Name: A Human 3D Extracellular Matrix-Adipocyte Culture Model for Studying Matrix-Cell Metabolic Crosstalk (Video) | JoVE Journal
Uploaded: 2019-11-07T15:00:00.000+00:00
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Description: Learn a 3D human ECM-adipocyte culture model for studying matrix-cell metabolic crosstalk in adipose tissue. This method enables robust examination of adipocyte-ECM interactions relevant to type 2 diabetes research.

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We thank Danielle Berger, Marilyn Woodruff, Simone Correa, and Retha Geiss for assistance with study coordination. SEM was performed by University of Michigan Microscopy &#38; 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 &#38; Image Analysis Laboratory Biomedical Research Core Facility. Figure 4 of this manuscript was originally published in Baker et al., J Clin Endo Metab 2017; Mar 1;102 (3), 1032-1043. doi: 10.1210/jc.2016-2915, and has been reproduced by permission of Oxford University Press [https://academic.oup.com/jcem/article/102/3/1032/2836329]. For permission to reuse this material, please visit http://global.oup.com/academic/rights.

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The authors declare no conflicting interests.

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...

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, defects in adipocyte cellular metabolism, and adipose tissue endoplasmic reticulum and oxidative stress and inflammation. While poorly understood, these complex processes conspire to impair adipose tissue nutrient buffering capacity, leading to nutrient overflow from adipose tissue, toxicity in multiple tissues, and systemic metabolic disease3,4,5.The sequence of events and specific mechanisms that underlie adipose tissue failure are poorly understood, but alterations in adipose tissue ECM have been implicated. The ECM composition is altered within adipose tissue in human and murine obesity, with increased deposition of ECM protein along with qualitative biochemical and structural differences in the adipose tissue ECM associated with human metabolic disease, including type 2 diabetes and hyperlipidemia6,7,8,9,10,11.
Despite these observations, the role of adipose tissue ECM in mediating adipose tissue dysfunction is not well-defined. This is in part due to a lack of tractable experimental models that permit dissection of the specific roles of ECM and adipocytes in regulating ultimate adipose tissue function. ECM-adipocyte culture better simulates the in vivo environment of native adipose tissue in at least two respects. Firstly, ECM culture provides a molecular environment similar to native adipose tissue, including native collagens, elastins, and other matrix proteins absent in standard 2D culture. Secondly, culture on 2D plastic has been shown to alter adipocyte metabolism via mechanical effects due to decreased elasticity of plastic substrate12, which ECM-culture eliminates.
Methods to engineer biological scaffolds by isolation of ECM from decellularized adipose and other tissues have been studied in the context of regenerative and reconstructive medicine and tissue engineering13,14,15,16,17,18. We have previously published methodology in which we adapted these methods to develop an in vitro 3D model of human ECM-adipocyte culture, using ECM and adipocyte stem cells (preadipocytes) derived from human visceral adipose tissues11. In the present article, we describe these methods in detail. The decellularization procedure for human adipose tissue is a four-day process that involves mechanical and enzymatic treatments to remove cells and lipid, leaving a biological scaffold that maintains characteristics of the tissue from which it is derived. Decellularized ECM supports adipogenic differentiation of human preadipocytes, and when reconstituted with adipocytes, maintains microarchitecture and biochemical and disease-specific characteristics of intact adipose tissue and engages in metabolic functions characteristic of native adipose tissue. This matrix can be studied alone or reseeded with cells, permitting study of interactions and crosstalk between the cellular and extra-cellular components of adipose tissue.

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St Louis, MO, USA</td><td>Cat#I5879</td><td></td></tr><tr><td>96-well tissue culture plates</td><td>VWR International LLC., Radnor, PA, USA</td><td>Cat#10861-666</td><td></td></tr><tr><td>Antibiotic-Antimycotic Solution (ABAM)</td><td>Gibco, ThermoFisher Scientific Inc., Waltham, MA, USA</td><td>Cat#15240062</td><td></td></tr><tr><td>Biotin</td><td>Sigma-Aldrich, Inc. St Louis, MO, USA</td><td>Cat#B4639</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Sigma-Aldrich, Inc., St Louis, MO, USA</td><td>Cat#A8806</td><td></td></tr><tr><td>Buffer RLT</td><td>Qiagen, Hilden, Germany</td><td>Cat#79216</td><td></td></tr><tr><td>Ciglitizone</td><td>Sigma-Aldrich, Inc. St Louis, MO, USA</td><td>Cat#C3974</td><td></td></tr><tr><td>Deoxy-D-glucose, 2-[1,2-3H (N)]-</td><td>PerkinElmer Inc., Waltham, MA, USA</td><td>Cat#NET328A250UC</td><td></td></tr><tr><td>Deoxyribonuclease I from bovine pancreas, type II-S</td><td>Sigma-Aldrich, Inc. St Louis, MO, USA</td><td>Cat#D4513</td><td></td></tr><tr><td>Dexamethasone</td><td>Sigma-Aldrich, Inc. St Louis, MO, USA</td><td>Cat#D4902</td><td></td></tr><tr><td>Dimethyl Sulfoxide</td><td>Fisher Scientific, ThermoFisher Scientific Inc., Waltham, MA USA</td><td>Cat#BP231</td><td>Flammable, caustic</td></tr><tr><td>Disodium EDTA</td><td>Fisher Scientific, ThermoFisher Scientific Inc., Waltham, MA USA</td><td>Cat#BP118</td><td></td></tr><tr><td>D-pantothenic acid hemicalcium salt</td><td>Sigma-Aldrich, Inc. St Louis, MO, USA</td><td>Cat#21210</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12</td><td>Gibco, ThermoFisher Scientific Inc., Waltham, MA USA</td><td>Cat#11320033</td><td></td></tr><tr><td>Ethanol</td><td>Decon Labs, Inc., King of Prussia, PA, USA</td><td>Cat#DSP-MD.43</td><td>Flammable</td></tr><tr><td>EVE Cell Counting Slides, NanoEnTek</td><td>VWR International LLC., Radnor, PA, USA</td><td>Cat#10027-446</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Gibco, ThermoFisher Scientific Inc., Waltham, MA, USA</td><td>Cat#10437028</td><td></td></tr><tr><td>Glutaraldehyde</td><td>Sigma-Aldrich, Inc., St Louis, MO, USA</td><td>Cat#G5882</td><td>Caustic</td></tr><tr><td>Hexamethyldisalizane</td><td>Sigma-Aldrich, Inc. St Louis, MO, USA</td><td>Cat#440191</td><td>Flammable, caustic</td></tr><tr><td>Human insulin solution</td><td>Sigma-Aldrich, Inc. St Louis, MO, USA</td><td>Cat#I9278</td><td></td></tr><tr><td>Isopropanol</td><td>Fisher Scientific, ThermoFisher Scientific Inc., Waltham, MA USA</td><td>Cat#A415</td><td>Flammable</td></tr><tr><td>Isoproterenol</td><td>Sigma-Aldrich, Inc., St Louis, MO, USA</td><td>Cat#I5627</td><td>Flammable</td></tr><tr><td>KCl</td><td>Sigma-Aldrich, Inc. St Louis, MO, USA</td><td>Cat#S25484</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich, Inc. St Louis, MO, USA</td><td>Cat#P5655</td><td></td></tr><tr><td>Lipase from porcine pancreas, type VI-S</td><td>Sigma-Aldrich, Inc. St Louis, MO, USA</td><td>Cat#L0382</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;*7H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich, Inc. St Louis, MO, USA</td><td>Cat#230391</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich, Inc. St Louis, MO, USA</td><td>Cat#S5136</td><td></td></tr><tr><td>NaCl</td><td>Sigma-Aldrich, Inc. St Louis, MO, USA</td><td>Cat#S3014</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Fisher Scientific, ThermoFisher Scientific Inc., Waltham, MA USA</td><td>Cat#S233</td><td></td></tr><tr><td>NH&lt;sub&gt;4&lt;/sub&gt;Cl</td><td>Fisher Scientific, ThermoFisher Scientific Inc., Waltham, MA USA</td><td>Cat#A661</td><td></td></tr><tr><td>Optimal cutting temperature (OCT) compound</td><td>Agar Scientific, Ltd., Stansted, Essex, UK</td><td>Cat# AGR1180</td><td></td></tr><tr><td>Oil Red-O Solution (ORO)</td><td>Sigma-Aldrich, Inc., St Louis, MO, USA</td><td>Cat#O1391</td><td></td></tr><tr><td>Oil Red-O Stain Kit</td><td>American Master Tech Scientific Inc., Lodi, CA, USA</td><td>Cat#KTORO-G</td><td></td></tr><tr><td>Osmium tetroxide</td><td>Sigma-Aldrich, Inc. St Louis, MO, USA</td><td>Cat#201030</td><td>Caustic</td></tr><tr><td>Phenylmethylsulfonyl fluoride (PMSF)</td><td>Sigma-Aldrich, Inc. St Louis, MO, USA</td><td>Cat#93482</td><td>Caustic</td></tr><tr><td>Phosphate Buffered Saline Solution (PBS)</td><td>Fisher Scientific, ThermoFisher Scientific Inc., Waltham, MA USA</td><td>Cat#SH3025601</td><td></td></tr><tr><td>Ribonuclease A from bovine pancreas, type III-A</td><td>Sigma-Aldrich, Inc. St Louis, MO, USA</td><td>Cat#R5125</td><td></td></tr><tr><td>RNAEasy Fibrous Tissue MiniKit</td><td>Qiagen, Hilden, Germany</td><td>Cat#74704</td><td></td></tr><tr><td>Scintillation Fluid</td><td>Fisher Scientific, ThermoFisher Scientific Inc., Waltham, MA USA</td><td>Cat#SX18</td><td></td></tr><tr><td>Scintillation Counter</td><td></td><td></td><td></td></tr><tr><td>Scissors, forceps, sterile</td><td></td><td></td><td></td></tr><tr><td>Sorensen&#039;s phosphate buffer</td><td>Thomas Scientific, Inc., Swedesboro, NJ</td><td>CAS #: 10049-21-5</td><td></td></tr><tr><td>T-150 culture flask</td><td>VWR International LLC., Radnor, PA, USA</td><td>Cat#10062-864</td><td></td></tr><tr><td>TaqMan Gene Expression Master Mix</td><td>ThermoFisher Scientific Inc., Waltham, MA USA</td><td>Cat#4369016</td><td></td></tr><tr><td>Temperature-controlled orbital shaker</td><td></td><td></td><td></td></tr><tr><td>Tissue Homogenizer, BeadBug Microtube Homogenizer</td><td>Benchmark Scientific</td><td>Cat#D1030</td><td></td></tr><tr><td>Transferrin</td><td>Sigma-Aldrich, Inc. St Louis, MO, USA</td><td>Cat#T3309</td><td></td></tr><tr><td>Triglyceride Determination Kit</td><td>Sigma-Aldrich, Inc., St Louis, MO, USA</td><td>Cat#TR0100</td><td></td></tr><tr><td>Trypan blue stain, 0.4%</td><td>VWR International LLC., Radnor, PA, USA</td><td>Cat#10027-446</td><td></td></tr><tr><td>Type II collagenase</td><td>Gibco, ThermoFisher Scientific Inc., Waltham, MA, USA</td><td>Cat#17101015</td><td></td></tr><tr><td>Whatman Reeve Angel filter paper, Grade 201, 150mm</td><td>Sigma-Aldrich, Inc., St Louis, MO, USA</td><td>Cat#WHA5201150</td><td></td></tr></tbody></table>

a human 3d extracellular matrix-adipocyte culture model for studying matrix-cell metabolic crosstalk

A Human 3D Extracellular Matrix-Adipocyte Culture Model for Studying Matrix-Cell Metabolic Crosstalk

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.

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...

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 ...

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JoVE Journal

Bioengineering

In standard culture methods cells are taken out of their physiological environment and grown on the plastic surface of a dish. To study the behavior of primary human bone marrow cells we created a 3-D culture system where cells are grown under conditions recapitulating the native microenvironment of the tissue.

A Three-dimensional Tissue Culture Model to Study Primary Human Bone Marrow and its Malignancies

Mature adipocytes may represent an abundant source of stem cells through dedifferentiation, which leads to a homogenous population of fibroblast-like cells. Collagenase digestion is used to isolate mature adipocytes from human fat. The goal of our protocol is to obtain multipotent, dedifferentiated fat cells from human mature adipocytes.

Generation of Human Adipose Stem Cells through Dedifferentiation of Mature Adipocytes in Ceiling Cultures

White adipose tissue (WAT) has critical deficiencies in its current primary culture models, hindering pharmacological development and metabolic studies. Here, we present a protocol to produce an adipose microphysiological system by sandwiching WAT between sheets of stromal cells. This construct provides a stable and adaptable platform for primary WAT culture.

A Microphysiologic Platform for Human Fat: Sandwiched White Adipose Tissue

The aim of this method is to obtain fibroblast-derived 3D matrices as a natural scaffold for subsequent cellular assays. Fibroblasts are seeded in a pre-treated culture plate and stimulated with ascorbic acid for matrix generation. Matrices are decellularized and blocked to culture relevant cells (e.g., endothelial cells).

Fibroblast-Derived 3D Matrix System Applicable to Endothelial Tube Formation Assay

Membrane mature adipocyte aggregate culture (MAAC) is a new method to culture mature human adipocytes. Here we detail how to isolate adipocytes from human adipose and how to set up MAAC.

Isolation and Culture of Human Mature Adipocytes Using Membrane Mature Adipocyte Aggregate Cultures (MAAC)

Presented here is a protocol to deliver oligonucleotides such as small-interfering RNA (siRNA), micro-RNA mimics (miRs), or anti-micro-RNA (anti-miR) into mature adipocytes to modulate protein and micro-RNA expression.

An Adipocyte Cell Culture Model to Study the Impact of Protein and Micro-RNA Modulation on Adipocyte Function

Here, we describe a protocol for isolating, culturing, and phenotyping microvascular endothelial cells from human subcutaneous adipose tissue (hSATMVECs). Additionally, we describe an experimental model of hSATMVEC-adipocyte cross-talk.

Studying Adipose Endothelial Cell/Adipocyte Cross-Talk in Human Subcutaneous Adipose Tissue

This protocol describes the fabrication of 3D bone constructs using human osteocytes embedded in methacrylated gelatin (GelMA) hydrogels supplemented with decellularized extracellular matrix. The method is low-cost, scalable, and compatible with basic laboratory equipment, providing a translational platform for studying musculoskeletal disease processes.

A 3D Bone Culture Platform Using Human Osteocytes and Decellularized Extracellular Matrix for Modeling Musculoskeletal Diseases

Using an ex vivo explant model, small pieces of the rostral portion of visceral white adipose tissue from lean mice are cultured for 7–10 days, enabling the induction and study of adipose tissue inflammation as it occurs during high-fat diet feeding.

An ex vivo Mouse Model for High-Fat Diet-Like Adipose Tissue Inflammation

We describe a simple, rapid method of generating 3D tissue-like spheroids and their potential application to quantify differences in cell-cell interactions.

A Simple Hanging Drop Cell Culture Protocol for Generation of 3D Spheroids

Learn a 3D human ECM-adipocyte culture model for studying matrix-cell metabolic crosstalk in adipose tissue. This method enables robust examination of adipocyte-ECM interactions relevant to type 2 diabetes research.

A Human 3D Extracellular Matrix-Adipocyte Culture Model for Studying Matrix-Cell Metabolic Crosstalk

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