Examination of ex-vivo viability of human adipose tissue slice culture

Obesity is associated with elevated inflammatory signals from various adipose tissue depots. This study aimed to evaluate release of regulated on activation, normal T cell expressed and secreted (RANTES) by human adipose tissue in vivo and ex vivo, in reference to monocyte chemoattractant protein-1 (MCP-1) and interleukin-6 (IL-6) release. Arteriovenous differences of RANTES, MCP-1, and IL-6 were studied in vivo across the abdominal subcutaneous adipose tissue in healthy Caucasian subjects with a wide range of adiposity. Systemic levels and ex vivo RANTES release were studied in abdominal subcutaneous, gastric fat pad, and omental adipose tissue from morbidly obese bariatric surgery patients and in thoracic subcutaneous and epicardial adipose tissue from cardiac surgery patients without coronary artery disease. Arteriovenous studies confirmed in vivo RANTES and IL-6 release in adipose tissue of lean and obese subjects and release of MCP-1 in obesity. However, in vivo release of MCP-1 and RANTES, but not IL-6, was lower than circulating levels. Ex vivo release of RANTES was greater from the gastric fat pad compared with omental ( P = 0.01) and subcutaneous ( P = 0.001) tissue. Epicardial adipose tissue released less RANTES than thoracic subcutaneous adipose tissue in lean ( P = 0.04) but not obese subjects. Indexes of obesity correlated with epicardial RANTES but not with systemic RANTES or its release from other depots. In conclusion, RANTES is released by human subcutaneous adipose tissue in vivo and in varying amounts by other depots ex vivo. While it appears unlikely that the adipose organ contributes significantly to circulating levels, local implications of this chemokine deserve further investigation.

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Secretion of neuropeptide Y in human adipose tissue and its role in maintenance of adipose tissue mass

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00333.2007 ◽

2007 ◽

Vol 293 (5) ◽

pp. E1335-E1340 ◽

Cited By ~ 57

Author(s):

Katarina Kos ◽

Alison L. Harte ◽

Sean James ◽

David R. Snead ◽

Joseph P. O'Hare ◽

...

Keyword(s):

Adipose Tissue ◽

Protein Expression ◽

Elective Surgery ◽

Ex Vivo ◽

Human Adipose Tissue ◽

Tissue Mass ◽

Paracrine Effects ◽

Tnf Α ◽

Adiposity Signals

NPY is an important central orexigenic hormone, but little is known about its peripheral actions in human adipose tissue (AT) or its potential paracrine effects. Our objective was to examine NPY's role in AT, specifically addressing NPY protein expression, the effect of NPY on adipokine secretion, and the influence of insulin and rosiglitazone (RSG) on adipocyte-derived NPY in vitro. Ex vivo human AT was obtained from women undergoing elective surgery [age: 42.7 ± 1.5 yr (mean ± SE), BMI: 26.2 ± 0.7 kg/m2; n = 38]. Western blot analysis was used to determine NPY protein expression in AT depots. Abdominal subcutaneous (AbSc) adipocytes were isolated and treated with recombinant (rh) NPY, insulin, and RSG. NPY and adipokine levels were measured by ELISA. Our results were that NPY was localized in human AT and adipocytes and confirmed by immunohistochemistry. Depot-specific NPY expression was noted as highest in AbSc AT (1.87 ± 0.23 ODU) compared with omental (Om; 1.03 ± 0.15 ODU, P = 0.029) or thigh AT (Th; 1.0 ± 0.29 ODU, P = 0.035). Insulin increased NPY secretion (control: 0.22 ± 0.024 ng/ml; 1 nM insulin: 0.26 ± 0.05 ng/ml; 100 nM insulin: 0.29 ± 0.04 ng/ml; 1,000 nM insulin: 0.3 ± 0.04 ng/ml; P < 0.05, n = 13), but cotreatment of RSG (10 nM) with insulin (100 nM) had no effect on NPY secretion. Furthermore, adipocyte treatment with rh-NPY downregulated leptin secretion (control: 6.99 ± 0.89 ng/ml; 1 nmol/l rh-NPY: 4.4 ± 0.64 ng/ml; 10 nmol/l rh-NPY: 4.3 ± 0.61 ng/ml, 100 nmol/l rh-NPY: 4.2 ± 0.67 ng/ml; P < 0.05, n = 10) but had no effect on adiponectin or TNF-α secretion. We conclude that NPY is expressed and secreted by human adipocytes. NPY secretion is stimulated by insulin, but this increment was limited by cotreatment with RSG. NPY's antilipolytic action may promote an increase in adipocyte size in hyperinsulinemic conditions. Adipose-derived NPY mediates reduction of leptin secretion and may have implications for central feedback of adiposity signals.

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Transduction of rat and human adipose-tissue derived mesenchymal stromal cells by adeno-associated viral vector serotype DJ

Biology Open ◽

10.1242/bio.058461 ◽

2021 ◽

Author(s):

E.S. Zubkova ◽

I.B. Beloglazova ◽

E.I. Ratner ◽

D.T. Dyikanov ◽

K.V. Dergilev ◽

...

Keyword(s):

Adipose Tissue ◽

Cellular Response ◽

Viral Vectors ◽

Viral Vector ◽

Ex Vivo ◽

Human Adipose Tissue ◽

Cell Types ◽

Specific Cell ◽

Therapeutic Benefits ◽

Rat Adipose Tissue

Ex vivo, gene therapy is a powerful approach holding great promises for the treatment of both genetic and acquired diseases. Adeno-associated virus (AAV) vectors are safe and efficient delivery system for modification of mesenchymal stem cells (MSC) that could maximize their therapeutic benefits. Assessment to MSC viability and functional activity after infection with new AAV serotypes is necessary, due to AAV tropism to specific cell types. We infected human and rat adipose-tissue MSC with hybrid AAV-DJ serotype vectors carrying GFP and SCF genes. GFP expression from AAV-DJ was about 1.5-fold superior to that observed with AAV-2 and lasted for at least 21 days as was evaluated by flow cytometry and fluorescence microscopy. AAV-DJ proves to be suitable for the infection of rat and human MSC with a similar efficiency. Infected MSC were still viable however showing 25-30%. growth rate slowdown. Moreover, we found increase of SERPINB2 mRNA expression in human MSC whereas expression of other oxidative stress markers and extracellular matrix proteins was not affected. These results suggest that there is a differential cellular response in MSC infected with AAV viral vectors, which should be taken into account as it can affect the expected outcome for the therapeutic application.

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Optimized polyethylenimine (PEI)-based nanoparticles for siRNA delivery, analyzed in vitro and in an ex vivo tumor tissue slice culture model

Drug Delivery and Translational Research ◽

10.1007/s13346-016-0306-y ◽

2016 ◽

Vol 7 (2) ◽

pp. 206-216 ◽

Cited By ~ 15

Author(s):

Alexander Ewe ◽

Sabrina Höbel ◽

Claudia Heine ◽

Lea Merz ◽

Sonja Kallendrusch ◽

...

Keyword(s):

Tumor Tissue ◽

Ex Vivo ◽

Sirna Delivery ◽

Tissue Slice ◽

Slice Culture ◽

Culture Model

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Sex and depot differences in ex vivo adipose tissue fatty acid storage and glycerol-3-phosphate acyltransferase activity

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00424.2014 ◽

2015 ◽

Vol 308 (9) ◽

pp. E830-E846 ◽

Cited By ~ 10

Author(s):

Maria Morgan-Bathke ◽

Liang Chen ◽

Elisabeth Oberschneider ◽

Debra Harteneck ◽

Michael D. Jensen

Keyword(s):

Adipose Tissue ◽

Fatty Acid ◽

Ex Vivo ◽

Human Adipose Tissue ◽

Adipocyte Size ◽

Enzyme Activity Assay ◽

Acyltransferase Activity ◽

Adipose Tissue Fatty Acid ◽

Tissue Fatty Acid ◽

Relative Contribution

Adipose tissue fatty acid storage varies according to sex, adipose tissue depot, and degree of fat gain. However, the mechanism(s) for these variations is not completely understood. We examined whether differences in adipose tissue glycerol-3-phosphate acyltransferase (GPAT) might play a role in these variations. We optimized an enzyme activity assay for total GPAT and GPAT1 activity in human adipose tissue and measured GPAT activity. Omental and subcutaneous adipose tissue was collected from obese and nonobese adults for measures of GPAT and GPAT1 activities, ex vivo palmitate storage, acyl-CoA synthetase (ACS) and diacylglycerol-acyltransferase (DGAT) activities, and CD36 protein. Total GPAT and GPAT1 activities decreased as a function of adipocyte size in both omental ( r = −0.71, P = 0.003) and subcutaneous ( r = −0.58, P = 0.04) fat. The relative contribution of GPAT1 to total GPAT activity increased as a function of adipocyte size, accounting for up to 60% of GPAT activity in those with the largest adipocytes. We found strong, positive correlations between ACS, GPAT, and DGAT activities for both sexes and depots ( r values 0.58–0.91) and between these storage factors and palmitate storage rates into TAG ( r values 0.55–0.90). We conclude that: 1) total GPAT activity decreases as a function of adipocyte size; 2) GPAT1 can account for over half of adipose GPAT activity in hypertrophic obesity; and 3) ACS, GPAT, and DGAT are coordinately regulated.

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A Histopathologic and Immunohistochemical Study on Liquification of Human Adipose Tissue Ex Vivo

Aesthetic Plastic Surgery ◽

10.1007/s00266-014-0371-x ◽

2014 ◽

Vol 38 (5) ◽

pp. 976-984 ◽

Cited By ~ 4

Author(s):

Nina-Fee Hübner ◽

Raymund E. Horch ◽

Elias Polykandriotis ◽

Tilman T. Rau ◽

Adrian Dragu

Keyword(s):

Adipose Tissue ◽

Immunohistochemical Study ◽

Ex Vivo ◽

Human Adipose Tissue

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Infusing Mesenchymal Stromal Cells into Porcine Kidneys during Normothermic Machine Perfusion: Intact MSCs Can Be Traced and Localised to Glomeruli

International Journal of Molecular Sciences ◽

10.3390/ijms20143607 ◽

2019 ◽

Vol 20 (14) ◽

pp. 3607 ◽

Cited By ~ 14

Author(s):

Merel Pool ◽

Tim Eertman ◽

Jesus Sierra Parraga ◽

Nils ’t Hart ◽

Marieke Roemeling-van Rhijn ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Adipose Tissue ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Ex Vivo ◽

Human Adipose Tissue ◽

Machine Perfusion ◽

Normothermic Machine Perfusion ◽

Glomerular Capillaries

Normothermic machine perfusion (NMP) of kidneys offers the opportunity to perform active interventions, such as the addition of mesenchymal stromal cells (MSCs), to an isolated organ prior to transplantation. The purpose of this study was to determine whether administering MSCs to kidneys during NMP is feasible, what the effect of NMP is on MSCs and whether intact MSCs are retained in the kidney and to which structures they home. Viable porcine kidneys were obtained from a slaughterhouse. Kidneys were machine perfused during 7 h at 37 °C. After 1 h of perfusion either 0, 105, 106 or 107 human adipose tissue derived MSCs were added. Additional ex vivo perfusions were conducted with fluorescent pre-labelled bone-marrow derived MSCs to assess localisation and survival of MSCs during NMP. After NMP, intact MSCs were detected by immunohistochemistry in the lumen of glomerular capillaries, but only in the 107 MSC group. The experiments with fluorescent pre-labelled MSCs showed that only a minority of glomeruli were positive for infused MSCs and most of these glomeruli contained multiple MSCs. Flow cytometry showed that the number of infused MSCs in the perfusion circuit steeply declined during NMP to approximately 10%. In conclusion, the number of circulating MSCs in the perfusate decreases rapidly in time and after NMP only a small portion of the MSCs are intact and these appear to be clustered in a minority of glomeruli.

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PAI-1 Produced Ex Vivo by Human Adipose Tissue Is Relevant to PAI-1 Blood Level

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/01.atv.19.5.1361 ◽

1999 ◽

Vol 19 (5) ◽

pp. 1361-1365 ◽

Cited By ~ 61

Author(s):

P. E. Morange ◽

M. C. Alessi ◽

M. Verdier ◽

D. Casanova ◽

G. Magalon ◽

...

Keyword(s):

Adipose Tissue ◽

Blood Level ◽

Ex Vivo ◽

Human Adipose Tissue ◽

Pai 1

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Reference Gene Optimization for Circadian Gene Expression Analysis in Human Adipose Tissue

Journal of Biological Rhythms ◽

10.1177/0748730419883043 ◽

2019 ◽

Vol 35 (1) ◽

pp. 84-97 ◽

Cited By ~ 1

Author(s):

Jeremy M. White ◽

Matthew J. Piron ◽

Vittobai R. Rangaraj ◽

Erin C. Hanlon ◽

Ronald N. Cohen ◽

...

Keyword(s):

Adipose Tissue ◽

Circadian Rhythms ◽

Reference Gene ◽

Reference Genes ◽

Messenger Rna ◽

Ex Vivo ◽

Human Adipose Tissue ◽

Morbidly Obese ◽

Obese Women ◽

Gene Optimization

A hallmark of biology is the cyclical nature of organismal physiology driven by networks of biological, including circadian, rhythms. Unsurprisingly, disruptions of the circadian rhythms through sleep curtailment or shift work have been connected through numerous studies to positive associations with obesity, insulin resistance, and diabetes. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) measures oscillation in messenger RNA expression, an essential foundation for the study of the physiological circadian regulatory network. Primarily, measured oscillations have involved the use of reference gene normalization. However, the validation and identification of suitable reference genes is a significant challenge across different biological systems. This study focuses on adipose tissue of premenopausal, otherwise healthy, morbidly obese women voluntarily enrolled after being scheduled for laparoscopic sleeve gastrectomy surgery. Acquisition of tissue was accomplished by aspiratory needle biopsies of subcutaneous adipose tissue 1 to 2 weeks prior to surgery and 12 to 13 weeks following surgery and an in-surgery scalpel-assisted excision of mesenteric adipose tissue. Each biopsy was sterile cultured ex vivo and serially collected every 4 h over approximately 36 h. The candidate reference genes that were tested were 18S rRNA, GAPDH, HPRT1, RPII, RPL13α, and YWHAZ. Three analytic tools were used to test suitability, and the candidate reference genes were used to measure oscillation in expression of a known circadian clock element (Dbp). No gene was deemed suitable as an individual reference gene control, which indicated that the optimal reference gene set was the geometrically averaged 3-gene panel composed of YWHAZ, RPL13α, and GAPDH. These methods can be employed to identify optimal reference genes in other systems.

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