scholarly journals Persistent Effect of Advanced Glycated Albumin Driving Inflammation and Disturbances in Cholesterol Efflux in Macrophages

Nutrients ◽  
2021 ◽  
Vol 13 (10) ◽  
pp. 3633
Author(s):  
Carlos André Minanni ◽  
Adriana Machado-Lima ◽  
Rodrigo Tallada Iborra ◽  
Lígia Shimabukuro Okuda ◽  
Raphael de Souza de Souza Pinto ◽  
...  
Keyword(s):  
Deleterious Effect ◽  
Cholesterol Efflux ◽  
Culture Media ◽  
Glycated Albumin ◽  
Cholesterol Homeostasis ◽  
Intracellular Lipid ◽  
Persistent Effect ◽  
Abca1 Protein ◽  
Effect Of Age

Advanced glycated albumin (AGE-albumin) impairs cholesterol efflux and contributes to inflammation in macrophages. The current study evaluated: (1) the persistence of the deleterious effect of AGE-albumin in cholesterol efflux and in inflammation, and (2) how metabolic control in diabetes mellitus (DM) contributes to attenuate the deleterious role of AGE-albumin in macrophage cholesterol homeostasis. Methods: AGE-albumin was produced in vitro or isolated from uncontrolled DM subjects’ serum before (bGC) and after improved glycemic control (aGC). Albumin samples were incubated with bone marrow-derived macrophages and 14C-cholesterol efflux or LPS- induced cytokine secretion were determined immediately, or after cell resting in culture media alone. The ABCA-1 degradation rate was determined after cell incubation with cycloheximide, and ABCA1 protein level by immunoblot. Oil Red O staining was used to assess intracellular lipid accumulation. Results: A persistent effect of AGE-albumin was observed in macrophages in terms of the secretion of inflammatory cytokines and reduced cholesterol efflux. HDL-mediated 14C-cholesterol efflux was at least two times higher in macrophages treated with aCG-albumin as compared to bGC-albumin, and intracellular lipid content was significantly reduced in aGC-albumin-treated cells. As compared to bGC-albumin, the ABCA-1 protein content in whole cell bulk was 94% higher in aCG-albumin. A 20% increased ABCA-1 decay rate was observed in macrophages treated with albumin from poorly controlled DM. AGE-albumin has a persistent deleterious effect on macrophage lipid homeostasis and inflammation. The reduction of AGEs in albumin ameliorates cholesterol efflux.

scholarly journals RAGE Mediates Cholesterol Efflux Impairment in Macrophages Caused by Human Advanced Glycated Albumin

2020 ◽  
Vol 21 (19) ◽  
pp. 7265
Author(s):  
Adriana Machado-Lima ◽  
Raquel López-Díez ◽  
Rodrigo Tallada Iborra ◽  
Raphael de Souza Pinto ◽  
Gurdip Daffu ◽  
...  
Keyword(s):  
Cholesterol Efflux ◽  
Glycated Albumin ◽  
Wild Type ◽  
Intracellular Lipid ◽  
Glycation End Products ◽  
Macrophage Cholesterol Efflux ◽  
Cellular Cholesterol Efflux

We addressed the involvement of the receptor for advanced glycation end products (RAGE) in the impairment of the cellular cholesterol efflux elicited by glycated albumin. Albumin was isolated from type 1 (DM1) and type 2 (DM2) diabetes mellitus (HbA1c > 9%) and non-DM subjects (C). Moreover, albumin was glycated in vitro (AGE-albumin). Macrophages from Ager null and wild-type (WT) mice, or THP-1 transfected with siRNA-AGER, were treated with C, DM1, DM2, non-glycated or AGE-albumin. The cholesterol efflux was reduced in WT cells exposed to DM1 or DM2 albumin as compared to C, and the intracellular lipid content was increased. These events were not observed in Ager null cells, in which the cholesterol efflux and lipid staining were, respectively, higher and lower when compared to WT cells. In WT, Ager, Nox4 and Nfkb1, mRNA increased and Scd1 and Abcg1 diminished after treatment with DM1 and DM2 albumin. In Ager null cells treated with DM-albumin, Nox4, Scd1 and Nfkb1 were reduced and Jak2 and Abcg1 increased. In AGER-silenced THP-1, NOX4 and SCD1 mRNA were reduced and JAK2 and ABCG1 were increased even after treatment with AGE or DM-albumin. RAGE mediates the deleterious effects of AGE-albumin in macrophage cholesterol efflux.

Abstract 5: Lipid Droplet Associated Hydrolase: A New Player in Cholesterol Mobilization From Foam Cells

2016 ◽  
Vol 36 (suppl_1) ◽  
Author(s):  
Younghwa Goo ◽  
Pradip Saha ◽  
Larry Chan ◽  
Antoni Paul
Keyword(s):  
Lipid Droplet ◽  
Cholesterol Efflux ◽  
Bone Marrow Cells ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Foam Cells ◽  
Peritoneal Macrophages ◽  
Cholesterol Homeostasis

Lipid laden macrophages/foam cells are a hallmark of atherosclerotic lesions from early to late stages of development. Macrophages take-up modified low-density lipoprotein (mLDL) particles and store surplus mLDL-derived cholesterol as cholesterol ester (CE) in cytoplasmic lipid droplets (LDs). Accelerating CE hydrolysis from the LDs is a plausible strategy to promote reverse cholesterol transport from the atheroma. However, the identity of the CE hydrolases that function on LDs remains unknown. Previously we identified lipid droplet-associated hydrolase (LDAH) in LDs purified from macrophages and reported that in vitro LDAH regulates CE levels by increasing CE hydrolysis. To determine the relevance of LDAH in atherogenesis, we have generated LDAH knockout (LDAH-/-) mice. Mouse peritoneal macrophages (MPM) isolated from LDAH-/- mice had increased cytoplasmic LDs, increased net CE content, and decreased cholesterol efflux. In atherosclerosis studies, both male and female LDAH-/- mice crossed with apolipoprotein E knockout (apoE-/-) mice fed a Western diet developed larger lesions. Lesions of LDAH-/-/ apoE-/- mice were characterized by increased areas of macrophages containing enlarged cytoplasms with large LDs. Supporting a direct atheroprotective role of LDAH in macrophages, lesions of apoE-/- mice that received bone marrows from LDAH-/-/apoE-/- mice progressed faster than those that received bone marrow cells from LDAH+/+/apoE-/- mice. In qPCR analyses of genes involved in cholesterol homeostasis in macrophages, we found that ABC binding cassette transporters ABCA1 and ABCG1, which mediate cholesterol efflux through the plasma membrane, were consistently decreased in LDAH-/- MPM. Further in vivo gene expression studies on macrophages selectively obtained from lesions using laser capture microdissection are underway. In conclusion, our study suggests that LDAH promotes LD CE hydrolysis and cholesterol efflux from foam cells within the atheroma, and uncovers a potential target to promote reverse cholesterol from arteries as a means of ameliorating atherosclerosis development.

scholarly journals Advanced glycation end products affect cholesterol homeostasis by impairing ABCA1 expression on macrophages

2017 ◽  
Vol 95 (8) ◽  
pp. 977-984 ◽  
Author(s):  
Olivier Kamtchueng Simo ◽  
Souade Ikhlef ◽  
Hicham Berrougui ◽  
Abdelouahed Khalil
Keyword(s):  
Advanced Glycation End Products ◽  
Cholesterol Homeostasis ◽  
High Density Lipoproteins ◽  
Advanced Glycation ◽  
Cholesterol Levels ◽  
Glycation End Products ◽  
End Products ◽  
Prevention Of Atherosclerosis ◽  
Abca1 Protein

Reverse cholesterol transport (RCT), which is intimately linked to high-density lipoproteins (HDLs), plays a key role in cholesterol homeostasis and the prevention of atherosclerosis. The goal of the present study was to investigate the effect of aging and advanced glycation end products (AGEs) on RCT as well as on other factors that may affect the antiatherogenic property of HDLs. The transfer of macrophage-derived cholesterol to the plasma and liver and then to the feces for elimination was significantly lower in aged mice than in young mice. Chronic injection of d -galactose (D-gal) or AGEs also significantly reduced RCT (65.3% reduction in [3H]cholesterol levels in the plasma of D-gal-treated mice after 48 h compared with control mice, P < 0.01). The injection of both D-gal and aminoguanidine hydrochloride increased [3H]cholesterol levels in the plasma, although the levels were lower than those of control mice. The in vitro incubation of HDLs with dicarbonyl compounds increased the carbonyl and conjugated diene content of HDLs and significantly reduced PON1 paraoxonase activity (87.4% lower than control HDLs, P < 0.0001). Treating J774A.1 macrophages with glycated fetal bovine serum increased carbonyl formation (39.5% increase, P < 0.003) and reduced ABCA1 protein expression and the capacity of macrophages to liberate cholesterol (69.1% decrease, P < 0.0001). Our results showed, for the first time, that RCT is altered with aging and that AGEs contribute significantly to this alteration.

scholarly journals Lactobacillus plantarum CUL66 can impact cholesterol homeostasis in Caco-2 enterocytes

2016 ◽  
Vol 7 (3) ◽  
pp. 443-451 ◽  
Author(s):  
D.R. Michael ◽  
J.W.E. Moss ◽  
D. Lama Calvente ◽  
I. Garaiova ◽  
S.F. Plummer ◽  
...  
Keyword(s):  
Lactobacillus Plantarum ◽  
Cholesterol Metabolism ◽  
Culture Media ◽  
Cholesterol Homeostasis ◽  
In Vivo Studies ◽  
Western Society ◽  
Bile Salt Hydrolase ◽  
Atp Binding ◽  
Atp Binding Cassette

Hypercholesterolemia drives the development of cardiovascular disease, the leading cause of mortality in western society. Supplementation with probiotics that interfere with cholesterol metabolism may provide a contribution to disease prevention. Lactobacillus plantarum CUL66 (NCIMB 30280) has been assessed in vitro for its ability to impact cholesterol absorption. L. plantarum CUL66 tested positive for bile salt hydrolase activity and the ability to assimilate cholesterol from culture media. RT-qPCR analysis showed that the bacterium significantly decreased the expression of Niemann-Pick C1-like 1 and ATP-binding cassette transporter-1 in polarised Caco-2 cells after 6 h exposure. Conversely, the expression of ATP-binding cassette sub-family G member (ABCG)-5 and ABCG-8, and 3-hydroxy-3-methylglutaryl-CoA reductase were significantly increased. Using a radiolabelled assay, we also observed significant reductions in the uptake and basolateral efflux of cholesterol by Caco-2 cells exposed to L. plantarum CUL66. This in vitro study identified L. plantarum CUL66 as a cholesterol lowering bacteria by highlighting its ability to beneficially regulate multiple in vitro events associated with intestinal cholesterol metabolism and provides evidence of efficacy for its inclusion in future in vivo studies.

scholarly journals Compounds targeting OSBPL7 increase ABCA1-dependent cholesterol efflux preserving kidney function in two models of kidney disease

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Matthew B. Wright ◽  
Javier Varona Santos ◽  
Christian Kemmer ◽  
Cyrille Maugeais ◽  
Jean-Philippe Carralot ◽  
...  
Keyword(s):  
Kidney Disease ◽  
Cholesterol Efflux ◽  
Cholesterol Homeostasis ◽  
Renal Diseases ◽  
Alternative Mechanism ◽  
Cellular Cholesterol ◽  
Oxysterol Binding Protein ◽  
Key Factor ◽  
Cellular Cholesterol Efflux

AbstractImpaired cellular cholesterol efflux is a key factor in the progression of renal, cardiovascular, and autoimmune diseases. Here we describe a class of 5-arylnicotinamide compounds, identified through phenotypic drug discovery, that upregulate ABCA1-dependent cholesterol efflux by targeting Oxysterol Binding Protein Like 7 (OSBPL7). OSBPL7 was identified as the molecular target of these compounds through a chemical biology approach, employing a photoactivatable 5-arylnicotinamide derivative in a cellular cross-linking/immunoprecipitation assay. Further evaluation of two compounds (Cpd A and Cpd G) showed that they induced ABCA1 and cholesterol efflux from podocytes in vitro and normalized proteinuria and prevented renal function decline in mouse models of proteinuric kidney disease: Adriamycin-induced nephropathy and Alport Syndrome. In conclusion, we show that small molecule drugs targeting OSBPL7 reveal an alternative mechanism to upregulate ABCA1, and may represent a promising new therapeutic strategy for the treatment of renal diseases and other disorders of cellular cholesterol homeostasis.

Abstract 460: Characterization of Glycated Albumin Isolated From Poorly Controlled Diabetic Patients and Its Role in Macrophage Cholesterol Efflux

2014 ◽  
Vol 34 (suppl_1) ◽  
Author(s):  
Adriana Machado-Lima ◽  
Erika R Oliveira ◽  
Rodrigo T Iborra ◽  
Gabriela Castilho ◽  
Edna R Nakandakare ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Diabetes Mellitus ◽  
Cholesterol Efflux ◽  
Glycated Albumin ◽  
Maldi Mass Spectrometry ◽  
Diabetic Patients ◽  
Advanced Glycation ◽  
Macrophage Cholesterol Efflux

Advanced glycation end products (AGE) are elevated in diabetes mellitus (DM) and predict the development of atherosclerosis. In vitro produced AGE-albumin induces oxidative stress that is linked to the reduction in ABCA-1 levels and cholesterol efflux mediated by apo A-I and HDL-subfractions, leading to macrophage cholesterol accumulation. We characterized the glycation level/profile of human serum albumin (HSA) isolated by fast protein liquid chromatography from poorly controlled type 1 (DM1) and type 2 (DM2) diabetes mellitus patients (HbA1c > 8%) in comparison to control (C) individuals, and how these AGE-albumin can interfere in macrophage lipid accumulation. The glycation level of HSA from C, DM1 and DM2 was analyzed by MALDI mass spectrometry and was similar between DM1 and DM2-HSA. An increased mean mass was observed in DM1-HSA (68,544 ± 192 Da; n=6) and DM2-HSA (68,547 ± 132 Da; n=6) compared to C-HSA (67,846 ± 301 Da; n=6), reflecting the condensation of at least 8 and 5 units of glucose, respectively. The tryptic digestion of C-HSA generated a number of peptide species higher than those originated from DM1 and DM2-HSA. Macrophages isolated from peritoneal wild-type mice were treated for 18 h with C, DM1 or DM2-HSA in order to measure the 14C-cholesterol efflux and the mRNA expression of NOX-4 (NADPHoxidase4), ABCA-1 (Abca1) and ABCG-1 (Abcg1). Data were compared by one-way ANOVA and Dunnet′s post test. In comparison to cells treated with C-HSA the expression of NADPHoxidase4 (p<0.05; n=3) mRNA was increased after cell treatment with DM1 (3.2x) and DM2-HSA (0.7x), confirming oxidative stress. Abcg1 mRNA was reduced by DM2-HSA (26%; p<0.05; n=3); Abca1 mRNA was unchanged but ABCA-1 protein content was greatly reduced (82 and 25%, respectively in DM1 and DM2-HAS; p<0.05; n=12). The % of apo A-I mediated cholesterol efflux was impaired in DM1 (1.3 ± 0.3) and DM2-HSA-treated cells (2.4 ± 0.5) as compared to C-HSA (4.4 ± 0.5; n= 5; p<0.05). The level of advanced glycation that takes place in vivo was similar between DM1 and DM2-HSA and induced macrophage oxidative stress and impairment in cholesterol efflux that may contribute to atherogenesis in DM. Funding: FAPESP, Brazil (2012/19112-0)

92 UTILISATION OF ENDOGENOUS FATTY ACID STORES FOR ENERGY PRODUCTION IN BOVINE PRE-IMPLANTATION EMBRYOS

2012 ◽  
Vol 24 (1) ◽  
pp. 158
Author(s):  
M. Sutton-McDowall ◽  
D. Feil ◽  
R. Robker ◽  
J. Thompson ◽  
K. Dunning
Keyword(s):  
Embryo Development ◽  
Lipid Content ◽  
Culture Media ◽  
Bovine Embryo ◽  
Carnitine Supplementation ◽  
Intracellular Lipid ◽  
Endogenous Fatty Acid ◽  
Developmental Potential ◽  
Morula Stage

Current embryo culture media are based on the carbohydrate metabolism of embryos. However, little is known about the metabolism of endogenous lipids. This is surprising given the high intracellular lipid densities of embryos of some species and the potential for ATP production via β-oxidation. L-carnitine is a β-oxidation co-factor that is absent in most culture media. The aim of this study is to investigate the influence of carnitine supplementation ± carbohydrates on bovine embryo development. Abattoir-derived cattle cumulus–oocyte complexes were cultured and fertilised (Sutton-McDowall et al. 2006 Biol. Reprod. 74, 881–888). Post-fertilisation (24 h), presumptive zygotes were transferred into an amino acid-free cleavage media ± carbohydrates (glucose, lactate and pyruvate) ±5 mM carnitine and cultured for 4 days. The absence of carbohydrates during culture resulted in embryos arresting at the 2- and 4-cell stages. Remarkably, +carnitine significantly increased development to the morula stage compared with +carbohydrates alone (20.4 ± 3% vs 4.7 ± 2.5% morula development; P < 0.001). The combination of carbohydrates and carnitine supplementation further improved embryo development, with 14-fold more embryos reaching the morula stage after culture in the +carbohydrates +carnitine group compared with the +carbohydrates group (+carbohydrates = 3.1 ± 1.9 vs +carbohydrates +carnitine = 43.8 ± 9.1% morula development; P < 0.05). The beneficial effects of carnitine supplementation on embryo development were reversed when embryos were cultured in presence of etomoxir, a non-reversible inhibitor of the rate-limiting enzyme of β-oxidation (development to 8-cell stage; +carnitine = 33.9 ± 8% vs +carnitine +etomoxir = 19.2 ± 4.9%; P < 0.05). Intracellular lipid content of embryos +carnitine was determined by culturing presumptive zygotes in media -carbohydrates ± carnitine for 24 h. Lipid content of embryos was determined by measuring BODIPY 493/503 dye fluorescence. Carnitine supplementation reduced fluorescence intensity 1.8-fold (P < 0.001). Adenosine triphosphate and ATP:ADP levels were measured in embryos after 24 h of culture ± carbohydrates ± carnitine. While there was a trend for +carnitine to increase ATP levels (P = 0.09), ADP levels were higher and ATP:ADP ratio were 1.9-fold lower (main effect, P < 0.05) compared with embryos cultured in –carnitine. This indicates +carnitine embryos were more metabolically active, with higher rates of ATP-ADP conversion. We have shown carnitine supplementation supports pre-compaction embryo development and there is an additive effect of +carnitine +carbohydrate on early embryo development. This is most likely through increased β-oxidation levels within embryos. Current disparities between in vivo and in vitro embryo production, in particular increased lipid content (Romek et al. 2010 Theriogenology 74, 265–276) and decreased developmental potential of in vitro-produced embryos, may be an artefact resulting from limited lipid oxidation in vitro.

Abstract 62: Cholesterol Efflux Pathways in Dendritic Cells Suppress Autoimmune Responses

2014 ◽  
Vol 34 (suppl_1) ◽  
Author(s):  
Marit Westerterp ◽  
Emmanuel L Gautier ◽  
Anjali Ganda ◽  
Gwendalyn J Randolph ◽  
Vivette D D'Agati ◽  
...  
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
Antigen Presentation ◽  
Cholesterol Efflux ◽  
Cholesterol Homeostasis ◽  
Apolipoprotein A1 ◽  
Cholesterol Depletion ◽  
Chow Diet ◽  
Type I

Patients with auto-immune disorders have low HDL levels. Mice deficient in genes regulating cholesterol homeostasis, such as liver X receptor (LXR) or apolipoprotein A1 (apoA1), show an autoimmune phenotype. LXR regulates the expression of ATP Binding Cassette A1 and G1 (ABCA1 and ABCG1) that mediate cholesterol efflux to apoA1 and HDL. ABCG1 is highly expressed in dendritic cells (DCs). We hypothesized that ABCA1 and ABCG1 regulate autoimmunity. On a chow diet, 40 weeks old Abca1-/-Abcg1-/- mice showed enlarged lymph nodes (LNs), increased plasma auto-antibodies to dsDNA, and glomerulonephritis, with characteristics typical for lupus nephritis. Using the Cre loxP system, we investigated whether these effects were due to Abca1/g1 deficiency in T-cells, macrophages, or DCs. Only Abca1/g1 deficiency in DCs in CD11cCreAbca1fl/flAbcg1fl/fl mice replicated the auto-immune phenotype found in Abca1-/-Abcg1-/- mice. This suggests a major role for DC cholesterol homeostasis in autoimmunity. DCs present antigens to T-cells, leading to their activation. CD11cCreAbca1fl/flAbcg1fl/fl mice showed increased T-cell activation in blood, spleen, and LNs. After immunization, DCs from CD11cCreAbca1fl/flAbcg1fl/fl mice showed increased antigen presentation to T-cells in vitro and in vivo. CD11cCreAbca1fl/flAbcg1fl/fl mice had increased CD80+ DCs. CD80 is a co-stimulatory molecule required for antigen presentation. Abca1/g1 deficiency in DCs increased endosomal cholesterol accumulation in vitro. Ligands for Toll like receptor (TLR) 3 and 4 increased CD80 mRNA in CD11cCreAbca1fl/flAbcg1fl/fl compared to Abca1fl/flAbcg1fl/fl DCs, where the effect of ligands for TLR3>TLR4. Cholesterol depletion by cyclodextrin decreased CD80 mRNA and antigen presentation in CD11cCreAbca1fl/flAbcg1fl/fl DCs in vitro. The increased CD80 mRNA in CD11cCreAbca1fl/flAbcg1fl/fl DCs was also reversed by a type I interferon (IFN) antibody, suggesting excessive signaling by the known TLR3-IFN-CD80 axis. These studies show for the first time a role for cholesterol efflux pathways in DCs in maintaining immune tolerance.

A potent soluble epoxide hydrolase inhibitor, t-AUCB, modulates cholesterol balance and oxidized low density lipoprotein metabolism in adipocytes in vitro

2014 ◽  
Vol 395 (4) ◽  
pp. 443-451 ◽  
Author(s):  
Li Shen ◽  
Hongchun Peng ◽  
Shuiping Zhao ◽  
Danyan Xu
Keyword(s):  
Cholesterol Efflux ◽  
Epoxide Hydrolase ◽  
Cholesterol Metabolism ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Soluble Epoxide Hydrolase ◽  
Cholesterol Homeostasis ◽  
Low Density ◽  
Oxidized Low Density Lipoprotein

Abstract The cholesterol metabolism in adipose tissue is dependent on the balance between cholesterol uptake and efflux. Adipocytes dysfunction and its cholesterol imbalance are associated with obesity. Adipocytes are the site for clearance of oxidized low density lipoprotein (oxLDL) in blood. Soluble epoxide hydrolase (sEH) is highly expressed in adipocytes. sEH converts epoxyeicosatrienoic acids (EETs) into less bioactive dihydroxyeicosatrienoic acids, which regulate cholesterol metabolism in adipocytes and block the development of atherosclerosis. In vitro, 3T3-L1 differentiated adipocytes were incubated with the sEH inhibitor t-AUCB (0, 1, 10, 50 or 100 mmol/l) for 24 h with or without the PPARγ inhibitor GW9662. To determine the effect of t-AUCB on oxLDL endocytosis, degradation and cholesterol efflux from adipocytes, we demonstrated that t-AUCB enhances the CD36-mediated recognition and degradation of oxLDL and improves cholesterol efflux via the upregulation of ABCA1 expression. Furthermore, t-AUCB blocked TNF-α secretion and increased adiponectin levels found in adipocytes culture medium. We provide evidence that these effects are PPARγ-dependent. These results suggest that an increase in EETs because of sEH inhibition could maintain cellular cholesterol homeostasis by the regulation of oxLDL clearance and cholesterol efflux via the EETs–PPARγ pathway.

Structural integrity after cold storage of human epidermal model in hypothermosol: A correlative ultrastructural and fluorescent assay

1994 ◽  
Vol 52 ◽  
pp. 270-271
Author(s):  
Henry H. Eichelberger ◽  
John G. Baust ◽  
Robert G. Van Buskirk
Keyword(s):  
Cold Storage ◽  
Structural Integrity ◽  
Culture Media ◽  
Cell Structure ◽  
Natural State ◽  
Sodium Cacodylate ◽  
Ruthenium Tetroxide ◽  
Cacodylate Buffer ◽  
Before And After

For research in cell differentiation and in vitro toxicology it is essential to provide a natural state of cell structure as a benchmark for interpreting results. Hypothermosol (Cryomedical Sciences, Rockville, MD) has proven useful in insuring the viability of synthetic human epidermis during cold-storage and in maintaining the epidermis’ ability to continue to differentiate following warming.Human epidermal equivalent, EpiDerm (MatTek Corporation, Ashland, MA) consisting of fully differentiated stratified human epidermal cells were grown on a microporous membrane. EpiDerm samples were fixed before and after cold-storage (4°C) for 5 days in Hypothermosol or skin culture media (MatTek Corporation) and allowed to recover for 7 days at 37°C. EpiDerm samples were fixed 1 hour in 2.5% glutaraldehyde in sodium cacodylate buffer (pH 7.2). A secondary fixation with 0.2% ruthenium tetroxide (Polysciences, Inc., Warrington, PA) in sodium cacodylate was carried out for 3 hours at 4°C. Other samples were similarly fixed, but with 1% Osmium tetroxide in place of ruthenium tetroxide. Samples were dehydrated through a graded acetone series, infiltrated with Spurrs resin (Polysciences Inc.) and polymerized at 70°C.

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