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.

<table><tbody><tr><td>0.25% trypsin-EDTA</td><td>Gibco, ThermoFisher Scientific Inc., Waltham, MA, USA</td><td>Cat#25200056</td><td></td></tr><tr><td>1.5 mL cryovial tube</td><td>Fisher Scientific, ThermoFisher Scientific Inc., Waltham, MA USA</td><td>Cat#02-682-557</td><td></td></tr><tr><td>10% Neutral Buffered Formalin</td><td>VWR International LLC., Radnor, PA, USA</td><td>Cat#89370-094</td><td></td></tr><tr><td>100 &amp;micro;m nylon mesh filter</td><td>Corning Inc., Corning, NY, USA</td><td>Cat#352360</td><td></td></tr><tr><td>2-Deoxy-D-glucose</td><td>Sigma-Aldrich, Inc., St Louis, MO, USA</td><td>Cat#D8375</td><td></td></tr><tr><td>2 nM 3,3&amp;rsquo;,5-Triiodo-L-thyronine sodium salt (T3)</td><td>Sigma-Aldrich, Inc. St Louis, MO, USA</td><td>Cat#T6397</td><td></td></tr><tr><td>24-well tissue culture plates</td><td>VWR International LLC., Radnor, PA, USA</td><td>Cat#10861-700</td><td></td></tr><tr><td>3-Isobutyl-1-methylxanthine (IBMX)</td><td>Sigma-Aldrich, Inc. 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

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...

Watch this Scientific Journal Video about A Human 3D Extracellular Matrix-Adipocyte Culture Model for Studying Matrix-Cell Metabolic Crosstalk at JoVE.com

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

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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9.2K Views.  University of Michigan Medical School. Ons protocol toont aan dat de ECM adipogene differentiatie kan bevorderen bij afwezigheid van klassieke adipogene bemiddelaars en is de eerste die ziektespecifieke regulatie van cellulair metabolisme door de ECM aantoont. Onze techniek biedt een hulpmiddel voor het ontleden van de rol van de ECM en adipocyten in vetweefsel metabolisme en laat studie van de rol van de ECM bij het reguleren van vetweefsel homeostase. Vergeleken met de standaard 2D-celcultuur, ons 3D-model zorgt voor meer robuus...

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

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

Mature adipocytes have been shown to reverse their phenotype into fibroblast-like cells in vitro through a technique called ceiling culture. Mature adipocytes can also be isolated from fresh adipose tissue for depot-specific characterization of their function and metabolic properties. Here, we describe a well-established protocol to isolate mature adipocytes from adipose tissues using collagenase digestion, and subsequent steps to perform ceiling cultures. Briefly, adipose tissues are incubated in a Krebs-Ringer-Henseleit buffer containing collagenase to disrupt tissue matrix. Floating mature adipocytes are collected on the top surface of the buffer. Mature cells are plated in a T25-flask completely filled with media and incubated upside down for a week. An alternative 6-well plate culture approach allows the characterization of adipocytes undergoing dedifferentiation. Adipocyte morphology drastically changes over time of culture. Immunofluorescence can be easily performed on slides cultivated in 6-well plates as demonstrated by FABP4 immunofluorescence staining. FABP4 protein is present in mature adipocytes but down-regulated through dedifferentiation of fat cells. Mature adipocyte dedifferentiation may represent a new avenue for cell therapy and tissue engineering.

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.

Mature adipocytes have been shown to reverse their phenotype into fibroblast-like cells in vitro through a technique called ceiling culture. Mature ...

Developmental Biology

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

The extracellular matrix (ECM) is a three-dimensional scaffold that acts as the main support for cells in tissues. Besides its structural function, the ECM also participates in cell migration, proliferation, and differentiation. Fibroblasts are the main type of cells modifying ECM fiber arrangement and production. In cancer, CAFs (cancer associated fibroblasts) are in permanent activation status, participating in ECM remodeling, facilitating tumor cell migration, and stimulating tumor-associated angiogenesis, among other pro-tumorigenic roles. The objective of this method is to create a three-dimensional matrix with a fiber composition that is similar to in vivo matrices, using immortalized fibroblasts or human primary CAFs. Fibroblasts are cultured in pre-treated cell culture plates and grown under ascorbic acid stimulation. Then, fibroblasts are removed and matrices are blocked for further cell seeding. In this ECM model, fibroblasts can be activated or modified to generate different kinds of matrix, whose effects can be studied in cell culture. 3D matrices are also shaped by cell signals, like degradation or cross-linking enzymes that might modify fiber distribution. In this context, angiogenesis can be studied, along with other cell types such as epithelial tumor cells.

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).

The extracellular matrix (ECM) is a three-dimensional scaffold that acts as the main support for cells in tissues. Besides its structural function, the ...

Cancer Research

A Microphysiologic Platform for Human Fat: Sandwiched White Adipose Tissue

White adipose tissue (WAT) plays a crucial role in regulating weight and everyday health. Still, there are significant limitations to available primary culture models, all of which have failed to faithfully recapitulate the adipose microenvironment or extend WAT viability beyond two weeks. The lack of a reliable primary culture model severely impedes research in WAT metabolism and drug development. To this end we have utilized NIH's standards of a microphysiologic system to develop a novel platform for WAT primary culture called 'SWAT' (sandwiched white adipose tissue). We overcome the natural buoyancy of adipocytes by sandwiching minced WAT clusters between sheets of adipose-derived stromal cells. In this construct, WAT samples are viable over eight weeks in culture. SWAT maintains the intact ECM, cell-to-cell contacts, and physical pressures of in vivo WAT conditions; additionally, SWAT maintains a robust transcriptional profile, sensitivity to exogenous chemical signaling, and whole tissue function. SWAT represents a simple, reproducible, and effective method of primary adipose culture. Potentially, it is a broadly applicable platform for research in WAT physiology, pathophysiology, metabolism, and pharmaceutical development.

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.

White adipose tissue (WAT) plays a crucial role in regulating weight and everyday health.  Still, there are significant limitations to available primary ...

Behavior

A Hormone-responsive 3D Culture Model of the Human Mammary Gland Epithelium

The process of mammary epithelial morphogenesis is influenced by hormones. The study of hormone action on the breast epithelium using 2D cultures is limited to cell proliferation and gene expression endpoints. However, in the organism, mammary morphogenesis occurs in a 3D environment. 3D culture systems help bridge the gap between monolayer cell culture (2D) and the complexity of the organism. Herein, we describe a 3D culture model of the human breast epithelium that is suitable to study hormone action. It uses the commercially available hormone-responsive human breast epithelial cell line, T47D, and rat tail collagen type 1 as a matrix. This 3D culture model responds to the main mammotropic hormones: estradiol, progestins and prolactin. The influence of these hormones on epithelial morphogenesis can be observed after 1- or 2-week treatment according to the endpoint. The 3D cultures can be harvested for analysis of epithelial morphogenesis, cell proliferation and gene expression.

We describe a 3D culture model of the human breast epithelium that is suitable to study hormone action.

The process of mammary epithelial morphogenesis is influenced by hormones. The study of hormone action on the breast epithelium using 2D cultures is ...

Fabrication of Extracellular Matrix-derived Foams and Microcarriers as Tissue-specific Cell Culture and Delivery Platforms

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus of this article is on the methods for fabricating ECM-derived porous foams and microcarriers for use as biologically-relevant substrates in advanced 3D in vitro cell culture models or as pro-regenerative scaffolds and cell delivery systems for tissue engineering and regenerative medicine. Using decellularized tissues or purified insoluble collagen as a starting material, the techniques can be applied to synthesize a broad array of tissue-specific bioscaffolds with customizable geometries. The approach involves mechanical processing and mild enzymatic digestion to yield an ECM suspension that is used to fabricate the three-dimensional foams or microcarriers through controlled freezing and lyophilization procedures. These pure ECM-derived scaffolds are highly porous, yet stable without the need for chemical crosslinking agents or other additives that may negatively impact cell function. The scaffold properties can be tuned to some extent by varying factors such as the ECM suspension concentration, mechanical processing methods, or synthesis conditions. In general, the scaffolds are robust and easy to handle, and can be processed as tissues for most standard biological assays, providing a versatile and user-friendly 3D cell culture platform that mimics the native ECM composition. Overall, these straightforward methods for fabricating customized ECM-derived foams and microcarriers may be of interest to both biologists and biomedical engineers as tissue-specific cell-instructive platforms for in vitro and in vivo applications.

The tissue-specific extracellular matrix (ECM) is a key mediator of cell function. This article describes methods for synthesizing pure ECM-derived foams and microcarriers that are stable in culture without the need for chemical crosslinking for applications in advanced 3D in vitro cell culture models or as pro-regenerative bioscaffolds.

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus ...

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

White adipose tissue (WAT) dysregulation plays a central role in development of insulin resistance and type 2 diabetes (T2D). To develop new treatments for T2D, more physiologically relevant in vitro adipocyte models are required. This study describes a new technique to isolate and culture mature human adipocytes. This method is entitled MAAC (membrane mature adipocyte aggregate culture), and compared to other adipocyte in vitro models, MAAC possesses an adipogenic gene signature that is the closest to freshly isolated mature adipocytes. Using MAAC, adipocytes can be cultured from lean and obese patients, different adipose depots, co-cultured with different cell types, and importantly, can be kept in culture for 2 weeks. Functional experiments can also be performed on MAAC including glucose uptake, lipogenesis, and lipolysis. Moreover, MAAC responds robustly to diverse pharmacological agonism and can be used to study adipocyte phenotypic changes, including the transdifferentiation of white adipocytes into brown-like fat cells.

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

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.

White adipose tissue (WAT) dysregulation plays a central role in development of insulin resistance and type 2 diabetes (T2D). To develop new treatments ...

Biology

Human SATMVECs: Isolation and Adipocyte Cross-Talk Studies

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

Microvascular endothelial cells (MVECs) have many critical roles, including control of vascular tone, regulation of thrombosis, and angiogenesis. Significant heterogeneity in endothelial cell (EC) genotype and phenotype depends on their vascular bed and host disease state. The ability to isolate MVECs from tissue-specific vascular beds and individual patient groups offers the opportunity to directly compare MVEC function in different disease states. Here, using subcutaneous adipose tissue (SAT) taken at the time of insertion of cardiac implantable electronic devices (CIED), we describe a method for the isolation of a pure population of functional human subcutaneous adipose tissue MVEC (hSATMVEC) and an experimental model of hSATMVEC-adipocyte cross-talk.
hSATMVEC were isolated following enzymatic digestion of SAT by incubation with anti-CD31 antibody-coated magnetic beads and passage through magnetic columns. hSATMVEC were grown and passaged on gelatin-coated plates. Experiments used cells at passages 2-4. Cells maintained classic features of EC morphology until at least passage 5. Flow cytometric assessment showed 99.5% purity of isolated hSATMVEC, defined as CD31+/CD144+/CD45-. Isolated hSATMVEC from controls had a population doubling time of approximately 57 h, and active proliferation was confirmed using a cell proliferation imaging kit. Isolated hSATMVEC function was assessed using their response to insulin stimulation and angiogenic tube-forming potential. We then established an hSATMVEC-subcutaneous adipocyte co-culture model to study cellular cross-talk and demonstrated a downstream effect of hSATMVEC on adipocyte function.
hSATMVEC can be isolated from SAT taken at the time of CIED insertion and are of sufficient purity to both experimentally phenotype and study hSATMVEC-adipocyte cross-talk.

Author Spotlight: Insights into Cardiometabolic Diseases with Subcutaneous Adipose Tissue Microvasculature Studies 

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.

Microvascular endothelial cells (MVECs) have many critical roles, including control of vascular tone, regulation of thrombosis, and angiogenesis. ...

Medicine

Three-Dimensional Adipocyte Culture as a Model to Study Cachexia-Induced White Adipose Tissue Remodeling

Cancer cachexia (CC) presents itself as a syndrome with multiple manifestations, causing a marked multi-organ metabolic imbalance. Recently, cachectic wasting has been proposed to be stimulated by several inflammatory mediators, which may disrupt the integrative physiology of adipose tissues and other tissues such as the brain and muscle. In this scenario, the tumor can survive at the host&#39;s expense. In recent clinical research, the intensity of depletion of the different fat deposits has been negatively correlated with the patient&#39;s survival outcome. Studies have also shown that various metabolic disorders can alter white adipose tissue (WAT) remodeling, especially in the early stages of cachexia development. WAT dysfunction resulting from tissue remodeling is a contributor to overall cachexia, with the main modifications in WAT consisting of morpho-functional changes, increased adipocyte lipolysis, accumulation of immune cells, reduction of adipogenesis, changes in progenitor cell population, and the increase of &#34;niches&#34; containing beige/brite cells.
To study the various facets of cachexia-induced WAT remodeling, particularly the changes&#160;progenitor cells and beige remodeling, two-dimensional (2D) culture has been the first option for in vitro studies. However, this approach does not adequately summarize WAT complexity. Improved assays for the reconstruction of functional AT ex vivo&#160;help the comprehension of physiological interactions between the distinct cell populations. This protocol describes an efficient three-dimensional (3D) printing tissue culture system based on magnetic nanoparticles. The protocol is optimized for investigating WAT remodeling induced by cachexia induced factors (CIFs). The results show that a 3D culture is an appropriate tool for studying WAT modeling ex vivo and may be useful for functional screens to identify bioactive molecules for individual adipose cell populations applications and aid the discovery of WAT-based cell anticachectic therapy.

This protocol describes a three-dimensional (3D) magnetic printing culture system that permits dissection of white adipose tissue (WAT) remodeling induced by a conditioned medium from cancer cells. Using a 3D culture system of UCP1+ adipocytes that express a green fluorescent protein (GFP) allows the study of beige adipocytes contributing to adipose tissue remodeling.

Cancer cachexia (CC) presents itself as a syndrome with multiple manifestations, causing a marked multi-organ metabolic imbalance. Recently, cachectic ...

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

Alteration of adipocyte function contributes to the pathogenesis of metabolic diseases including Type 2 diabetes and insulin resistance. This highlights the need to better understand the molecular mechanism involved in adipocyte dysfunction to develop new therapies against obesity-related diseases. Modulating the expression of proteins and micro-RNAs in adipocytes remains highly challenging. This paper describes a protocol to differentiate murine fibroblasts into mature adipocytes and to modulate the expression of proteins and micro-RNAs in mature adipocytes through reverse-transfection using small-interfering RNA (siRNA) and micro-RNA mimicking (miR mimic) oligonucleotides. This reverse-transfection protocol involves the incubation of the transfection reagent and the oligonucleotides to form a complex in the cell culture plate to which the mature adipocytes are added. The adipocytes are then allowed to reattach to the adherent plate surface in the presence of the oligonucleotides/transfection reagent complex. Functional analyses such as the study of insulin signaling, glucose uptake, lipogenesis, and lipolysis can be performed on the transfected 3T3-L1 mature adipocytes to study the impact of protein or micro-RNA manipulation on adipocyte function.

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.

Alteration of adipocyte function contributes to the pathogenesis of metabolic diseases including Type 2 diabetes and insulin resistance. This highlights ...

A Human Bone Marrow 3D Model to Investigate the Dynamics and Interactions Between Resident Cells in Physiological or Tumoral Contexts

The medullary niche is a complex ecosystem that is essential to maintain homeostasis for resident cells. Indeed, the bone marrow, which includes a complex extracellular matrix and various cell types, such as mesenchymal stem cells, osteoblasts, and endothelial cells, is deeply involved in hematopoietic stem cell regulation through direct cell-cell interactions, as well as cytokine production. To closely mimic this in vivo structure and conduct experiments reflecting the responses of the human bone marrow, several 3D models have been created based on biomaterials, relying primarily on primary stromal cells. Here, a protocol is described to obtain a minimal and standardized system that is easy to set up and provides features of bone marrow-like structure, which combines different cell populations including endothelial cells, and reflects the heterogeneity of in vivo bone marrow tissue. This 3D bone marrow-like structure-assembled using calcium phosphate-based particles and human cell lines, representative of the bone marrow microenvironment-allows the monitoring of a wide variety of biological processes by combining or replacing different primary cell populations within the system. The final 3D structures can then either be harvested for image analysis after fixation, paraffin-embedding, and histological/immunohistochemical staining for cell localization within the system, or dissociated to collect each cellular component for molecular or functional characterization.

Here, we describe an easy-to-implement, standardized, microphysiological system that reflects the complexity of the human bone marrow's in vivo structure, providing a pertinent model to finely study a broad range of normal and pathological events.

The medullary niche is a complex ecosystem that is essential to maintain homeostasis for resident cells. Indeed, the bone marrow, which includes a complex ...

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

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