Name: three-dimensional culture of vascularized thermogenic adipose tissue from microvascular fragments
Uploaded: 2023-02-03T13:30:00.000+00:00
Duration: 11 min 24 s
Description: Watch this Scientific Journal Video about Three-Dimensional Culture of Vascularized Thermogenic Adipose Tissue from Microvascular Fragments at JoVE.com

Dr. Acosta is supported by the National Institutes of Health grants CA148724 and TL1TR002647. Dr. Gonzalez Porras is supported by the National Institute of Diabetes and Digestive and Kidney Diseases of the National Institutes of Health, under Award Number F32-0DK122754. This work was supported, in part, by the National Institutes of Health (5SC1DK122578) and the University of Texas at San Antonio Department of Biomedical Engineering. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Figures were partially created with Biorender.com.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The field of brown/beige adipose tissue engineering is largely immature22,23,24,25,26,27,28, with the bulk of adipose models being developed for white adipose tissue8,22,31. Engineered brown/beige m...

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 diabetes1,2,3,4,5,6,7. There is significant interest in brown/beige fat as a therapeutic target for preventing the harmful metabolic effects linked with obesity and its comorbidities8,9,10,11,12.
MVFs are vessel structures that can be directly isolated from adipose tissue, cultured, and maintained in a three-dimensional configuration for extended periods of time13,14,15. 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 formation16,17,18. 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 tissue22,23,24,25,26,27,28, 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.

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

three-dimensional culture of vascularized thermogenic adipose tissue from microvascular fragments

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.

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 &#34;brownish&#34; 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...

Watch this Scientific Journal Video about Three-Dimensional Culture of Vascularized Thermogenic Adipose Tissue from Microvascular Fragments at JoVE.com

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

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.

Engineering thermogenic adipose tissue (e.g., beige or brown adipose tissues) has been investigated as a potential therapy for metabolic diseases or for ...

three-dimensional-culture-vascularized-thermogenic-adipose-tissue

Research

JoVE Journal

Bioengineering

1.7K Views.  University of Texas Health Science Center. 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.

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

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

Isolation of Murine Adipose Tissue-derived Microvascular Fragments as Vascularization Units for Tissue Engineering

A functional microvascular network is of pivotal importance for the survival and integration of engineered tissue constructs. For this purpose, several angiogenic and prevascularization strategies have been established. However, most cell-based approaches include time-consuming in vitro steps for the formation of a microvascular network. Hence, they are not suitable for intraoperative one-step procedures. Adipose tissue-derived microvascular fragments (ad-MVF) represent promising vascularization units. They can be easily isolated from fat tissue and exhibit a functional microvessel morphology. Moreover, they rapidly reassemble into new microvascular networks after in vivo implantation. In addition, ad-MVF have been shown to induce lymphangiogenesis. Finally, they are a rich source of mesenchymal stem cells, which may further contribute to their high vascularization potential. In previous studies we have demonstrated the remarkable vascularization capacity of ad-MVF in engineered bone and skin substitutes. In the present study, we report on a standardized protocol for the enzymatic isolation of ad-MVF from murine fat tissue.

We present a protocol to isolate adipose tissue-derived microvascular fragments that represent promising vascularization units. They can be rapidly isolated, do not require in vitro processing and, thus, may be used for one-step prevascularization in different fields of tissue engineering.

A functional microvascular network is of pivotal importance for the survival and integration of engineered tissue constructs. For this purpose, several ...

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.

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 ...

Assessment of Human Adipose Tissue Microvascular Function Using Videomicroscopy

While obesity is closely linked to the development of metabolic and cardiovascular disease, little is known about mechanisms that govern these processes. It is hypothesized that pro-atherogenic mediators released from fat tissues particularly in association with central/visceral adiposity may promote pathogenic vascular changes locally and systemically, and the notion that cardiovascular disease may be the consequence of adipose tissue dysfunction continues to evolve. Here, we describe a unique method of videomicroscopy that involves analysis of vasodilator and vasoconstrictor responses of intact small human arterioles removed from the adipose depot of living human subjects. Videomicroscopy is used to examine functional properties of isolated microvessels in response to pharmacological or physiological stimuli using a pressured system that mimics in vivo conditions. The technique is a useful approach to gain understanding of the pathophysiology and molecular mechanisms that contribute to vascular dysfunction locally within the adipose tissue milieu. Moreover, abnormalities in the adipose tissue microvasculature have also been linked with systemic diseases. We applied this technique to examine depot-specific vascular responses in obese subjects. We assessed endothelium-dependent vasodilation to both increased flow and acetylcholine in adipose arterioles (50 - 350 &#181;m internal diameter, 2 - 3 mm in length) isolated from two different adipose depots during bariatric surgery from the same individual. We demonstrated that arterioles from visceral fat exhibit impaired endothelium-dependent vasodilation compared to vessels isolated from the subcutaneous depot. The findings suggest that the visceral microenvironment is associated with vascular endothelial dysfunction which may be relevant to clinical observation linking increased visceral adiposity to systemic disease mechanisms. The videomicroscopy technique can be used to examine vascular phenotypes from different fat depots as well as compare findings across individuals with different degrees of obesity and metabolic dysfunction. The method can also be used to examine vascular responses longitudinally in response to clinical interventions.

Videomicroscopy systems are used to examine functional properties of isolated adipose tissue arterioles in response to physiological and pharmacological stimuli. This technique can be used to examine microvascular phenotypes in different adipose tissue domains in obese humans.

While obesity is closely linked to the development of metabolic and cardiovascular disease, little is known about mechanisms that govern these processes. ...

Medicine

Adipo-Clear: A Tissue Clearing Method for Three-Dimensional Imaging of Adipose Tissue

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte precursors, immune cells, fibroblasts, blood vessels, and nerve projections. Although the molecular control of cell type specification and how these cells interact have been increasingly delineated, a more comprehensive understanding of these adipose-resident cells can be achieved by visualizing their distribution and architecture throughout the whole tissue. Existing immunohistochemistry and immunofluorescence approaches to analyze adipose histology rely on thin paraffin-embedded sections. However, thin sections capture only a small portion of tissue; as a result, the conclusions can be biased by what portion of tissue is analyzed. We have therefore developed an adipose tissue clearing technique, Adipo-Clear, to permit comprehensive three-dimensional visualization of molecular and cellular patterns in whole adipose tissues. Adipo-Clear was adapted from iDISCO/iDISCO+, with specific modifications made to completely remove the lipid stored in the tissue while preserving native tissue morphology. In combination with light-sheet fluorescence microscopy, we demonstrate here the use of the Adipo-Clear method to obtain high-resolution volumetric images&#160;of an entire adipose tissue.

Due to the high lipid content, adipose tissue has been challenging to visualize using traditional histological methods. Adipo-Clear is a tissue clearing technique that allows robust labeling and high-resolution volumetric fluorescent imaging of adipose tissue. Here, we describe the methods for sample preparation, pretreatment, staining, clearing, and mounting for imaging.

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte ...

Biology

Visualization of 3D White Adipose Tissue Structure Using Whole-mount Staining

Adipose tissue is an important metabolic organ with high plasticity and is responsive to environmental stimuli and nutrient status. As such, various techniques have been developed to study the morphology and biology of adipose tissue. However, conventional visualization methods are limited to studying the tissue in 2D sections, failing to capture the 3D architecture of the whole organ. Here we present whole-mount staining, an immunohistochemistry method that preserves intact adipose tissue morphology with minimal processing steps. Hence, the structures of adipocytes and other cellular components are maintained without distortion, achieving the most representative 3D visualization of the tissue. In addition, whole-mount staining can be combined with lineage tracing methods to determine cell fate decisions. However, this technique has some limitations to providing accurate information regarding deeper parts of adipose tissue. To overcome this limitation, whole-mount staining can be further combined with tissue clearing techniques to remove the opaqueness of tissue and allow for complete visualization of entire adipose tissue anatomy using light-sheet fluorescent microscopy. Therefore, a higher resolution and more accurate representation of adipose tissue structures can be captured with the combination of these techniques.

The focus of the present study is to demonstrate the whole-mount immunostaining and visualization technique as an ideal method for 3D imaging of adipose tissue architecture and cellular component.

Adipose tissue is an important metabolic organ with high plasticity and is responsive to environmental stimuli and nutrient status. As such, various ...

Human Adipose Tissue Micro-fragmentation for Cell Phenotyping and Secretome Characterization

In the past decade, adipose tissue transplants have been widely used in plastic surgery and orthopaedics to enhance tissue repletion and/or regeneration. Accordingly, techniques for harvesting and processing human adipose tissue have evolved in order to quickly and efficiently obtain large amounts of tissue. Among these, the closed system technology represents an innovative and easy-to-use system to harvest, process, and re-inject refined fat tissue in a short time and in the same intervention (intra-operatively). Adipose tissue is collected by liposuction, washed, emulsified, rinsed and minced mechanically into cell clusters of 0.3 to 0.8 mm. Autologous transplantation of mechanically fragmented adipose tissue has shown remarkable efficacy in different therapeutic indications such as aesthetic medicine and surgery, orthopedic and general surgery. Characterization of micro-fragmented adipose tissue revealed the presence of intact small vessels within the adipocyte clusters; hence, the perivascular niche is left unperturbed. These clusters are enriched in perivascular cells (i.e., mesenchymal stem cell (MSC) ancestors) and in vitro analysis showed an increased release of growth factors and cytokines involved in tissue repair and regeneration, compared to enzymatically derived MSCs. This suggests that the superior therapeutic potential of microfragmented adipose tissue is explained by a higher frequency of presumptive MSCs and enhanced secretory activity. Whether these added pericytes directly contribute to higher growth factor and cytokine production is not known. This clinically approved procedure allows the transplantation of presumptive MSCs without the need for expansion and/or enzymatic treatment, thus bypassing the requirements of GMP guidelines, and reducing the costs for cell-based therapies.

Here, we present human adipose tissue enzyme-free micro-fragmentation using a closed system device. This new method allows the obtainment of sub-millimeter clusters of adipose tissue suitable for in vivo transplantation, in vitro culture, and further cell isolation and characterization.

In the past decade, adipose tissue transplants have been widely used in plastic surgery and orthopaedics to enhance tissue repletion and/or regeneration. ...

Exploring Adipose Tissue Structure by Methylsalicylate Clearing and 3D Imaging

Obesity is a major worldwide public health issue that increases the risk to develop cardiovascular diseases, type-2 diabetes, and liver diseases. Obesity is characterized by an increase in adipose tissue (AT) mass due to adipocyte hyperplasia and/or hypertrophia, leading to profound remodeling of its three-dimensional structure. Indeed, the maximal capacity of AT to expand during obesity is pivotal to the development of obesity-associated pathologies. This AT expansion is an important homeostatic mechanism to enable adaptation to an excess of energy intake and to avoid deleterious lipid spillover to other metabolic organs, such as muscle and liver. Therefore, understanding the structural remodeling that leads to the failure of AT expansion is a fundamental question with high clinical applicability. In this article, we describe a simple and fast clearing method that is routinely used in our laboratory to explore the morphology of mouse and human white adipose tissue by fluorescent imaging. This optimized AT clearing method is easily performed in any standard laboratory equipped with a chemical hood, a temperature-controlled orbital shaker and a fluorescent microscope. Moreover, the chemical compounds used are readily available. Importantly, this method allows one to resolve the 3D AT structure by staining various markers to specifically visualize the adipocytes, the neuronal and vascular networks, and the innate and adaptive immune cells distribution.

Here, we describe a simple, inexpensive and fast clearing method to resolve the 3D structure of both mouse and human white adipose tissue using a combination of markers to visualize vasculature, nuclei, immune cells, neurons, and lipid-droplet coat proteins by fluorescent imaging.

Obesity is a major worldwide public health issue that increases the risk to develop cardiovascular diseases, type-2 diabetes, and liver diseases. Obesity ...

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. ...

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 ...