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Silk is a natural polymer sourced mainly from spiders and silkworms. Due to its biocompatibility, biodegradability, and mechanical properties, it has been heavily investigated for biomedical applications. It can be processed into a number of formats, such as scaffolds, films, and nanoparticles. Common methods of production create constructs with limited complexity. 3D printing allows silk to be printed into more intricate designs, increasing its potential applications. Extrusion and inkjet printing are the primary ways silk has been 3D printed, though other methods are beginning to be investigated. Silk has been integrated into bioink with other polymers, both natural and synthetic. The addition of silk is primarily done to offer more desirable viscosity characteristics and mechanical properties for printing. Silk-based bioinks have been used to fabricate medical devices and tissues. This article discusses recent research and printing parameters important for 3D printing with silk.
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Materiais Biocompatíveis/química , Matriz Extracelular/química , Impressão Tridimensional , Seda/química , Engenharia Tecidual , Alicerces Teciduais/químicaRESUMO
Increases in adipocyte volume and tissue mass due to obesity can result in inflammation, further dysregulation in adipose tissue function, and eventually adipose tissue fibrosis. Like other fibrotic diseases, adipose tissue fibrosis is the accumulation and increased production of extracellular matrix (ECM) proteins. Adipose tissue fibrosis has been linked to decreased insulin sensitivity, poor bariatric surgery outcomes, and difficulty in weight loss. With the rising rates of obesity, it is important to create accurate models for adipose tissue fibrosis to gain mechanistic insights and develop targeted treatments. This article discusses recent research in modeling adipose tissue fibrosis using in vivo and in vitro (2D and 3D) methods with considerations for biomaterial selections. Additionally, this article outlines the importance of adipose tissue in treating other fibrotic diseases and methods used to detect and characterize adipose tissue fibrosis.
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Tecido Adiposo/patologia , Técnicas de Cultura de Tecidos/métodos , Adipócitos/patologia , Animais , Materiais Biocompatíveis , Estudos Epidemiológicos , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Fibrose , Humanos , RoedoresRESUMO
Adipose tissue engineered models are needed to enhance our understanding of disease mechanisms and for soft tissue regenerative strategies. Perfusion systems generate more physiologically relevant and sustainable adipose tissue models, however adipocytes have unique properties that make culturing them in a perfusion environment challenging. In this paper we describe the methods involved in the development of two perfusion culture systems (2D and 3D) to test their applicability for long term in vitro adipogenic cultures. It was hypothesized that a silk protein biomaterial scaffold would provide a 3D framework, in combination with perfusion flow, to generate a more physiologically relevant sustainable adipose tissue engineered model than 2D cell culture. Consistent with other studies evaluating 2D and 3D culture systems for adipogenesis we found that both systems successfully model adipogenesis, however 3D culture systems were more robust, providing the mechanical structure required to contain the large, fragile adipocytes that were lost in 2D perfused culture systems. 3D perfusion also stimulated greater lipogenesis and lipolysis and resulted in decreased secretion of LDH compared to 2D perfusion. Regardless of culture configuration (2D or 3D) greater glycerol was secreted with the increased nutritional supply provided by perfusion of fresh media. These results are promising for adipose tissue engineering applications including long term cultures for studying disease mechanisms and regenerative approaches, where both acute (days to weeks) and chronic (weeks to months) cultivation are critical for useful insight.
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Adipogenia/fisiologia , Adipócitos/citologia , Células-Tronco Adultas/citologia , Animais , Materiais Biocompatíveis/química , Técnicas de Cultura de Células/métodos , Humanos , Teste de Materiais , Perfusão/instrumentação , Seda/química , Engenharia Tecidual/métodos , Alicerces Teciduais/químicaRESUMO
Obesity is a significant public health concern that is closely associated with various comorbidities such as heart disease, stroke, type II diabetes (T2D), and certain cancers. Due to the central role of adipose tissue in many disease etiologies and the pervasive nature in the body, engineered adipose tissue models are essential for drug discovery and studying disease progression. This study validates a fat-on-a-chip (FOAC) model derived from primary mature adipocytes. Our FOAC model uses a Micronit perfusion device and introduces a novel approach for collecting continuous data by using two non-invasive readout techniques, resazurin and glucose uptake. The Micronit platform proved to be a reproducible model that can effectively maintain adipocyte viability, metabolic activity, and basic functionality, and is capable of mimicking physiologically relevant responses such as adipocyte hypertrophy and insulin-mediated glucose uptake. Importantly, we demonstrate that adipocyte size is highly dependent on extracellular matrix properties, as adipocytes derived from different patients with variable starting lipid areas equilibrate to the same size in the hyaluronic acid hydrogel. This model can be used to study T2D and monitor adipocyte responses to insulin for longitudinally tracking therapeutic efficacy of novel drugs or drug combinations.
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Conventional therapies to address critically sized defects in subcutaneous adipose tissue remain a reconstructive challenge for surgeons, largely due to the lack of graft pre-vascularization. Adipose tissue relies on a dense microvasculature network to deliver nutrients, oxygen, nonadipose tissue-derived growth factors, cytokines, and hormones, as well as transporting adipose tissue-derived endocrine signals to other organ systems. This chapter addresses these vascularization issues by combining decellularized lung matrices with a step-wise seeding of patient-specific adipose-derived stem cells and endothelial cells to develop large-volume, perfusable, and pre-vascularized adipose grafts.
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Engenharia Tecidual , Alicerces Teciduais , Humanos , Células Endoteliais , Tecido Adiposo , AdipócitosRESUMO
Adipose tissue is a dynamic regulatory organ that has profound effects on the overall health of patients. Unfortunately, inconsistencies in human adipose tissues are extensive and multifactorial, including large variability in cellular sizes, lipid content, inflammation, extracellular matrix components, mechanics, and cytokines secreted. Given the high human variability, and since much of what is known about adipose tissue is from animal models, we sought to establish correlations and patterns between biological, mechanical, and epidemiological properties of human adipose tissues. To do this, twenty-six independent variables were cataloged for twenty patients, which included patient demographics and factors that drive health, obesity, and fibrosis. A factorial analysis for mixed data (FAMD) was used to analyze patterns in the dataset (with BMI > 25), and a correlation matrix was used to identify interactions between quantitative variables. Vascular endothelial growth factor A (VEGFA) and actin alpha 2, smooth muscle (ACTA2) gene expression were the highest loadings in the first two dimensions of the FAMD. The number of adipocytes was also a key driver of patient-related differences, where a decrease in the density of adipocytes was associated with aging. Aging was also correlated with a decrease in overall lipid percentage of subcutaneous tissue, with lipid deposition being favored extracellularly, an increase in transforming growth factor-ß1 (TGFß1), and an increase in M1 macrophage polarization. An important finding was that self-identified race contributed to variance between patients in this study, where Black patients had significantly lower gene expression levels of TGFß1 and ACTA2. This finding supports the urgent need to account for patient ancestry in biomedical research to develop better therapeutic strategies for all patients. Another important finding was that TGFß induced factor homeobox 1 (TGIF1), an understudied signaling molecule, which is highly correlated with leptin signaling, was correlated with metabolic inflammation. Furthermore, this study draws attention to what we define as "extracellular lipid droplets", which were consistently found in collagen-rich regions of the obese adipose tissues evaluated here. Reduced levels of TGIF1 were correlated with higher numbers of extracellular lipid droplets and an inability to suppress fibrotic changes in adipose tissue. Finally, this study indicated that M1 and M2 macrophage markers were correlated with each other and leptin in patients with a BMI > 25. This finding supports growing evidence that macrophage polarization in obesity involves a complex, interconnecting network system rather than a full switch in activation patterns from M2 to M1 with increasing body mass. Overall, this study reinforces key findings in animal studies and identifies important areas for future research, where human and animal studies are divergent. Understanding key drivers of human patient variability is required to unravel the complex metabolic health of unique patients.
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Gordura Subcutânea , Humanos , Gordura Subcutânea/metabolismo , Masculino , Feminino , Pessoa de Meia-Idade , Adulto , Adipócitos/metabolismo , Obesidade/metabolismo , Obesidade/patologia , Actinas/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , IdosoRESUMO
Obesity and its co-morbidities including type 2 diabetes are increasing at epidemic rates in the U.S. and worldwide. Brown adipose tissue (BAT) is a potential therapeutic to combat obesity and type 2 diabetes. Increasing BAT mass by transplantation improves metabolic health in rodents, but its clinical translation remains a challenge. Here, we investigated if transplantation of 2-4 million differentiated brown pre-adipocytes from mouse BAT stromal fraction (SVF) or human pluripotent stem cells (hPSCs) could improve metabolic health. Transplantation of differentiated brown pre-adipocytes, termed "committed pre-adipocytes" from BAT SVF from mice or derived from hPSCs improves glucose homeostasis and insulin sensitivity in recipient mice under conditions of diet-induced obesity, and this improvement is mediated through the collaborative actions of the liver transcriptome, tissue AKT signaling, and FGF21. These data demonstrate that transplantation of a small number of brown adipocytes has significant long-term translational and therapeutic potential to improve glucose metabolism.
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In areolar "loose" connective tissue, fibroblasts remodel their cytoskeleton within minutes in response to static stretch resulting in increased cell body cross-sectional area that relaxes the tissue to a lower state of resting tension. It remains unknown whether the loosely arranged collagen matrix, characteristic of areolar connective tissue, is required for this cytoskeletal response to occur. The purpose of this study was to evaluate cytoskeletal remodeling of fibroblasts in, and dissociated from, areolar and dense connective tissue in response to 2 h of static stretch in both native tissue and collagen gels of varying crosslinking. Rheometric testing indicated that the areolar connective tissue had a lower dynamic modulus and was more viscous than the dense connective tissue. In response to stretch, cells within the more compliant areolar connective tissue adopted a large "sheet-like" morphology that was in contrast to the smaller dendritic morphology in the dense connective tissue. By adjusting the in vitro collagen crosslinking, and the resulting dynamic modulus, it was demonstrated that cells dissociated from dense connective tissue are capable of responding when seeded into a compliant matrix, while cells dissociated from areolar connective tissue can lose their ability to respond when their matrix becomes stiffer. This set of experiments indicated stretch-induced fibroblast expansion was dependent on the distinct matrix material properties of areolar connective tissues as opposed to the cells' tissue of origin. These results also suggest that disease and pathological processes with increased crosslinks, such as diabetes and fibrosis, could impair fibroblast responsiveness in connective tissues.
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Citoesqueleto/fisiologia , Fibroblastos/citologia , Fibroblastos/fisiologia , Estresse Fisiológico/fisiologia , Animais , Técnicas de Cultura de Células , Sobrevivência Celular , Células Cultivadas , Colágeno/química , Colágeno/fisiologia , Tecido Conjuntivo/fisiologia , Tecido Conjuntivo/ultraestrutura , Fibroblastos/ultraestrutura , Imuno-Histoquímica , Masculino , Camundongos , ReologiaRESUMO
STUDY DESIGN: Cells isolated from moderately and severely degenerated human intervertebral disks (IVDs) cultured in an alginate scaffold. OBJECTIVE: To compare the regenerative potential of moderately versus severely degenerated cells using 3 proanabolic stimulants. SUMMARY OF BACKGROUND DATA: Injection of soluble cell signaling factors has potential to slow the progression of IVD degeneration. Although degenerative grade is thought to be an important factor in targeting therapeutic interventions it remains unknown whether cells in severely degenerated IVDs have impaired metabolic functions compared to lesser degenerative levels or if they are primarily influenced by the altered microenvironment. METHODS: Nucleus pulposus (NP) cells were cultured in alginate for 21 days and treated with 3 different proanabolic stimulants: a growth factor/anti-inflammatory combination of transforming growth factor ß3 (TGFß3)+dexamethasone (Dex), or matricellular proteins connective tissue growth factor (CTGF) or Link-N. They were assayed for metabolic activity, DNA content, glycosaminoglycan, and qRT-PCR gene profiling. RESULTS: Moderately degenerated cells responded to stimulation with increased proliferation, decreased IL-1ß, MMP9, and COL1A1 expression, and upregulated HAS1 as compared with severely degenerated cells. TGFßR1 (ALK5) receptors were expressed at greater levels in moderately than severely degenerated cells. TGFß3+Dex had a notable stimulatory effect on moderately degenerated NP cells with increased anabolic gene expression and decreased COL1A1 and ADAMTS5 gene expression. Link-N and CTGF had similar responses in all assays, and both treatments upregulated IL-1ß expression and had a more catabolic response than TGFß3+Dex, particularly in the more severely degenerated group. All groups, including different degenerative grades, produced similar amounts of glycosaminoglycan. CONCLUSIONS: Proanabolic stimulants alone had limited capacity to overcome the catabolic and proinflammatory cytokine expression of severely degenerated NP cells and likely require additional anti-inflammatory treatments. Moderately degenerated NP cells had greater TGFß receptor 1 expression and better responded to anabolic stimulation.
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Degeneração do Disco Intervertebral/patologia , Disco Intervertebral/efeitos dos fármacos , Adulto , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Cadeia alfa 1 do Colágeno Tipo I , Fator de Crescimento do Tecido Conjuntivo/farmacologia , Dexametasona/farmacologia , Feminino , Humanos , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Disco Intervertebral/metabolismo , Disco Intervertebral/patologia , Degeneração do Disco Intervertebral/metabolismo , Masculino , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Pessoa de Meia-Idade , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo I , Receptores de Fatores de Crescimento Transformadores beta/genética , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Índice de Gravidade de Doença , Fator de Crescimento Transformador beta3/farmacologiaRESUMO
Obesity is an ever-increasing phenomenon, with 42% of Americans being considered obese (BMI ≥ 30) and 9.2% being considered morbidly obese (BMI ≥ 40) as of 2016. With obesity being characterized by an abundance of adipose tissue expansion, abnormal tissue remodeling is a typical consequence. Importantly, this pathological tissue expansion is associated with many alterations in the cellular populations and phenotypes within the tissue, lending to cellular, paracrine, mechanical, and metabolic alterations that have local and systemic effects, including diabetes and cardiovascular disease. In particular, vascular dynamics shift during the progression of obesity, providing signaling cues that drive metabolic dysfunction. In this review, paracrine-, autocrine-, and matrix-dependent signaling between adipocytes and endothelial cells is discussed in the context of the development and progression of obesity and its consequential diseases, including adipose fibrosis, diabetes, and cardiovascular disease.
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Doenças Cardiovasculares , Obesidade Mórbida , Humanos , Obesidade Mórbida/metabolismo , Células Endoteliais/metabolismo , Doenças Cardiovasculares/metabolismo , Tecido Adiposo/metabolismo , Adipócitos/metabolismoRESUMO
A push for environmentally friendly approaches to biomaterials fabrication has emerged from growing conservational concerns in recent years. Different stages in silk fibroin scaffold production, including sodium carbonate (Na2CO3)-based degumming and 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP)-based fabrication, have drawn attention for their associated environmental concerns. Environmentally friendly alternatives have been proposed for each processing stage; however, an integrated green fibroin scaffold approach has not been characterized or used for soft tissue applications. Here, we show that the combination of sodium hydroxide (NaOH) as a substitute degumming agent with the popular "aqueous-based" alternative silk fibroin gelation method yields fibroin scaffolds with comparable properties to traditional Na2CO3-degummed aqueous-based scaffolds. The more environmentally friendly scaffolds were found to have comparable protein structure, morphology, compressive modulus, and degradation kinetics, with increased porosity and cell seeding density relative to traditional scaffolds. Human adipose-derived stem cells showed high viability after three days of culture while seeded in each scaffold type, with uniform cell attachment to pore walls. Adipocytes from human whole adipose tissue seeded into scaffolds were found to have similar levels of lipolytic and metabolic function between conditions, in addition to a healthy unilocular morphology. Results indicate that our more environmentally friendly methodology for silk scaffold production is a viable alternative and well suited to soft tissue applications.
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High matrix strains in the intervertebral disc occur during physiological motions and are amplified around structural defects in the annulus fibrosus (AF). It remains unknown if large matrix strains in the human AF result in localized cell death. This study investigated strain amplitudes and substrate conditions where AF cells were vulnerable to stretch-induced apoptosis. Human degenerated AF cells were subjected to 1 Hz-cyclic tensile strains for 24h on uniformly collagen coated substrates and on substrates with 40 µm stripes of collagen that restricted cellular reorientation. AF cells were capable of responding to stretch (stress fibers and focal adhesions aligned perpendicular to the direction of stretch), but were vulnerable to stretch-induced apoptosis when cytoskeletal reorientation was restricted, as could occur in degenerated states due to fibrosis and crosslink accumulation and at areas where high strains occur (around structural defects, delaminations, and herniations).
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Apoptose , Disco Intervertebral/citologia , Estresse Mecânico , Estresse Fisiológico , Resistência à Tração , Células Cultivadas , Adesões Focais/ultraestrutura , Humanos , Disco Intervertebral/fisiologia , Fibras de Estresse/ultraestruturaRESUMO
Despite developing prenatally, the adipose tissue is unique in its ability to undergo drastic growth even after reaching its mature size. This development and subsequent maintenance rely on the proper coordination between the vascular niche and the adipose compartment. In this review, the process of adipose tissue development is broken down to explain (1) the ultrastructural matrix remodeling that is undertaken during simultaneous adipogenesis and angiogenesis, (2) the paracrine crosstalk involved during adipose development, (3) the mechanical regulators involved in adipose growth, and (4) the proteolytic and paracrine oversight for matrix remodeling during adipose development. It is crucial to gain a better understanding of the complex relationships that exist between adipose tissue and the vasculature during tissue development to provide insights into the pathological tissue expansion of obesity and to develop improved soft-tissue reconstruction techniques.
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Biophysical properties of the extracellular environment dynamically regulate cellular fates. In this review, we highlight silk, an indispensable polymeric biomaterial, owing to its unique mechanical properties, bioactive component sequestration, degradability, well-defined architectures, and biocompatibility that can regulate temporospatial biochemical and biophysical responses. We explore how the materiobiology of silks, both mulberry and non-mulberry based, affect cell behaviors including cell adhesion, cell proliferation, cell migration, and cell differentiation. Keeping in mind the novel biophysical properties of silk in film, fiber, or sponge forms, coupled with facile chemical decoration, and its ability to match functional requirements for specific tissues, we survey the influence of composition, mechanical properties, topography, and 3D geometry in unlocking the body's inherent regenerative potential.
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Silk fibroin has been explored as a suitable biomaterial due to its biocompatibility, tunable degradability, low toxicity, and mechanical properties. To harness silk fibroin's innate properties, it is purified from native silkworm cocoons by removing proteins and debris that have the potential to cause inflammatory responses. Typically, within the purification and fabrication steps, chemical solvents, energy-intensive equipment, and large quantities of water are used to reverse engineer silk fibroin into an aqueous solution and then process into the final material format. Gentler, green methods for extraction and fabrication have been developed that reduce or remove the need for harmful chemical additives and energy-inefficient equipment while still producing mechanically robust biomaterials. This review will focus on the alternative green processing and fabrication methods that have proven useful in creating silk fibroin materials for a range of applications including consumer and medical materials.
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Bombyx , Fibroínas , Animais , Materiais Biocompatíveis/efeitos adversos , SedaRESUMO
A patient's capacity for tissue regeneration varies based on age, nutritional status, disease state, lifestyle, and gender. Because regeneration cannot be predicted prior to biomaterial implantation, there is a need for responsive biomaterials with adaptive, personalized degradation profiles to improve regenerative outcomes. This study reports a new approach to use therapeutic ultrasound as a means of altering the degradation profile of silk fibroin biomaterials noninvasively postimplantation. By evaluating changes in weight, porosity, surface morphology, compressive modulus, and chemical structure, it is concluded that therapeutic ultrasound can trigger enhanced degradation of silk fibroin scaffolds noninvasively. By removing microbubbles on the scaffold surface, it is found that acoustic cavitation is the mechanism responsible for changing the degradation profile. This method is proved to be safe for human cells with no negative effects on cell viability or metabolism. Sonication through human skin also effectively triggers scaffold degradation, increasing the clinical relevance of these results. These findings suggest that silk is an ultrasound-responsive biomaterial, where the degradation profile can be adjusted noninvasively to improve regenerative outcomes.
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Fibroínas , Materiais Biocompatíveis , Humanos , Porosidade , Seda , Engenharia Tecidual , Alicerces TeciduaisRESUMO
Critically sized defects in subcutaneous white adipose tissue result in extensive disfigurement and dysfunction and remain a reconstructive challenge for surgeons; as larger defect sizes are correlated with higher rates of complications and failure due to insufficient vascularization following implantation. Our study demonstrates, for the first time, a method to engineer perfusable, pre-vascularized, high-density adipose grafts that combine patient-derived adipose cells with a decellularized lung matrix (DLM). The lung is one of the most vascularized organs with high flow, low resistance, and a large blood-alveolar interface separated by a thin basement membrane. For our work, the large volume capacity within the alveolar compartment was repurposed for high-density adipose cell filling, while the acellular vascular bed provided efficient graft perfusion throughout. Both adipocytes and hASCs were successfully delivered and remained in the alveolar space even after weeks of culture. While adipose-derived cells maintained their morphology and functionality in both static and perfusion DLM cultures, perfusion culture offered enhanced outcomes over static culture. Furthermore, we demonstrate that endothelial cells seamlessly integrate into the acellular vascular tree of the DLM with adipocytes. These results support that the DLM is a unique platform for creating vascularized adipose tissue grafts for large defect filling.
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There is increasing evidence for the role of environmental endocrine disrupting contaminants, coined obesogens, in exacerbating the rising obesity epidemic. Obesogens can be found in everyday items ranging from pesticides to food packaging. Although research shows that obesogens can have effects on adipocyte size, phenotype, metabolic activity, and hormone levels, much remains unknown about these chemicals. This review will discuss what is currently known about the mechanisms of obesogens, including expression of the PPARs, hormone interference, and inflammation. Strategies for identifying obesogenic chemicals and their mechanisms through chemical characteristics and model systems will also be discussed. Ultimately, research should focus on improving models to discern precise mechanisms of obesogenic action and to test therapeutics targeting these mechanisms.
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The obesity epidemic and its associated comorbidities present a looming challenge to health care delivery throughout the world. Obesity is characterized as a sterile inflammatory process within adipose tissues leading to dysregulated secretion of bioactive adipokines such as adiponectin and leptin, as well as systemic metabolic dysfunction. The majority of current obesity research has focused primarily on preclinical animal models in vivo and two-dimensional cell culture models in vitro. Neither of these generalized approaches is optimal due to interspecies variability, insufficient accuracy with respect to predicting human outcomes, and failure to recapitulate the three-dimensional (3D) microenvironment. Consequently, there is a growing demand and need for more sophisticated microphysiological systems to reproduce more physiologically accurate human white and brown/beige adipose depots. To address this research need, human and murine cell lines and primary cultures are being combined with bioscaffolds to create functional 3D environments that are suitable for metabolically active adipose organoids in both static and perfusion bioreactor cultures. The development of these technologies will have considerable impact on the future pace of discovery for novel small molecules and biologics designed to prevent and treat metabolic syndrome and obesity in humans. Furthermore, when these adipose tissue models are integrated with other organ systems they will have applicability to obesity-related disorders such as diabetes, nonalcoholic fatty liver disease, and osteoarthritis. Impact statement The current review article summarizes the advances made within the organ-onchip field, as it pertains to adipose tissue models of obesity and obesity-related syndromes, such as diabetes, non-alcoholic fatty liver disease, and osteoarthritis. As humanized 3D adipose-derived constructs become more accessible to the research community, it is anticipated that they will accelerate and enhance the drug discovery pipeline for obesity, diabetes, and metabolic diseases by reducing the preclinical evaluation process and improving predictive accuracy. Such developments, applications, and usages of existing technologies can change the paradigm of personalized medicine and create substantial progress in our approach to modern medicine.
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Tecido Adiposo Marrom , Dispositivos Lab-On-A-Chip , Animais , Humanos , Camundongos , Obesidade/complicaçõesRESUMO
Obesity, defined as a body mass index of 30 kg/m2 or above, has increased considerably in incidence and frequency within the United States and globally. Associated comorbidities including cardiovascular disease, type 2 diabetes mellitus, metabolic syndrome, and nonalcoholic fatty liver disease have led to a focus on the mechanisms promoting the prevention and treatment of obesity. Commonly utilized in vitro models employ human or mouse preadipocyte cell lines in a 2-dimensional (2D) format. Due to the structural, biochemical, and biological limitations of these models, increased attention has been placed on "organ on a chip" technologies for a 3-dimensional (3D) culture. Herein, we describe a method employing cryopreserved primary human stromal vascular fraction (SVF) cells and a human blood product-derived biological scaffold to create a 3D adipose depot in vitro. The "fat-on-chip" 3D cultures have been validated relative to 2D cultures based on proliferation, flow cytometry, adipogenic differentiation, confocal microscopy/immunofluorescence, and functional assays (adipokine secretion, glucose uptake, and lipolysis). Thus, the in vitro culture system demonstrates the critical characteristics required for a humanized 3D white adipose tissue (WAT) model.