFisher Scientific Inc., Waltham, MA USA | Cat#4369016 | |
Temperature-controlled orbital shaker | |||
Tissue Homogenizer, BeadBug Microtube Homogenizer | Benchmark Scientific | Cat#D1030 | |
Transferrin | Sigma-Aldrich, Inc. St Louis, MO, USA | Cat#T3309 | |
Triglyceride Determination Kit | Sigma-Aldrich, Inc., St Louis, MO, USA | Cat#TR0100 | |
Trypan blue stain, 0.4% | VWR International LLC., Radnor, PA, USA | Cat#10027-446 | |
Type II collagenase | Gibco, ThermoFisher Scientific Inc., Waltham, MA, USA | Cat#17101015 | |
Whatman Reeve Angel filter paper, Grade 201, 150mm | Sigma-Aldrich, Inc., St Louis, MO, USA | Cat#WHA5201150 |
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