Fibronectin and Kupffer cell function in fulminant hepatic failure

International Nuclear Information System (INIS)

Imawari, M.; Hughes, R.D.; Gove, C.D.; Williams, R.

1985-01-01

The relationship between plasma fibronectin, in vitro plasma opsonic activity, which measures the biological activity of fibronectin, and in vivo Kupffer cell function, as assessed by the systemic clearance of microaggregated [ 125 I]albumin, were determined simultaneously in 15 patients with fulminant hepatic failure and 12 normal subjects. Both the plasma fibronectin and plasma opsonic activity were significantly reduced in patients with fulminant hepatic failure, while the systemic clearance of microaggregated albumin was decreased. There was a significant correlation between plasma fibronectin and the plasma opsonic activity on admission, but no correlation could be detected between either parameter and the clearance of microaggregated albumin. A gelatin-derived plasma expander was shown to block the plasma opsonic activity both in vitro and in vivo. The low plasma fibronectin and decreased clearance of microaggregated albumin in fulminant hepatic failure reflect different aspects of the overall impairment of Kupffer cell function

Hepatocellular proliferation in response to agonists of peroxisome proliferator-activated receptor alpha: a role for kupffer cells?

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Cunningham Michael

2006-01-01

Full Text Available Abstract Background It has been proposed that PPARα agonists stimulate Kupffer cells in rodents which in turn, release mitogenic factors leading to hepatic hyperplasia, and eventually cancer. However, Kupffer cells do not express PPARα receptors, and PPARα agonists stimulate hepatocellular proliferation in both TNFα- and TNFα receptor-null mice, casting doubt on the involvement of Kupffer cells in the mitogenic response to PPARα agonists. This study was therefore designed to investigate whether the PPARα agonist PFOA and the Kupffer cell inhibitor methylpalmitate produce opposing effects on hepatocellular proliferation and Kupffer cell activity in vivo, in a manner that would implicate these cells in the mitogenic effects of PPARα agonists. Methods Male Sprague-Dawley rats were treated intravenously via the tail vein with methylpalmitate 24 hrs prior to perfluorooctanoic acid (PFOA, and were sacrificed 24 hrs later, one hr after an intraperitoneal injection of bromodeoxyuridine (BrdU. Sera were analyzed for TNFα and IL-1β. Liver sections were stained immunohistochemically and quantified for BrdU incorporated into DNA. Results Data show that PFOA remarkably stimulated hepatocellular proliferation in the absence of significant changes in the serum levels of either TNFα or IL-1β. In addition, methylpalmitate did not alter the levels of these mitogens in PFOA-treated animals, despite the fact that it significantly blocked the hepatocellular proliferative effect of PFOA. Correlation between hepatocellular proliferation and serum levels of TNFα or IL-1β was extremely poor. Conclusion It is unlikely that mechanisms involving Kupffer cells play an eminent role in the hepatic hyperplasia, and consequently hepatocarcinogenicity attributed to PPARα agonists. This conclusion is based on the above mentioned published data and the current findings showing animals treated with PFOA alone or in combination with methylpalmitate to have similar

Kupffer cells are activated in cirrhotic portal hypertension and not normalised by TIPS

DEFF Research Database (Denmark)

Holland-Fischer, Peter; Grønbæk, Henning; Sandahl, Thomas Damgaard

2011-01-01

INTRODUCTION: Hepatic macrophages (Kupffer cells) undergo inflammatory activation during the development of portal hypertension in experimental cirrhosis; this activation may play a pathogenic role or be an epiphenomenon. Our objective was to study serum soluble CD163 (sCD163), a sensitive marker...... in the patients (52.2 vs 30.4 μg/l, pportal hypertension. The activation was not alleviated by the mechanical...... reduction of portal hypertension and the decreasing signs of endotoxinaemia. The findings suggest that Kupffer cell activation is a constitutive event that may play a pathogenic role for portal hypertension....

Immunomodulation by gadolinium chloride-induced Kupffer cell phagocytosis blockade

International Nuclear Information System (INIS)

Lazar, G.; Husztik, E.; Kiss, I.; Szakacs, J.; Olah, J.

1998-01-01

Gadolinium chloride (GdCl 3 ), a rare earth metal salt, depresses macrophage activity, and is commonly used to study the physiology of the reticuloendothelial system. In the present work, the effect of GdCl 3 -induced Kupffer cell blockade on the humoral immune response in mice to sheep red blood cells (SRBC) was investigated. Kupffer cell phagocytosis blockade was found to increase both the primary and secondary immune responses to SRBC. The primary immune response was significantly augmented in animals injected intravenously with GdCl 3 2, 3 or 4 days before injection of the cellular antigen, but GdCl 3 injected 7 days before the antigen did not modify the immune response. Increased secondary humoral immune responses were also observed. When GdCl 3 was injected 2 days before the second dose of antigen, the numbers of both IgM and IgG-producing plaque forming cells were augmented. GdCl 3 injected 2 days before the first dose of SRBC did not modify the humoral immune response. Earlier studies with 51 Cr-labelled foreign red blood cells suggested that the augmentation of the humoral immune response in GdCl 3 -pretreated mice is a consequence of the spillover of the antigen from the liver into the spleen and other extrahepatic reticuloendothelial organs. (orig.)

Role of Kupffer Cells in Thioacetamide-Induced Cell Cycle Dysfunction

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Mirandeli Bautista

2011-09-01

Full Text Available It is well known that gadolinium chloride (GD attenuates drug-induced hepatotoxicity by selectively inactivating Kupffer cells. In the present study the effect of GD in reference to cell cycle and postnecrotic liver regeneration induced by thioacetamide (TA in rats was studied. Two months male rats, intraveously pretreated with a single dose of GD (0.1 mmol/Kg, were intraperitoneally injected with TA (6.6 mmol/Kg. Samples of blood and liver were obtained from rats at 0, 12, 24, 48, 72 and 96 h following TA intoxication. Parameters related to liver damage were determined in blood. In order to evaluate the mechanisms involved in the post-necrotic regenerative state, the levels of cyclin D and cyclin E as well as protein p27 and Proliferating Cell Nuclear Antigen (PCNA were determined in liver extracts because of their roles in the control of cell cycle check-points. The results showed that GD significantly reduced the extent of necrosis. Noticeable changes were detected in the levels of cyclin D1, cyclin E, p27 and PCNA when compared to those induced by thioacetamide. Thus GD pre-treatment reduced TA-induced liver injury and accelerated the postnecrotic liver regeneration. These results demonstrate that Kupffer cells are involved in TA-induced liver and also in the postnecrotic proliferative liver states.

Kupffer cells promote hepatic steatosis via interleukin-1beta-dependent suppression of peroxisome proliferator-activated receptor alpha activity

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Stienstra, Rinke; Saudale, Fredy; Duval, Caroline; Keshtkar, Shohreh; Groener, Johanna E. M.; van Rooijen, Nico; Staels, Bart; Kersten, Sander; Müller, Michael

2010-01-01

Kupffer cells have been implicated in the pathogenesis of various liver diseases. However, their involvement in metabolic disorders of the liver, including fatty liver disease, remains unclear. The present study sought to determine the impact of Kupffer cells on hepatic triglyceride storage and to

Non-invasive imaging of kupffer cell status using radiolabelled mannosylated albumin

NARCIS (Netherlands)

Mahajan, V.; Hartimath, S.; Comley, R.; Stefan-Gueldner, M.; Roth, A.; Poelstra, K.; Reker-Smit, C.; Kamps, J.; Dierckx, R.; de Vries, Erik

2014-01-01

Background and Aims: Kupffer cells are responsible for maintaining liver homeostasis and have a vital role in chronic hepatotoxicity and various liver diseases. Positron Imaging Tomography (PET) is a non-invasive imaging technique that allows quantification and visualization of biochemical processes

GPBAR1/TGR5 mediates bile acid-induced cytokine expression in murine Kupffer cells.

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Guiyu Lou

Full Text Available GPBAR1/TGR5 is a novel plasma membrane-bound G protein-coupled bile acid (BA receptor. BAs are known to induce the expression of inflammatory cytokines in the liver with unknown mechanism. Here we show that without other external stimuli, TGR5 activation alone induced the expression of interleukin 1β (IL-1β and tumor necrosis factor-α (TNF-α in murine macrophage cell line RAW264.7 or murine Kupffer cells. The TGR5-mediated increase of pro-inflammatory cytokine expression was suppressed by JNK inhibition. Moreover, the induced pro-inflammatory cytokine expression in mouse liver by 1% cholic acid (CA diet was blunted in JNK-/- mice. TGR5 activation by its ligands enhanced the phosphorylation levels, DNA-binding and trans-activities of c-Jun and ATF2 transcription factors. Finally, the induced pro-inflammatory cytokine expression in Kupffer cells by TGR5 activation correlated with the suppression of Cholesterol 7α-hydroxylase (Cyp7a1 expression in murine hepatocytes. These results suggest that TGR5 mediates the BA-induced pro-inflammatory cytokine production in murine Kupffer cells through JNK-dependent pathway. This novel role of TGR5 may correlate to the suppression of Cyp7a1 expression in hepatocytes and contribute to the delicate BA feedback regulation.

Fumonisin B1 hepatotoxicity in mice is attenuated by depletion of Kupffer cells by gadolinium chloride

International Nuclear Information System (INIS)

He, Quanren; Kim, Jiyoung; Sharma, Raghubir P.

2005-01-01

Fumonisin B 1 (FB 1 ) is a toxic and carcinogenic mycotoxin produced by Fusarium verticillioides found on corn worldwide. The biological effects of FB 1 are attributed to sphingolipid metabolism disruption as a result of ceramide synthase inhibition. Tumor necrosis factor α (TNFα) is an important modulator of FB 1 hepatotoxicity. Kupffer cells are major source of cytokine production in liver. In the present study we investigated the effects of Kupffer cell depletion by gadolinium on FB 1 hepatotoxicity in female BALB/c mice. Mice were given saline or 50 mg/kg of gadolinium chloride once via the tail vein; 16 h later they were treated with subcutaneous injections of vehicle or 2.25 mg/kg/day FB 1 in saline for three successive days. Gadolinium significantly attenuated FB 1 -induced increases in the activities of circulating alanine aminotransferase and aspartate aminotransferase and reduced the FB 1 -induced hepatocyte apoptosis and free sphinganine accumulation in liver. Both gadolinium and FB 1 treatments individually increased the expression of selected cell signal factors; e.g., TNFα, TNF receptor 1, TNF-related apoptosis-inducing ligand, lymphotoxin β, interferon γ, and transforming growth factor β1; gadolinium chloride did not alter FB 1 -induced expression of the above genes. Results indicated that Kupffer cells play a role in FB 1 hepatotoxicity. Decreased FB 1 -induced sphinganine accumulation and increased protective TNFα signaling by gadolinium chloride may in part account for its ameliorating effect on FB 1 liver damage

Kupffer cells are activated in cirrhotic portal hypertension and not normalised by TIPS.

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Holland-Fischer, Peter; Grønbæk, Henning; Sandahl, Thomas Damgaard; Moestrup, Søren K; Riggio, Oliviero; Ridola, Lorenzo; Aagaard, Niels Kristian; Møller, Holger Jon; Vilstrup, Hendrik

2011-10-01

Hepatic macrophages (Kupffer cells) undergo inflammatory activation during the development of portal hypertension in experimental cirrhosis; this activation may play a pathogenic role or be an epiphenomenon. Our objective was to study serum soluble CD163 (sCD163), a sensitive marker of macrophage activation, before and after reduction of portal venous pressure gradient by insertion of a transjugular intrahepatic portosystemic shunt (TIPS) in patients with cirrhosis. sCD163 was measured in 11 controls and 36 patients before and 1, 4 and 26 weeks after TIPS. We used lipopolysaccharide binding protein (LBP) levels as a marker of endotoxinaemia. Liver function and clinical status of the patients were assessed by galactose elimination capacity and Model for End Stage Liver Disease score. The sCD163 concentration was more than threefold higher in the patients than in the controls (median 5.22 mg/l vs 1.45 mg/l, pportal venous pressure gradient (r(2)=0.24, pportal vein (pportal hypertension. The activation was not alleviated by the mechanical reduction of portal hypertension and the decreasing signs of endotoxinaemia. The findings suggest that Kupffer cell activation is a constitutive event that may play a pathogenic role for portal hypertension.

Can technetium-labelled millimicrospheres be used to measure Kupffer-cell function

International Nuclear Information System (INIS)

Pearson, H.J.; Chamberlain, J.; Anderson, J.; Bowry, V.; Bell, P.R.F.

1985-01-01

It has been suggested that sodium pertechnetate sup(99m)Tc millimicrospheres can be used to measure Kupffercell function. We studied animals and humans to show whether the clearance and catabolism of sup(99m)Tc-labelled millimicrospheres can be used as a measure of Kupffer-cell function. Comparison with albumin 125 I-microaggregates clearance of human serum albumin failed to demonstrate that they can be used for this purpose. We suggest that their blood clearance is mainly an expression of liver blood flow. (orig.)

Fumonisin B{sub 1} hepatotoxicity in mice is attenuated by depletion of Kupffer cells by gadolinium chloride

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He, Quanren [Department of Physiology and Pharmacology, College of Veterinary Medicine, The University of Georgia, Athens, GA 30602-7389 (United States); Kim, Jiyoung [Department of Physiology and Pharmacology, College of Veterinary Medicine, The University of Georgia, Athens, GA 30602-7389 (United States); Sharma, Raghubir P [Department of Physiology and Pharmacology, College of Veterinary Medicine, The University of Georgia, Athens, GA 30602-7389 (United States)

2005-02-01

Fumonisin B{sub 1} (FB{sub 1}) is a toxic and carcinogenic mycotoxin produced by Fusarium verticillioides found on corn worldwide. The biological effects of FB{sub 1} are attributed to sphingolipid metabolism disruption as a result of ceramide synthase inhibition. Tumor necrosis factor {alpha} (TNF{alpha}) is an important modulator of FB{sub 1} hepatotoxicity. Kupffer cells are major source of cytokine production in liver. In the present study we investigated the effects of Kupffer cell depletion by gadolinium on FB{sub 1} hepatotoxicity in female BALB/c mice. Mice were given saline or 50 mg/kg of gadolinium chloride once via the tail vein; 16 h later they were treated with subcutaneous injections of vehicle or 2.25 mg/kg/day FB{sub 1} in saline for three successive days. Gadolinium significantly attenuated FB{sub 1}-induced increases in the activities of circulating alanine aminotransferase and aspartate aminotransferase and reduced the FB{sub 1}-induced hepatocyte apoptosis and free sphinganine accumulation in liver. Both gadolinium and FB{sub 1} treatments individually increased the expression of selected cell signal factors; e.g., TNF{alpha}, TNF receptor 1, TNF-related apoptosis-inducing ligand, lymphotoxin {beta}, interferon {gamma}, and transforming growth factor {beta}1; gadolinium chloride did not alter FB{sub 1}-induced expression of the above genes. Results indicated that Kupffer cells play a role in FB{sub 1} hepatotoxicity. Decreased FB{sub 1}-induced sphinganine accumulation and increased protective TNF{alpha} signaling by gadolinium chloride may in part account for its ameliorating effect on FB{sub 1} liver damage.

Splenic red pulp macrophages are intrinsically superparamagnetic and contaminate magnetic cell isolates.

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Franken, Lars; Klein, Marika; Spasova, Marina; Elsukova, Anna; Wiedwald, Ulf; Welz, Meike; Knolle, Percy; Farle, Michael; Limmer, Andreas; Kurts, Christian

2015-08-11

A main function of splenic red pulp macrophages is the degradation of damaged or aged erythrocytes. Here we show that these macrophages accumulate ferrimagnetic iron oxides that render them intrinsically superparamagnetic. Consequently, these cells routinely contaminate splenic cell isolates obtained with the use of MCS, a technique that has been widely used in immunological research for decades. These contaminations can profoundly alter experimental results. In mice deficient for the transcription factor SpiC, which lack red pulp macrophages, liver Kupffer cells take over the task of erythrocyte degradation and become superparamagnetic. We describe a simple additional magnetic separation step that avoids this problem and substantially improves purity of magnetic cell isolates from the spleen.

CD205-TLR9-IL-12 axis contributes to CpG-induced oversensitive liver injury in HBsAg transgenic mice by promoting the interaction of NKT cells with Kupffer cells.

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Hou, Xin; Hao, Xiaolei; Zheng, Meijuan; Xu, Congfei; Wang, Jun; Zhou, Rongbin; Tian, Zhigang

2017-08-01

Gut-derived bacterial products contribute to liver inflammation and injury during chronic hepatitis B virus infection; however, the underlying mechanisms remain obscure. In this study, hepatitis B surface antigen transgenic (HBs-Tg) mice and their wild-type (WT) control C57BL/6 mice were injected with CpG-oligodeoxynucleotides (ODNs) to mimic the translocation of gut microbial products into the systemic circulation. We found that, compared with the WT mice, the HBs-Tg mice were oversensitive to CpG-ODN-induced liver injury, which was dependent on natural killer T (NKT) cells. CpG-ODN injection enhanced the expression of Fas ligand (FasL) on NKT cells. In addition, hepatocytes from the HBs-Tg mice expressed higher levels of Fas than did those from the WT mice, which was further augmented by CpG-ODN. Interaction of Fas and FasL was involved in the cytotoxicity of NKT cells against hepatocytes in the HBs-Tg mice. Moreover, Kupffer cells in the HBs-Tg mice expressed higher levels of CD205 and produced greater amounts of interleukin (IL)-12 than did those in the WT mice. Finally, the depletion of Kupffer cells, neutralization of IL-12 or specific silencing of CD205 on Kupffer cells significantly inhibited CpG-ODN-induced liver injury and NKT activation in the HBs-Tg mice. Our data suggest that CD205-expressing Kupffer cells respond to CpG-ODNs and subsequently release IL-12 to promote NKT cell activation. Activated NKT cells induce liver damage through the Fas signaling pathway in HBs-Tg mice.

Thyroid Hormone-Induced Cytosol-to-Nuclear Translocation of Rat Liver Nrf2 Is Dependent on Kupffer Cell Functioning

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Luis A. Videla

2012-01-01

Full Text Available L-3,3′,5-triiodothyronine (T3 administration upregulates nuclear factor-E2-related factor 2 (Nrf2 in rat liver, which is redox-sensitive transcription factor mediating cytoprotection. In this work, we studied the role of Kupffer cell respiratory burst activity, a process related to reactive oxygen species generation and liver homeostasis, in Nrf2 activation using the macrophage inactivator gadolinium chloride (GdCl3; 10 mg/kg i.v. 72 h before T3 [0.1 mg/kg i.p.] or NADPH oxidase inhibitor apocynin (1.5 mmol/L added to the drinking water for 7 days before T3, and determinations were performed 2 h after T3. T3 increased nuclear/cytosolic Nrf2 content ratio and levels of heme oxygenase 1 (HO-1, catalytic subunit of glutamate cysteine ligase, and thioredoxin (Western blot over control values, proteins whose gene transcription is induced by Nrf2. These changes were suppressed by GdCl3 treatment prior to T3, an agent-eliciting Kupffer-cell depletion, inhibition of colloidal carbon phagocytosis, and the associated respiratory burst activity, with enhancement in nuclear inhibitor of Nrf2 kelch-like ECH-associated protein 1 (Keap1/Nrf2 content ratios suggesting Nrf2 degradation. Under these conditions, T3-induced tumor necrosis factor-α (TNF-α response was eliminated by previous GdCl3 administration. Similar to GdCl3, apocynin given before T3 significantly reduced liver Nrf2 activation and HO-1 expression, a NADPH oxidase inhibitor eliciting abolishment of colloidal carbon-induced respiratory burst activity without altering carbon phagocytosis. It is concluded that Kupffer cell functioning is essential for upregulation of liver Nrf2-signaling pathway by T3. This contention is supported by suppression of the respiratory burst activity of Kupffer cells and the associated reactive oxygen species production by GdCl3 or apocynin given prior to T3, thus hindering Nrf2 activation.

Activation of Kupffer Cells Is Associated with a Specific Dysbiosis Induced by Fructose or High Fat Diet in Mice.

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Gladys Ferrere

Full Text Available The increase consumption of fructose in diet is associated with liver inflammation. As a specific fructan substrate, fructose may modify the gut microbiota which is involved in obesity-induced liver disease. Here, we aimed to assess whether fructose-induced liver damage was associated with a specific dysbiosis, especially in mice fed a high fat diet (HFD. To this end, four groups of mice were fed with normal and HFD added or not with fructose. Body weight and glucose sensitivity, liver inflammation, dysbiosis and the phenotype of Kupffer cells were determined after 16 weeks of diet. Food intake was increased in the two groups of mice fed with the HFD. Mice fed with HFD and fructose showed a higher infiltration of lymphocytes into the liver and a lower inflammatory profile of Kupffer cells than mice fed with the HFD without fructose. The dysbiosis associated with diets showed that fructose specifically prevented the decrease of Mouse intestinal bacteria in HFD fed mice and increased Erysipelotrichi in mice fed with fructose, independently of the amount of fat. In conclusion, fructose, used as a sweetener, induced a dysbiosis which is different in presence of fat in the diet. Consequently, the activation of Kupffer cells involved in mice model of HFD-induced liver inflammation was not observed in an HFD/fructose combined diet. These data highlight that the complexity of diet composition could highly impact the development of liver lesions during obesity. Specific dysbiosis associated with the diet could explain that the progressions of liver damage are different.

Tattoo Pigments Are Observed in the Kupffer Cells of the Liver Indicating Blood-Borne Distribution of Tattoo Ink

DEFF Research Database (Denmark)

Sepehri, Mitra; Steen Sejersen, Tobias; Qvortrup, Klaus

2017-01-01

AIM: Tattoo pigments are deposited in the skin and known to distribute to regional lymph nodes. Tattoo pigments are small particles and may be hypothesized to reach the blood stream and become distributed to peripheral organs. This has not been studied in the past. The aim of the study was to trace....... Mice were sacrificed after 1 year. Samples were isolated from tattooed skin, lymph nodes, liver, spleen, kidney, and lung. Samples were examined for deposits of tattoo pigments by light microscopy and transmission electron microscopy (TEM). RESULTS: TEM identified intracellular tattoo pigments...... in the skin and in lymph nodes. TEM in both groups of tattooed mice showed tattoo pigment deposits in the Kupffer cells in the liver, which is a new observation. TEM detected no pigment in other internal organs. Light microscopy showed dense pigment in the skin and in lymph nodes but not in internal organs...

The cachectic mediator proteolysis inducing factor activates NF-kappaB and STAT3 in human Kupffer cells and monocytes

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Watchorn, T.M.; Dowidar, N.; Dejong, C.H.; Waddell, I.D.; Garden, O.J.; Ross, J.A.

2005-01-01

A novel proteoglycan, proteolysis inducing factor (PIF), is capable of inducing muscle proteolysis during the process of cancer cachexia, and of inducing an acute phase response in human hepatocytes. We investigated whether PIF is able to activate pro-inflammatory pathways in human Kupffer cells,

Kupffer cells hasten resolution of liver immunopathology in mouse models of viral hepatitis.

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Giovanni Sitia

2011-06-01

Full Text Available Kupffer cells (KCs are widely considered important contributors to liver injury during viral hepatitis due to their pro-inflammatory activity. Herein we utilized hepatitis B virus (HBV-replication competent transgenic mice and wild-type mice infected with a hepatotropic adenovirus to demonstrate that KCs do not directly induce hepatocellular injury nor do they affect the pathogenic potential of virus-specific CD8 T cells. Instead, KCs limit the severity of liver immunopathology. Mechanistically, our results are most compatible with the hypothesis that KCs contain liver immunopathology by removing apoptotic hepatocytes in a manner largely dependent on scavenger receptors. Apoptotic hepatocytes not readily removed by KCs become secondarily necrotic and release high-mobility group box 1 (HMGB-1 protein, promoting organ infiltration by inflammatory cells, particularly neutrophils. Overall, these results indicate that KCs resolve rather than worsen liver immunopathology.

Predominance of Th1 response, increase of megakaryocytes and Kupffer cells are related to survival in Trypanosoma cruzi infected mice treated with Lycopodium clavatum.

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Falkowski-Temporini, Gislaine Janaina; Lopes, Carina Ribeiro; Massini, Paula Fernanda; Brustolin, Camila Fernanda; Sandri, Patricia Flora; Ferreira, Érika Cristina; Aleixo, Denise Lessa; Pala, Nelson Roberto; de Araújo, Silvana Marques

2016-12-01

We investigated the number of megakaryocytes, Kupffer cells and ratios of Th1/Th2 and Th1/Th17 cytokines in survival of mice infected with Y strain of Trypanosoma cruzi and treated with Lycopodium clavatum. In a blind, randomized and controlled assay, Swiss male mice, 8weeks-old, infected with 1400 trypomastigotes (Y strain) were divided into groups and treated with: GLy - Lycopodium clavatum dynamization13c and GCI - alcohol solution 7° GL (vehicle medicine). The treatment was offered two days before infection and on the 2nd, 4th and 6th days after infection, overnight (1mL/100mL) and ad libitum. Parameters assessed were: survival rate, number of megakaryocytes and Kupffer cells, cytokines dosage (TNF-α, IFN-γ, IL-2, IL-4, IL-6, IL-10, IL-17), Th1/Th2 and Th1/Th17 ratios. The increase in megakaryocytes, Kupffer cells, predominance of Th1 response, with increased TNF-α, IL-10, TNF-α/IL-4, TNF-α/IL-17 and decreased IL-6 IL-6/IL-4, are related to increased survival in mice infected with T. cruzi and treated with Lycopodium clavatum 13c. This result demonstrates the possibility of an alternative approach for the treatment of Chagas disease with dynamized drugs. Copyright © 2016 Elsevier Ltd. All rights reserved.

Subtoxic Concentrations of Hepatotoxic Drugs Lead to Kupffer Cell Activation in a Human In Vitro Liver Model: An Approach to Study DILI

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Victoria Kegel

2015-01-01

Full Text Available Drug induced liver injury (DILI is an idiosyncratic adverse drug reaction leading to severe liver damage. Kupffer cells (KC sense hepatic tissue stress/damage and therefore could be a tool for the estimation of consequent effects associated with DILI. Aim of the present study was to establish a human in vitro liver model for the investigation of immune-mediated signaling in the pathogenesis of DILI. Hepatocytes and KC were isolated from human liver specimens. The isolated KC yield was 1.2±0.9×106 cells/g liver tissue with a purity of >80%. KC activation was investigated by the measurement of reactive oxygen intermediates (ROI, DCF assay and cell activity (XTT assay. The initial KC activation levels showed broad donor variability. Additional activation of KC using supernatants of hepatocytes treated with hepatotoxic drugs increased KC activity and led to donor-dependent changes in the formation of ROI compared to KC incubated with supernatants from untreated hepatocytes. Additionally, a compound- and donor-dependent increase in proinflammatory cytokines or in anti-inflammatory cytokines was detected. In conclusion, KC related immune signaling in hepatotoxicity was successfully determined in a newly established in vitro liver model. KC were able to detect hepatocyte stress/damage and to transmit a donor- and compound-dependent immune response via cytokine production.

Subtoxic Concentrations of Hepatotoxic Drugs Lead to Kupffer Cell Activation in a Human In Vitro Liver Model: An Approach to Study DILI

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Kegel, Victoria; Pfeiffer, Elisa; Burkhardt, Britta; Liu, Jia L.; Zeilinger, Katrin; Nüssler, Andreas K.; Seehofer, Daniel; Damm, Georg

2015-01-01

Drug induced liver injury (DILI) is an idiosyncratic adverse drug reaction leading to severe liver damage. Kupffer cells (KC) sense hepatic tissue stress/damage and therefore could be a tool for the estimation of consequent effects associated with DILI. Aim of the present study was to establish a human in vitro liver model for the investigation of immune-mediated signaling in the pathogenesis of DILI. Hepatocytes and KC were isolated from human liver specimens. The isolated KC yield was 1.2 ± 0.9 × 106 cells/g liver tissue with a purity of >80%. KC activation was investigated by the measurement of reactive oxygen intermediates (ROI, DCF assay) and cell activity (XTT assay). The initial KC activation levels showed broad donor variability. Additional activation of KC using supernatants of hepatocytes treated with hepatotoxic drugs increased KC activity and led to donor-dependent changes in the formation of ROI compared to KC incubated with supernatants from untreated hepatocytes. Additionally, a compound- and donor-dependent increase in proinflammatory cytokines or in anti-inflammatory cytokines was detected. In conclusion, KC related immune signaling in hepatotoxicity was successfully determined in a newly established in vitro liver model. KC were able to detect hepatocyte stress/damage and to transmit a donor- and compound-dependent immune response via cytokine production. PMID:26491234

Biofilm-Forming Methicillin-Resistant Staphylococcus aureus Survive in Kupffer Cells and Exhibit High Virulence in Mice

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Takuto Oyama

2016-06-01

Full Text Available Although Staphylococcus aureus is part of the normal body flora, heavy usage of antibiotics has resulted in the emergence of methicillin-resistant strains (MRSA. MRSA can form biofilms and cause indwelling foreign body infections, bacteremia, soft tissue infections, endocarditis, and osteomyelitis. Using an in vitro assay, we screened 173 clinical blood isolates of MRSA and selected 20 high-biofilm formers (H-BF and low-biofilm formers (L-BF. These were intravenously administered to mice and the general condition of mice, the distribution of bacteria, and biofilm in the liver, lung, spleen, and kidney were investigated. MRSA count was the highest in the liver, especially within Kupffer cells, which were positive for acid polysaccharides that are associated with intracellular biofilm. After 24 h, the general condition of the mice worsened significantly in the H-BF group. In the liver, bacterial deposition and aggregation and the biofilm-forming spot number were all significantly greater for H-BF group than for L-BF. CFU analysis revealed that bacteria in the H-BF group survived for long periods in the liver. These results indicate that the biofilm-forming ability of MRSA is a crucial factor for intracellular persistence, which could lead to chronic infections.

Pivotal roles of Kupffer cells in the progression and regression of DDC-induced chronic cholangiopathy.

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Jemail, Leila; Miyao, Masashi; Kotani, Hirokazu; Kawai, Chihiro; Minami, Hirozo; Abiru, Hitoshi; Tamaki, Keiji

2018-04-23

Kupffer cells (KCs) are key players in maintaining tissue homeostasis and are involved in various liver diseases. However, the roles of KCs in the pathogenesis of cholangiopathy are largely unknown. We aimed to investigate the precise roles of KCs in both the progression and regression phases of the 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-induced cholangiopathy model. In the early phase of DDC-induced cholangiopathy, the number of KCs significantly increased over time. Moreover, KCs were associated with abnormal phenotypic changes in other liver cells, such as hepatocytes, biliary epithelial cells, liver sinusoidal endothelial cells, and hepatic stellate cells. In contrast, KC depletion by clodronate administration suppressed the progression of the disease, and maintained the phenotypes of other cells. In the regression phase, the numbers of KCs significantly decreased, and the cells redifferentiated to their quiescent state. In contrast, KC depletion delayed the recovery of cells by maintaining other liver cells in an active state. These findings suggest that KCs play detrimental roles in the progression phase; however, they are beneficial in the regression phase by mediating interactions between other liver cells. Our data provide new insights into the roles of KCs in the pathogenesis of cholangiopathy.

All-In-One: Advanced preparation of Human Parenchymal and Non-Parenchymal Liver Cells.

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Melanie Werner

Full Text Available Liver cells are key players in innate immunity. Thus, studying primary isolated liver cells is necessary for determining their role in liver physiology and pathophysiology. In particular, the quantity and quality of isolated cells are crucial to their function. Our aim was to isolate a large quantity of high-quality human parenchymal and non-parenchymal cells from a single liver specimen.Hepatocytes, Kupffer cells, liver sinusoidal endothelial cells, and stellate cells were isolated from liver tissues by collagenase perfusion in combination with low-speed centrifugation, density gradient centrifugation, and magnetic-activated cell sorting. The purity and functionality of cultured cell populations were controlled by determining their morphology, discriminative cell marker expression, and functional activity.Cell preparation yielded the following cell counts per gram of liver tissue: 2.0 ± 0.4 × 10(7 hepatocytes, 1.8 ± 0.5 × 10(6 Kupffer cells, 4.3 ± 1.9 × 10(5 liver sinusoidal endothelial cells, and 3.2 ± 0.5 × 10(5 stellate cells. Hepatocytes were identified by albumin (95.5 ± 1.7% and exhibited time-dependent activity of cytochrome P450 enzymes. Kupffer cells expressed CD68 (94.5 ± 1.2% and exhibited phagocytic activity, as determined with 1 μm latex beads. Endothelial cells were CD146(+ (97.8 ± 1.1% and exhibited efficient uptake of acetylated low-density lipoprotein. Hepatic stellate cells were identified by the expression of α-smooth muscle actin (97.1 ± 1.5%. These cells further exhibited retinol (vitamin A-mediated autofluorescence.Our isolation procedure for primary parenchymal and non-parenchymal liver cells resulted in cell populations of high purity and quality, with retained physiological functionality in vitro. Thus, this system may provide a valuable tool for determining liver function and disease.

Failure to demonstrate a major role for Kupffer cells and radiosensitive leukocytes in immunoglobulin-mediated elimination of Trypanosoma musculi

International Nuclear Information System (INIS)

Kongshavn, P.A.; Shaw, K.; Ghadirian, E.; Ulczak, O.

1990-01-01

Previous studies have indicated that elimination of parasitemia in Trypanosoma musculi infection is brought about by immunoglobulin G2a antibodies, C3, and an effector cell. Experiments were designed to identify the putative effector cell by using several approaches. Infected C5-deficient or C5-sufficient mice treated with silica particles or given 900 rads of radiation 3 days earlier effectively eliminated trypanosomes following administration of immune plasma (IP). Silica-treated, noninfected mice given T. musculi preincubated with IP also cleared the parasites. Radiolabeling studies revealed that uptake of the cleared trypanosomes by the liver in normal mice was relatively low and fell only slightly (19%) in silica-treated mice. In contrast, uptake of radiolabeled sheep erythrocytes by the liver was normally much higher and fell drastically (7%) in silica-treated mice. Mice were then immunocompromised by 900 rads of radiation, silica particles, and anti-platelet serum combined before IP-sensitized trypanosomes were given. Leukocyte and platelet counts were both reduced by 95% and sheep erythrocyte uptake by the liver fell from 77 to 5%; however, greater than 99% of the injected trypanosomes were cleared in these mice and uptake of radiolabeled trypanosomes by the liver was similar to that of normal mice. Lastly, in anesthetized mice in which Kupffer cells were excluded surgically from the circulation, greater than 99% of the IP-sensitized trypanosomes disappeared rapidly from the blood. Only 7% of the radiolabel was found in the liver versus 60% in sham-operated mice. The results are interpreted as showing that hepatic Kupffer cells play a minor role in the immune elimination of T. musculi. Likewise, radiosensitive leukocytes and platelets are unlikely to be sole candidates for the putative effector cell that mediates a cure of murine trypanosomiasis

Dynamic imaging of experimental Leishmania donovani-induced hepatic granulomas detects Kupffer cell-restricted antigen presentation to antigen-specific CD8 T cells.

Directory of Open Access Journals (Sweden)

Lynette Beattie

2010-03-01

Full Text Available Kupffer cells (KCs represent the major phagocytic population within the liver and provide an intracellular niche for the survival of a number of important human pathogens. Although KCs have been extensively studied in vitro, little is known of their in vivo response to infection and their capacity to directly interact with antigen-specific CD8(+ T cells. Here, using a combination of approaches including whole mount and thin section confocal microscopy, adoptive cell transfer and intra-vital 2-photon microscopy, we demonstrate that KCs represent the only detectable population of mononuclear phagocytes within granulomas induced by Leishmania donovani infection that are capable of presenting parasite-derived peptide to effector CD8(+ T cells. This restriction of antigen presentation to KCs within the Leishmania granuloma has important implications for the identification of new candidate vaccine antigens and for the design of novel immuno-therapeutic interventions.

Gold nanoparticles administration induced prominent inflammatory, central vein intima disruption, fatty change and Kupffer cells hyperplasia

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Abdelhalim Mohamed

2011-08-01

Full Text Available Abstract Background Advances in nanotechnology have identified promising candidates for many biological, biomedical and biomedicine applications. They are being increasingly exploited for medical uses and other industrial applications. The aim of the present study was to investigate the effects of administration of gold nanoparticles (GNPs on inflammatory cells infiltration, central vein intima disruption, fatty change, and Kupffer cells hyperplasia in the hepatic tissue in an attempt to cover and understand the toxicity and the potential threat of their therapeutic and diagnostic use. Methods A total of 70 healthy male Wistar-Kyoto rats were exposed to GNPs received 50 or 100 μl of GNPs infusion of 10, 20 and 50 nm GNPs for 3 or 7 days. Animals were randomly divided into groups, 12 GNPs-treated rats groups and one control group (NG. Groups 1, 2 and 3 received infusion of 50 μl GNPs of size 10 nm (3 or 7 days, size 20 nm (3 or 7 days and 50 nm (3 or 7 days, respectively; while groups 4, 5 and 6 received infusion of 100 μl GNPs of size 10 nm, size 20 nm and 50 nm, respectively. Results In comparison with respective control rats, exposure to GNPs doses has produced alterations in the hepatocytes, portal triads and sinusoids. The alterations in the hepatocytes were mainly vacuolar to hydropic degeneration, cytopasmic hyaline vacuolation, polymorphism, binucleation, karyopyknosis, karyolysis, karyorrhexis and necrosis. In addition, inflammatory cell infiltration, Kupffer cells hyperplasia, central veins intima disruption, hepatic strands dilatation and occasional fatty change together with a loss of normal architechiture of hepatic strands were also seen. Conclusions The alterations induced by the administration of GNPs were size-dependent with smaller ones induced more affects and related with time exposure of GNPs. These alterations might be an indication of injured hepatocytes due to GNPs toxicity that became unable to deal with the

Soluble CD163, a marker of Kupffer cell activation, is related to portal hypertension in patients with liver cirrhosis

DEFF Research Database (Denmark)

Grønbaek, H; Sandahl, T D; Mortensen, C

2012-01-01

BACKGROUND: Activation of Kupffer cells may be involved in the pathogenesis of portal hypertension by release of vasoconstrictive substances and fibrosis due to co-activation of hepatic stellate cells. AIM: To study soluble plasma (s) CD163, a specific marker of activated macrophages......, as a biomarker for portal hypertension in patients with liver cirrhosis. METHODS: We measured sCD163 concentration and the hepatic venous pressure gradient (HVPG) by liver vein catheterisation in 81 cirrhosis patients (Child-Pugh CP-A: n = 26, CP-B: n = 29, CP-C: n = 26) and 22 healthy subjects. We also measured...... for HVPG. These findings support a primary role of macrophage activation in portal hypertension, and may indicate a target for biological intervention....

Protocol for Isolation of Primary Human Hepatocytes and Corresponding Major Populations of Non-parenchymal Liver Cells

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Pfeiffer, Elisa; Zeilinger, Katrin; Seehofer, Daniel; Damm, Georg

2016-01-01

Beside parenchymal hepatocytes, the liver consists of non-parenchymal cells (NPC) namely Kupffer cells (KC), liver endothelial cells (LEC) and hepatic Stellate cells (HSC). Two-dimensional (2D) culture of primary human hepatocyte (PHH) is still considered as the "gold standard" for in vitro testing of drug metabolism and hepatotoxicity. It is well-known that the 2D monoculture of PHH suffers from dedifferentiation and loss of function. Recently it was shown that hepatic NPC play a central role in liver (patho-) physiology and the maintenance of PHH functions. Current research focuses on the reconstruction of in vivo tissue architecture by 3D- and co-culture models to overcome the limitations of 2D monocultures. Previously we published a method to isolate human liver cells and investigated the suitability of these cells for their use in cell cultures in Experimental Biology and Medicine1. Based on the broad interest in this technique the aim of this article was to provide a more detailed protocol for the liver cell isolation process including a video, which will allow an easy reproduction of this technique. Human liver cells were isolated from human liver tissue samples of surgical interventions by a two-step EGTA/collagenase P perfusion technique. PHH were separated from the NPC by an initial centrifugation at 50 x g. Density gradient centrifugation steps were used for removal of dead cells. Individual liver cell populations were isolated from the enriched NPC fraction using specific cell properties and cell sorting procedures. Beside the PHH isolation we were able to separate KC, LEC and HSC for further cultivation. Taken together, the presented protocol allows the isolation of PHH and NPC in high quality and quantity from one donor tissue sample. The access to purified liver cell populations could allow the creation of in vivo like human liver models. PMID:27077489

Amphiphilic core shell nanoparticles containing dense polyethyleneimine shells for efficient delivery of microRNA to Kupffer cells

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Liu Z

2016-06-01

Full Text Available Zuojin Liu,1,* Dechao Niu,2,3,* Junyong Zhang,1 Wenfeng Zhang,1 Yuan Yao,2 Pei Li,2 Jianping Gong1 1Department of Hepatobiliary Surgery, The Second Affiliated Hospital of Chongqing Medical University, Chongqing, 2Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong, 3Lab of Low-Dimensional Materials Chemistry, School of Materials Science and Engineering, East China University of Science and Technology, Shanghai, People’s Republic of China *These authors contributed equally to this work Abstract: Efficient and targeted delivery approach to transfer exogenous genes into macrophages is still a great challenge. Current gene delivery methods often result in low cellular uptake efficiency in vivo in some types of cells, especially for the Kupffer cells (KCs. In this article, we demonstrate that amphiphilic core–shell nanoparticles (NPs consisting of well-defined hydrophobic poly(methyl methacrylate (PMMA cores and branched polyethyleneimine (PEI shells (denoted as PEI@PMMA NPs are efficient nanocarriers to deliver microRNA (miRNA-loaded plasmid to the KCs. Average hydrodynamic diameter of PEI@PMMA NPs was 279 nm with a narrow size distribution. The NPs also possessed positive surface charges up to +30 mV in water, thus enabling effective condensation of negatively charged plasmid DNA. Gel electrophoresis assay showed that the resultant PEI@PMMA NPs were able to completely condense miRNA plasmid at a weight ratio of 25:1 (N/P ratio equal to 45:1. The Cell Counting Kit-8 assay and flow cytometry results showed that the PEI@PMMA/miRNA NPs displayed low cytotoxicity and cell apoptosis activity against the KCs. The maximum cell transfection efficiency reached 34.7% after 48 hours, which is much higher than that obtained by using the commercial Lipofectamine™ 2000 (1.7%. Bio-transmission electron microscope observation revealed that the PEI@PMMA NPs were mainly distributed in

Mesenchymal stem cells improve mouse non-heart-beating liver graft survival by inhibiting Kupffer cell apoptosis via TLR4-ERK1/2-Fas/FasL-caspase3 pathway regulation

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Yang Tian

2016-10-01

Full Text Available Abstract Background Liver transplantation is the optimal treatment option for end-stage liver disease, but organ shortages dramatically restrict its application. Donation after cardiac death (DCD is an alternative approach that may expand the donor pool, but it faces challenges such as graft dysfunction, early graft loss, and cholangiopathy. Moreover, DCD liver grafts are no longer eligible for transplantation after their warm ischaemic time exceeds 30 min. Mesenchymal stem cells (MSCs have been proposed as a promising therapy for treatment of certain liver diseases, but the role of MSCs in DCD liver graft function remains elusive. Methods In this study, we established an arterialized mouse non-heart-beating (NHB liver transplantation model, and compared survival rates, cytokine and chemokine expression, histology, and the results of in vitro co-culture experiments in animals with or without MSC infusion. Results MSCs markedly ameliorated NHB liver graft injury and improved survival post-transplantation. Additionally, MSCs suppressed Kupffer cell apoptosis, Th1/Th17 immune responses, chemokine expression, and inflammatory cell infiltration. In vitro, PGE2 secreted by MSCs inhibited Kupffer cell apoptosis via TLR4-ERK1/2-caspase3 pathway regulation. Conclusion Our study uncovers a protective role for MSCs and elucidates the underlying immunomodulatory mechanism in an NHB liver transplantation model. Our results suggest that MSCs are uniquely positioned for use in future clinical studies owing to their ability to protect DCD liver grafts, particularly in patients for whom DCD organs are not an option according to current criteria.

Time course investigation of PPARα- and Kupffer cell-dependent effects of WY-14,643 in mouse liver using microarray gene expression

International Nuclear Information System (INIS)

Woods, Courtney G.; Kosyk, Oksana; Bradford, Blair U.; Ross, Pamela K.; Burns, Amanda M.; Cunningham, Michael L.; Qu Pingping; Ibrahim, Joseph G.; Rusyn, Ivan

2007-01-01

Administration of peroxisome proliferators to rodents causes proliferation of peroxisomes, induction of β-oxidation enzymes, hepatocellular hypertrophy and hyperplasia, with chronic exposure ultimately leading to hepatocellular carcinomas. Many responses associated with peroxisome proliferators are nuclear receptor-mediated events involving peroxisome proliferators-activated receptor alpha (PPARα). A role for nuclear receptor-independent events has also been shown, with evidence of Kupffer cell-mediated free radical production, presumably through NAPDH oxidase, induction of redox-sensitive transcription factors involved in cytokine production and cytokine-mediated cell replication following acute treatment with peroxisome proliferators in rodents. Recent studies have demonstrated, by using p47 phox -null mice which are deficient in NADPH oxidase, that this enzyme is not related to the phenotypic events caused by prolonged administration of peroxisome proliferators. In an effort to determine the timing of the transition from Kupffer cell-to PPARα-dependent modulation of peroxisome proliferator effects, gene expression was assessed in liver from Pparα-null, p47 phox -null and corresponding wild-type mice following treatment with 4-chloro-6-(2,3-xylidino)-pyrimidynylthioacetic acid (WY-14,643) for 8 h, 24 h, 72 h, 1 week or 4 weeks. WY-14,643-induced gene expression in p47 phox -null mouse liver differed substantially from wild-type mice at acute doses and striking differences in baseline expression of immune related genes were evident. Pathway mapping of genes that respond to WY-14,643 in a time- and dose-dependent manner demonstrates suppression of immune response, cell death and signal transduction and promotion of lipid metabolism, cell cycle and DNA repair. Furthermore, these pathways were largely dependent on PPARα, not NADPH oxidase demonstrating a temporal shift in response to peroxisome proliferators. Overall, this study shows that NADPH oxidase

Tattoo Pigments Are Observed in the Kupffer Cells of the Liver Indicating Blood-Borne Distribution of Tattoo Ink.

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Sepehri, Mitra; Sejersen, Tobias; Qvortrup, Klaus; Lerche, Catharina M; Serup, Jørgen

2017-01-01

Tattoo pigments are deposited in the skin and known to distribute to regional lymph nodes. Tattoo pigments are small particles and may be hypothesized to reach the blood stream and become distributed to peripheral organs. This has not been studied in the past. The aim of the study was to trace tattoo pigments in internal organs in mice extensively tattooed with 2 different tattoo ink products. Three groups of mice were studied, i.e., 10 tattooed black, 10 tattooed red, and 5 untreated controls. They were tattooed on the entire back with commercial tattoo inks, black and red. Mice were sacrificed after 1 year. Samples were isolated from tattooed skin, lymph nodes, liver, spleen, kidney, and lung. Samples were examined for deposits of tattoo pigments by light microscopy and transmission electron microscopy (TEM). TEM identified intracellular tattoo pigments in the skin and in lymph nodes. TEM in both groups of tattooed mice showed tattoo pigment deposits in the Kupffer cells in the liver, which is a new observation. TEM detected no pigment in other internal organs. Light microscopy showed dense pigment in the skin and in lymph nodes but not in internal organs. The study demonstrated black and red tattoo pigment deposits in the liver; thus, tattoo pigment distributed from the tattooed skin via the blood stream to this important organ of detoxification. The finding adds a new dimension to tattoo pigment distribution in the body, i.e., as observed via the blood in addition to the lymphatic pathway. © 2017 S. Karger AG, Basel.

Featured Article: Isolation, characterization, and cultivation of human hepatocytes and non-parenchymal liver cells

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Pfeiffer, Elisa; Kegel, Victoria; Zeilinger, Katrin; Hengstler, Jan G; Nüssler, Andreas K; Seehofer, Daniel

2015-01-01

Primary human hepatocytes (PHH) are considered to be the gold standard for in vitro testing of xenobiotic metabolism and hepatotoxicity. However, PHH cultivation in 2D mono-cultures leads to dedifferentiation and a loss of function. It is well known that hepatic non-parenchymal cells (NPC), such as Kupffer cells (KC), liver endothelial cells (LEC), and hepatic stellate cells (HSC), play a central role in the maintenance of PHH functions. The aims of the present study were to establish a protocol for the simultaneous isolation of human PHH and NPC from the same tissue specimen and to test their suitability for in vitro co-culture. Human PHH and NPC were isolated from tissue obtained by partial liver resection by a two-step EDTA/collagenase perfusion technique. The obtained cell fractions were purified by Percoll density gradient centrifugation. KC, LEC, and HSC contained in the NPC fraction were separated using specific adherence properties and magnetic activated cell sorting (MACS®). Identified NPC revealed a yield of 1.9 × 106 KC, 2.7 × 105 LEC and 4.7 × 105 HSC per gram liver tissue, showing viabilities >90%. Characterization of these NPC showed that all populations went through an activation process, which influenced the cell fate. The activation of KC strongly depended on the tissue quality and donor anamnesis. KC became activated in culture in association with a loss of viability within 4–5 days. LEC lost specific features during culture, while HSC went through a transformation process into myofibroblasts. The testing of different culture conditions for HSC demonstrated that they can attenuate, but not prevent dedifferentiation in vitro. In conclusion, the method described allows the isolation and separation of PHH and NPC in high quality and quantity from the same donor. PMID:25394621

Histones activate the NLRP3 Inflammasome in Kupffer Cells during Sterile Inflammatory Liver Injury

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Huang, Hai; Chen, Hui-Wei; Evankovich, John; Yan, Wei; Rosborough, Brian R.; Nace, Gary W.; Ding, Qing; Loughran, Patricia; Beer-Stolz, Donna; Billiar, Timothy R.; Esmon, Charles T.; Tsung, Allan

2013-01-01

Cellular processes that drive sterile inflammatory injury after hepatic ischemia/reperfusion (I/R) injury are not completely understood. Activation of the inflammasome plays a key role in response to invading intracellular pathogens, but mounting evidence suggests it also plays a role in inflammation driven by endogenous danger-associate molecular pattern (DAMP) molecules released after ischemic injury. The nucleotide-binding domain, leucine-rich repeat containing protein 3 (NLRP3) inflammasome is one such process, and the mechanism by which its activation results in damage and inflammatory responses following liver I/R is unknown. Here we report that both NLRP3 and its downstream target Caspase-1 are activated I/R and are essential for hepatic I/R injury as both NLRP3 and Caspase-1 KO mice are protected from injury. Furthermore, inflammasome-mediated injury is dependent on Caspase-1 expression in liver non-parenchymal cells. While upstream signals that activate the inflammasome during ischemic injury are not well characterized, we show that endogenous extracellular histones activate the NLRP3 inflammasome during liver I/R through Toll-like Receptor-9 (TLR9). This occurs through TLR9-dependent generation of reactive oxygen species. This mechanism is operant in resident liver Kupffer cells, which drive innate immune responses after I/R injury by recruiting additional cell types, including neutrophils and inflammatory monocytes. These novel findings illustrate a new mechanism by which extracellular histones and activation of NLRP3 inflammasome contribute to liver damage and activation of innate immunity during sterile inflammation. PMID:23904166

Klf8 regulates left-right asymmetric patterning through modulation of Kupffer's vesicle morphogenesis and spaw expression.

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Lin, Che-Yi; Tsai, Ming-Yuan; Liu, Yu-Hsiu; Lu, Yu-Fen; Chen, Yi-Chung; Lai, Yun-Ren; Liao, Hsin-Chi; Lien, Huang-Wei; Yang, Chung-Hsiang; Huang, Chang-Jen; Hwang, Sheng-Ping L

2017-07-17

Although vertebrates are bilaterally symmetric organisms, their internal organs are distributed asymmetrically along a left-right axis. Disruption of left-right axis asymmetric patterning often occurs in human genetic disorders. In zebrafish embryos, Kupffer's vesicle, like the mouse node, breaks symmetry by inducing asymmetric expression of the Nodal-related gene, spaw, in the left lateral plate mesoderm (LPM). Spaw then stimulates transcription of itself and downstream genes, including lft1, lft2, and pitx2, specifically in the left side of the diencephalon, heart and LPM. This developmental step is essential to establish subsequent asymmetric organ positioning. In this study, we evaluated the role of krüppel-like factor 8 (klf8) in regulating left-right asymmetric patterning in zebrafish embryos. Zebrafish klf8 expression was disrupted by both morpholino antisense oligomer-mediated knockdown and a CRISPR-Cas9 system. Whole-mount in situ hybridization was conducted to evaluate gene expression patterns of Nodal signalling components and the positions of heart and visceral organs. Dorsal forerunner cell number was evaluated in Tg(sox17:gfp) embryos and the length and number of cilia in Kupffer's vesicle were analyzed by immunocytochemistry using an acetylated tubulin antibody. Heart jogging, looping and visceral organ positioning were all defective in zebrafish klf8 morphants. At the 18-22 s stages, klf8 morphants showed reduced expression of genes encoding Nodal signalling components (spaw, lft1, lft2, and pitx2) in the left LPM, diencephalon, and heart. Co-injection of klf8 mRNA with klf8 morpholino partially rescued spaw expression. Furthermore, klf8 but not klf8△zf overexpressing embryos showed dysregulated bilateral expression of Nodal signalling components at late somite stages. At the 10s stage, klf8 morphants exhibited reductions in length and number of cilia in Kupffer's vesicle, while at 75% epiboly, fewer dorsal forerunner cells were observed

Exploring Kupffer's Vescicle Through Self Propelled Particle Simulations

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Lundy, Kassidy; Dasgupta, Agnik; Amack, Jeff; Manning, M. Lisa

Early development is an important stage in the formation of functional, relatively healthy organisms. In zebrafish embryos, a transient organ in the tailbud called Kupffer's Vescicle (KV) is responsible for the initial left-right (L-R) asymmetry that results in asymmetric organ and tissue placement in the adult zebrafish. Originating as a collection of symmetrically organized monociliated cells, the KV experiences a shift in cell shapes over time that leaves more cells on the anterior or top side of the KV. This arrangement helps to generate a stronger counter-clockwise fluid flow across the anterior side of the organ, which is required for L-R asymmetry. In seeking to understand the source of the shape changes occurring within the KV, we simulate a Self Propelled Particle (SPP) model that includes parameters for cell polarization and speed. We model the KV as a large particle moving in a straight line with constant velocity to mimic the physical forces of the notochord acting on this organ, and we model the surrounding tailbud cells as smaller, slower active particles with an orientation that changes over time due to rotational noise. Our goal is to calculate the forces exerted on the KV by the surrounding tissue, to see if they are sufficient to explain the shape changes we observe in the KV that lead to L-R asymmetry.

Long live the liver: immunohistochemical and stereological study of hepatocytes, liver sinusoidal endothelial cells, Kupffer cells and hepatic stellate cells of male and female rats throughout ageing.

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Marcos, Ricardo; Correia-Gomes, Carla

2016-12-01

Male/female differences in enzyme activity and gene expression in the liver are known to be attenuated with ageing. Nevertheless, the effect of ageing on liver structure and quantitative cell morphology remains unknown. Male and female Wistar rats aged 2, 6, 12 and 18 months were examined by means of stereological techniques and immunohistochemical tagging of hepatocytes (HEP), liver sinusoidal endothelial cells (LSEC), Kupffer cells (KC) and hepatic stellate cells (HSC) in order to assess the total number and number per gram of these cells throughout life. The mean cell volume of HEP and HSC, the lobular position and the collagen content of the liver were also evaluated with stereological techniques. The number per gram of HSC was similar for both genders and was maintained throughout ageing. The mean volume of HSC was also conserved but differences in the cell body and lobular location were observed. Statistically significant gender differences in HEP were noted in young rats (females had smaller and more binucleated HEP) but were attenuated with ageing. The same occurred for KC and LSEC, since the higher number per gram in young females disappeared in older animals. Liver collagen increased with ageing but only in males. Thus, the numbers of these four cell types are related throughout ageing, with well-defined cell ratios. The shape and lobular position of HSC change with ageing in both males and females. Gender dimorphism in HEP, KC and LSEC of young rat liver disappears with ageing.

Re-evaluation of thin layer chromatography as an alternative method for the quantification of prostaglandins from rat Kupffer cells.

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Pestel, Sabine; Jungermann, Kurt; Schieferdecker, Henrike L

2005-01-01

In contrast to conventionally used immunoassays, thin layer chromatography (TLC)--by prelabeling of cells with radioactive arachidonic acid (AA)--allows to differentiate between cellularly built and added prostanoids and thus to investigate feedback effects of prostanoids on their own release. PGD2, TXB2 and PGE2 released from zymosan-stimulated Kupffer cells were separated with distinct RF-values, corresponding to those of the pure substances. Quantification of PGD2 and PGE2 gave comparable results with TLC and immunoassays, but measurement in the presence of added prostanoids was only possible with TLC. Moreover TLC was superior to immunoassays in having a longer linear range while being comparably sensitive. Cellularly built TXB2 in its radioactively labeled form was not detectable by TLC. Inhibition of TXB2 release by externally added AA or technical artifacts were excluded, suggesting that the cellular AA-pools used for prostaglandin and thromboxane synthesis differ in their accessibility for added AA. Thus, TLC is a simple, sensitive and precise method for the quantification of cellularly built prostaglandins but not of thromboxane even in the presence of added prostanoids.

beta-oxidation modulates metabolic competition between eicosapentaenoic acid and arachidonic acid regulating prostaglandin E(2) synthesis in rat hepatocytes-Kupffer cells

DEFF Research Database (Denmark)

Du, Zhen-Yu; Ma, Tao; Winterthun, Synnøve

2010-01-01

and eicosapentaenoic acid (EPA) for PGE(2) synthesis in a rat hepatocyte-Kupffer cell (HPC/KC) co-culture system when the cellular oxidation capacity was enhanced by exogenous l-carnitine. We demonstrate that in the absence of l-carnitine, 1) beta-oxidation rates of EPA and AA were comparable in HPCs and in KCs; 2) AA...... and not EPA was preferentially incorporated into glycerolipids; and 3) addition of EPA significantly decreased AA-dependent PGE(2) synthesis in HPCs and cyclooxygenase-2 (COX-2) expression in co-cultured HPCs/KCs. However, enhancing the cellular oxidation capacity by the addition of l-carnitine 1...... inhibition of AA-dependent PGE(2) synthesis and COX-2 expression by EPA. Taken together, the results strongly suggest that l-carnitine affects competition between AA and EPA in PG synthesis in liver cells by enhancing oxidation of EPA in HPCs. This implies that the beneficial effects of n-3 PUFA, especially...

Transcriptome atlas of eight liver cell types uncovers effects of ...

Indian Academy of Sciences (India)

... types, and bioinformatic and systems biology approaches were employed to analyse the relationship between above genes and rat liver regeneration. The results showed that the urocanic acid (UA) was degraded from histidine in Kupffer cells, acts on Kupffer cells itself and dendritic cells to generate immune suppression ...

Curative Effects of Thiacremonone against Acetaminophen-Induced Acute Hepatic Failure via Inhibition of Proinflammatory Cytokines Production and Infiltration of Cytotoxic Immune Cells and Kupffer Cells

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Yu Ri Kim

2013-01-01

Full Text Available High doses of acetaminophen (APAP; N-acetyl-p-aminophenol cause severe hepatotoxicity after metabolic activation by cytochrome P450 2E1. This study was undertaken to examine the preventive effects of thiacremonone, a compound extracted from garlic, on APAP-induced acute hepatic failure in male C57BL/6J. Mice received with 500 mg/kg APAP after a 7-day pretreatment with thiacremonone (10–50 mg/kg. Thiacremonone inhibited the APAP-induced serum ALT and AST levels in a dose-dependent manner, and markedly reduced the restricted area of necrosis and inflammation by administration of APAP. Thiacremonone also inhibited the APAP-induced depletion of intracellular GSH, induction of nitric oxide, and lipid peroxidation as well as expression of P450 2E1. After APAP injection, the numbers of Kupffer cells, natural killer cells, and cytotoxic T cells were elevated, but the elevated cell numbers in the liver were reduced in thiacremonone pretreated mice. The expression levels of I-309, M-CSF, MIG, MIP-1α, MIP-1β, IL-7, and IL-17 were increased by APAP treatment, which were inhibited in thiacremonone pretreated mice. These data indicate that thiacremonone could be a useful agent for the treatment of drug-induced hepatic failure and that the reduction of cytotoxic immune cells as well as proinflammatory cytokine production may be critical for the prevention of APAP-induced acute liver toxicity.

Kupffer cell depletion attenuates leptin-mediated methoxamine-stimulated portal perfusion pressure and thromboxane A2 release in a rodent model of NASH-cirrhosis.

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Yang, Ying-Ying; Huang, Yi-Tsau; Tsai, Tung-Hu; Hou, Ming-Chih; Lee, Fa-Yauh; Lee, Shou-Dong; Lin, Han-Chieh

2012-12-01

Cirrhotic portal hypertension is characterized by increased hepatic oxidative stress, AA (arachidonic acid)-derived TXA(2) (thromboxane A(2)) release and exaggerated hepatic response to the α-adrenergic agonist MTX (methoxamine). Besides promoting hepatic fibrosis, the role of hyperleptinaemia in the modulation of vascular response in NASH (non-alcoholic steatohepatitis) rat livers remains unknown. The aim of the present study was to explore the possible links between hyperleptinaemia and the disarrangement in the hepatic microcirculation. NASH-cirrhosis with hyperleptinaemia was induced in lean rats by feeding with an HF/MCD (high-fat/methionine-choline-deficient) diet. Portal haemodynamics, various substances, protein and mRNA expression and PUFA (polyunsaturated fatty acid) composition were measured. Finally, the effects of leptin pre-infusion on TXA(2) release and concentration-PPP (portal perfusion pressure) curves in response to MTX were evaluated by simultaneously pre-treatment with the Kupffer cell inactivators GdCl(3) (gadolinium chloride) or EC (encapsulated clodronate), the TXS (TXA(2) synthase) inhibitor furegrelate, the TP receptor (TXA(2) receptor) antagonist SQ29548 and the dual TXS/TP receptor antagonist BM567. In HF/MCD+leptin-lean rats, cirrhosis-induced PPP and MTX hyper-responsiveness were associated with increased hepatic TXA(2) production, TBARS (thiobarbituric acid-reacting substances) levels and the AA (arachidonic acid)/n-3 PUFA ratio, and up-regulation of hepatic leptin, FAS (fatty acid synthase), NADPH oxidase subunits, TXS, TP receptor, TGFβ(1) (transforming growth factor β(1)) proteins and mRNAs. Pre-infusion of leptin significantly enhanced MTX-stimulated PPP elevation and TXA(2) release, which were attenuated by GdCl(3) and EC pre-treatment. Concomitantly pre-incubation with BM567, but not furegrelate or SQ29548, significantly abolished the leptin-enhanced MTX-stimulated increase in PPP in NASH-cirrhotic rats. Hyperleptinaemia

A transgenic rat hepatocyte - Kupffer cell co-culture model for evaluation of direct and macrophage-related effect of poly(amidoamine) dendrimers.

Science.gov (United States)

Jemnitz, Katalin; Bátai-Konczos, Attila; Szabó, Mónika; Ioja, Enikő; Kolacsek, Orsolya; Orbán, Tamás I; Török, György; Homolya, László; Kovács, Eszter; Jablonkai, István; Veres, Zsuzsa

2017-02-01

Increasing number of papers demonstrate that Kupffer cells (KCs) play a role in the development of drug induced liver injury (DILI). Furthermore, elevated intracellular Ca 2+ level of hepatocytes is considered as a common marker of DILI. Here we applied an in vitro model based on hepatocyte mono- and hepatocyte/KC co-cultures (H/KC) isolated from transgenic rats stably expressing the GCaMP2 fluorescent Ca 2+ sensor protein to investigate the effects of polycationic (G5), polyanionic (G4.5) and polyethylene-glycol coated neutral (G5 Peg) dendrimers known to accumulate in the liver, primarily in KCs. Following dendrimer exposure, hepatocyte homeostasis was measured by MTT cytotoxicity assay and by Ca 2+ imaging, while hepatocyte functions were studied by CYP2B1/2 inducibility, and bilirubin and taurocholate transport. G5 was significantly more cytotoxic than G4.5 for hepatocytes and induced Ca 2+ oscillation and sustained Ca 2+ signals at 1μM and10 μM, respectively both in hepatocytes and KCs. Dendrimer-induced Ca 2+ signals in hepatocytes were attenuated by macrophages. Activation of KCs by lipopolysaccharide and G5 decreased the inducibility of CYP2B1/2, which was restored by depleting the KCs with gadolinium-chloride and pentoxyphylline, suggesting a role of macrophages in the hindrance of CYP2B1/2 induction by G5 and lipopolysaccharide. In the H/KC, but not in the hepatocyte mono-culture, G5 reduced the canalicular efflux of bilirubin and stimulated the uptake and canalicular efflux of taurocholate. In conclusion, H/KC provides a good model for the prediction of hepatotoxic potential of drugs, especially of nanomaterials known to be trapped by macrophages, activation of which presumably contributes to DILI. Copyright © 2016 Elsevier B.V. All rights reserved.

Trapping of oxidized LDL in lysosomes of Kupffer cells is a trigger for hepatic inflammation.

Science.gov (United States)

Bieghs, Veerle; Walenbergh, Sofie M A; Hendrikx, Tim; van Gorp, Patrick J; Verheyen, Fons; Olde Damink, Steven W; Masclee, Ad A; Koek, Ger H; Hofker, Marten H; Binder, Christoph J; Shiri-Sverdlov, Ronit

2013-08-01

Non-alcoholic steatohepatitis (NASH) is characterized by steatosis and inflammation. The transition from steatosis towards NASH represents a key step in pathogenesis, as it will set the stage for further severe liver damage. Under normal conditions, lipoproteins that are endocytosed by Kupffer cells (KCs) are easily transferred from the lysosomes into the cytoplasm. Oxidized LDL (oxLDL) that is taken up by the macrophages in vitro is trapped within the lysosomes, while acetylated LDL (acLDL) is leading to normal lysosomal hydrolysis, resulting in cytoplasmic storage. We have recently demonstrated that hepatic inflammation is correlated with lysosomal trapping of lipids. So far, a link between lysosomal trapping of oxLDL and inflammation was not established. We hypothesized that lysosomal trapping of oxLDL in KCs will lead to hepatic inflammation. Ldlr(-/-) mice were injected with LDL, acLDL and oxLDL and sacrificed after 2, 6 and 24 h. Electron microscopy of KCs demonstrated that after oxLDL injection, small lipid inclusions were present inside the lysosomes after all time points and were mostly pronounced after 6 and 24 h. In contrast, no lipid inclusions were present inside KCs after LDL or acLDL injection. Hepatic expression of several inflammatory genes and scavenger receptors was higher after oxLDL injections compared with LDL or acLDL. These data suggest that trapping of oxLDL inside lysosomes of KCs in vivo is causally linked to increased hepatic inflammatory gene expression. Our novel observations provide new bases for prevention and treatment of NASH. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

The requirement for freshly isolated human colorectal cancer (CRC) cells in isolating CRC stem cells.

Science.gov (United States)

Fan, F; Bellister, S; Lu, J; Ye, X; Boulbes, D R; Tozzi, F; Sceusi, E; Kopetz, S; Tian, F; Xia, L; Zhou, Y; Bhattacharya, R; Ellis, L M

2015-02-03

Isolation of colorectal cancer (CRC) cell populations enriched for cancer stem cells (CSCs) may facilitate target identification. There is no consensus regarding the best methods for isolating CRC stem cells (CRC-SCs). We determined the suitability of various cellular models and various stem cell markers for the isolation of CRC-SCs. Established human CRC cell lines, established CRC cell lines passaged through mice, patient-derived xenograft (PDX)-derived cells, early passage/newly established cell lines, and cells directly from clinical specimens were studied. Cells were FAC-sorted for the CRC-SC markers CD44, CD133, and aldehyde dehydrogenase (ALDH). Sphere formation and in vivo tumorigenicity studies were used to validate CRC-SC enrichment. None of the markers studied in established cell lines, grown either in vitro or in vivo, consistently enriched for CRC-SCs. In the three other cellular models, CD44 and CD133 did not reliably enrich for stemness. In contrast, freshly isolated PDX-derived cells or early passage/newly established CRC cell lines with high ALDH activity formed spheres in vitro and enhanced tumorigenicity in vivo, whereas cells with low ALDH activity did not. PDX-derived cells, early passages/newly established CRC cell lines and cells from clinical specimen with high ALDH activity can be used to identify CRC-SC-enriched populations. Established CRC cell lines should not be used to isolate CSCs.

Nitric oxide inhibits glycogen synthesis in isolated rat hepatocytes

NARCIS (Netherlands)

Sprangers, F.; Sauerwein, H. P.; Romijn, J. A.; van Woerkom, G. M.; Meijer, A. J.

1998-01-01

There is increasing evidence for the existence of intrahepatic regulation of glucose metabolism by Kupffer cell products. Nitric oxide (NO) is known to inhibit gluconeogenic flux through pyruvate carboxylase and phosphoenolpyruvate carboxykinase. However, NO may also influence glucose metabolism at

Kupffer cells activation promoted binge drinking-induced fatty liver by activating lipolysis in white adipose tissues.

Science.gov (United States)

Zhao, Yu-Ying; Yang, Rui; Xiao, Mo; Guan, Min-Jie; Zhao, Ning; Zeng, Tao

2017-09-01

Kupffer cells (KCs) have been suggested to play critical roles in chronic ethanol induced early liver injury, but the role of KCs in binge drinking-induced hepatic steatosis remains unclear. This study was designed to investigate the roles of KCs inhibitor (GdCl 3 ) and TNF-α antagonist (etanercept) on binge drinking-induced liver steatosis and to explore the underlying mechanisms. C57BL/6 mice were exposed to three doses of ethanol (6g/kg body weight) to mimic binge drinking-induced fatty liver. The results showed that both GdCl 3 and etanercept partially but significantly alleviated binge drinking-induced increase of hepatic triglyceride (TG) level, and reduced fat droplets accumulation in mice liver. GdCl 3 but not etanercept significantly blocked binge drinking-induced activation of KCs. However, neither GdCl 3 nor etanercept could affect binge drinking-induced decrease of PPAR-α, ACOX, FAS, ACC and SCD protein levels, or increase of the LC3 II/LC3 I ratio and p62 protein level. Interestingly, both GdCl 3 and etanercept significantly suppressed binge drinking-induced phosphorylation of HSL in epididymal adipose tissues. Results of in vitro studies with cultured epididymal adipose tissues showed that TNF-α could increase the phosphorylation of HSL in adipose tissues and upgrade the secretion of free fatty acid (FFA) in the culture medium. Taken together, KCs inhibitor and TNF-α antagonist could partially attenuate binge drinking-induced liver steatosis, which might be attributed to the suppression of mobilization of white adipose tissues. These results suggest that KCs activation may promote binge drinking-induced fatty liver by TNF-α mediated activation of lipolysis in white adipose tissues. Copyright © 2017 Elsevier B.V. All rights reserved.

Claudin5a is required for proper inflation of Kupffer's vesicle lumen and organ laterality.

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Jeong-Gyun Kim

Full Text Available Left-right asymmetric organ development is critical to establish a proper body plan of vertebrates. In zebrafish, the Kupffer's vesicle (KV is a fluid-filled sac which controls asymmetric organ development, and a properly inflated KV lumen by means of fluid influx is a prerequisite for the asymmetric signal transmission. However, little is known about the components that support the paracellular tightness between the KV luminal epithelial cells to sustain hydrostatic pressure during KV lumen expansion. Here, we identified that the claudin5a (cldn5a is highly expressed at the apical surface of KV epithelial cells and tightly seals the KV lumen. Downregulation of cldn5a in zebrafish showed a failure in organ laterality that resulted from malformed KV. In addition, accelerated fluid influx into KV by combined treatment of forskolin and 3-isobutyl-1-methylxanthine failed to expand the partially-formed KV lumen in cldn5a morphants. However, malformed KV lumen and defective heart laterality in cldn5a morphants were significantly rescued by exogenous cldn5a mRNA, suggesting that the tightness between the luminal epithelial cells is important for KV lumen formation. Taken together, these findings suggest that cldn5a is required for KV lumen inflation and left-right asymmetric organ development.

Claudin5a is required for proper inflation of Kupffer's vesicle lumen and organ laterality.

Science.gov (United States)

Kim, Jeong-Gyun; Bae, Sung-Jin; Lee, Hye Shin; Park, Ji-Hyeon; Kim, Kyu-Won

2017-01-01

Left-right asymmetric organ development is critical to establish a proper body plan of vertebrates. In zebrafish, the Kupffer's vesicle (KV) is a fluid-filled sac which controls asymmetric organ development, and a properly inflated KV lumen by means of fluid influx is a prerequisite for the asymmetric signal transmission. However, little is known about the components that support the paracellular tightness between the KV luminal epithelial cells to sustain hydrostatic pressure during KV lumen expansion. Here, we identified that the claudin5a (cldn5a) is highly expressed at the apical surface of KV epithelial cells and tightly seals the KV lumen. Downregulation of cldn5a in zebrafish showed a failure in organ laterality that resulted from malformed KV. In addition, accelerated fluid influx into KV by combined treatment of forskolin and 3-isobutyl-1-methylxanthine failed to expand the partially-formed KV lumen in cldn5a morphants. However, malformed KV lumen and defective heart laterality in cldn5a morphants were significantly rescued by exogenous cldn5a mRNA, suggesting that the tightness between the luminal epithelial cells is important for KV lumen formation. Taken together, these findings suggest that cldn5a is required for KV lumen inflation and left-right asymmetric organ development.

Chronic Ethanol Feeding Modulates Inflammatory Mediators, Activation of Nuclear Factor-κB, and Responsiveness to Endotoxin in Murine Kupffer Cells and Circulating Leukocytes

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Miriam Maraslioglu

2014-01-01

Full Text Available Chronic ethanol abuse is known to increase susceptibility to infections after injury, in part, by modification of macrophage function. Several intracellular signalling mechanisms are involved in the initiation of inflammatory responses, including the nuclear factor-κB (NF-κB pathway. In this study, we investigated the systemic and hepatic effect of chronic ethanol feeding on in vivo activation of NF-κB in NF-κBEGFP reporter gene mice. Specifically, the study focused on Kupffer cell proinflammatory cytokines IL-6 and TNF-α and activation of NF-κB after chronic ethanol feeding followed by in vitro stimulation with lipopolysaccharide (LPS. We found that chronic ethanol upregulated NF-κB activation and increased hepatic and systemic proinflammatory cytokine levels. Similarly, LPS-stimulated IL-1β release from whole blood was significantly enhanced in ethanol-fed mice. However, LPS significantly increased IL-6 and TNF-α levels. These results demonstrate that chronic ethanol feeding can improve the responsiveness of macrophage LPS-stimulated IL-6 and TNF-α production and indicate that this effect may result from ethanol-induced alterations in intracellular signalling through NF-κB. Furthermore, LPS and TNF-α stimulated the gene expression of different inflammatory mediators, in part, in a NF-κB-dependent manner.

Loss of lysosomal membrane protein NCU-G1 in mice results in spontaneous liver fibrosis with accumulation of lipofuscin and iron in Kupffer cells

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Xiang Y. Kong

2014-03-01

Full Text Available Human kidney predominant protein, NCU-G1, is a highly conserved protein with an unknown biological function. Initially described as a nuclear protein, it was later shown to be a bona fide lysosomal integral membrane protein. To gain insight into the physiological function of NCU-G1, mice with no detectable expression of this gene were created using a gene-trap strategy, and Ncu-g1gt/gt mice were successfully characterized. Lysosomal disorders are mainly caused by lack of or malfunctioning of proteins in the endosomal-lysosomal pathway. The clinical symptoms vary, but often include liver dysfunction. Persistent liver damage activates fibrogenesis and, if unremedied, eventually leads to liver fibrosis/cirrhosis and death. We demonstrate that the disruption of Ncu-g1 results in spontaneous liver fibrosis in mice as the predominant phenotype. Evidence for an increased rate of hepatic cell death, oxidative stress and active fibrogenesis were detected in Ncu-g1gt/gt liver. In addition to collagen deposition, microscopic examination of liver sections revealed accumulation of autofluorescent lipofuscin and iron in Ncu-g1gt/gt Kupffer cells. Because only a few transgenic mouse models have been identified with chronic liver injury and spontaneous liver fibrosis development, we propose that the Ncu-g1gt/gt mouse could be a valuable new tool in the development of novel treatments for the attenuation of fibrosis due to chronic liver damage.

Isolation and functional aspects of free luteal cells

International Nuclear Information System (INIS)

Luborsky, J.L.; Berhrman, H.R.

1985-01-01

Methods of luteal cell isolation employ enzymatic treatment of luteal tissue with collagenase and deoxyribonuclease. Additional enzymes such as hyaluronidase or Pronase are also used in some instances. Isolated luteal cells retain the morphological characteristics of steroid secreting cells after isolation. They contain mitochondria, variable amounts of lipid droplets, and an extensive smooth endoplasmic reticulum. Isolated luteal cells have been used in numerous studies to examine the regulation of steriodogenesis by luteinizing hormone (LH). LH receptor binding studies were employed to quantitate specific properties of hormone-receptor interaction in relation to cellular function. Binding of [ 125 I]LH to bovine luteal cells and membranes was compared and it was concluded that the enzymatic treatment used to isolate cells did not change the LH receptor binding kinetics

Isolation and culture of larval cells from C. elegans.

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Sihui Zhang

Full Text Available Cell culture is an essential tool to study cell function. In C. elegans the ability to isolate and culture cells has been limited to embryonically derived cells. However, cells or blastomeres isolated from mixed stage embryos terminally differentiate within 24 hours of culture, thus precluding post-embryonic stage cell culture. We have developed an efficient and technically simple method for large-scale isolation and primary culture of larval-stage cells. We have optimized the treatment to maximize cell number and minimize cell death for each of the four larval stages. We obtained up to 7.8×10(4 cells per microliter of packed larvae, and up to 97% of adherent cells isolated by this method were viable for at least 16 hours. Cultured larval cells showed stage-specific increases in both cell size and multinuclearity and expressed lineage- and cell type-specific reporters. The majority (81% of larval cells isolated by our method were muscle cells that exhibited stage-specific phenotypes. L1 muscle cells developed 1 to 2 wide cytoplasmic processes, while L4 muscle cells developed 4 to 14 processes of various thicknesses. L4 muscle cells developed bands of myosin heavy chain A thick filaments at the cell center and spontaneously contracted ex vivo. Neurons constituted less than 10% of the isolated cells and the majority of neurons developed one or more long, microtubule-rich protrusions that terminated in actin-rich growth cones. In addition to cells such as muscle and neuron that are high abundance in vivo, we were also able to isolate M-lineage cells that constitute less than 0.2% of cells in vivo. Our novel method of cell isolation extends C. elegans cell culture to larval developmental stages, and allows use of the wealth of cell culture tools, such as cell sorting, electrophysiology, co-culture, and high-resolution imaging of subcellular dynamics, in investigation of post-embryonic development and physiology.

The isolated anatase for dye sensitized solar cell

Energy Technology Data Exchange (ETDEWEB)

Ilmi, Irfan, E-mail: irfan.ilmi149@gmail.com [Postgraduate Program, Department of Chemistry, Universitas Gadjah Mada, Yogyakarta 55281 Indonesia (Indonesia); Functional Coating Materials Research Group, Department of Chemistry, Universitas Gadjah Mada, Yogyakarta 55281 Indonesia (Indonesia); Kartin, Indriana; Suyanta [Functional Coating Materials Research Group, Department of Chemistry, Universitas Gadjah Mada, Yogyakarta 55281 Indonesia (Indonesia); Department of Chemistry,Universitas Gadjah Mada, Yogyakarta 55281 Indonesia (Indonesia); Ohtani, Bunsho; Wang, Kunlei [Graduate School of Environmental and Earth Science, Hokkaido University Japan (Japan)

2015-09-30

The isolation of crystallite anatase from commercial TiO{sub 2} P25 Degussa was investigated. The aim of this research was to study of isolated anatase based DSSC as an effort to develop industrial DSSC. The crystal phase, crystallite size and crystal shape both of original P25 and isolated anatase were characterized by XRD and TEM. By observing DSSC parameters such as FF, Jsc and Voc resulted in cell test, the efficiency of samples based DSSC was known. The isolation of anatase crystal was done by dissolving P25 in ammonia catalyzed hydrogen peroxide solution for 15 hours followed by washing and drying. DSSC cell performance was evaluated by applying the isolated anantase and original P25 as photoanode in the Gratzel cell system. The observation of cell efficiency was measured under 100 mW /cm{sup 2} with active area 1.5 cm{sup 2}. X-ray diffraction pattern showed obviously that no rutile contaminant in produced isolated anatase. TEM image shows typical anatase crystal with the particle size 21 nm. Surface area measurement exhibits that surface area of isolated anatase was 64.7m{sup 2}/g. I-V measurement showed that the efficiency of anatase based cell and P25 based cell is 0.79% and 0.51% respectively.

The characterization and metabolism of rat hepatic nascent HLD subfractions

International Nuclear Information System (INIS)

Winkler, K.E.

1988-01-01

Nascent HDL was isolated from recirculating rat liver perfusates and separated by heparin-sepharose chromatography into a non-retained fraction (A) and a fraction (B) that eluted with 0.5 M NaCl. Fractions A and B contained 70% and 30% of the nascent HDL protein, respectively. Livers perfused by the single-pass technique produced fractions A and B in the same ratio as livers perfused by recirculation. The apolipoprotein compositions were similar to those in the recirculating perfusion; however, both fractions A and B had more triglyceride (greater than 50% of total lipid). In a preliminary study designed to investigate whether nascent HDL-apo E was secreted by Kupffer cells or hepatocytes, label was targeted to Kupffer cells by perfusing rat livers with 3 H-acetylated LDL or 3 H-amino acids incorporated into large multilamellar vesicles. For metabolic studies, nascent HDL and nascent VLDL were isolated from rat livers that had been perfused with 3 H-glycerol to label the triglyceride

Technologies for Single-Cell Isolation

Science.gov (United States)

Gross, Andre; Schoendube, Jonas; Zimmermann, Stefan; Steeb, Maximilian; Zengerle, Roland; Koltay, Peter

2015-01-01

The handling of single cells is of great importance in applications such as cell line development or single-cell analysis, e.g., for cancer research or for emerging diagnostic methods. This review provides an overview of technologies that are currently used or in development to isolate single cells for subsequent single-cell analysis. Data from a dedicated online market survey conducted to identify the most relevant technologies, presented here for the first time, shows that FACS (fluorescence activated cell sorting) respectively Flow cytometry (33% usage), laser microdissection (17%), manual cell picking (17%), random seeding/dilution (15%), and microfluidics/lab-on-a-chip devices (12%) are currently the most frequently used technologies. These most prominent technologies are described in detail and key performance factors are discussed. The survey data indicates a further increasing interest in single-cell isolation tools for the coming years. Additionally, a worldwide patent search was performed to screen for emerging technologies that might become relevant in the future. In total 179 patents were found, out of which 25 were evaluated by screening the title and abstract to be relevant to the field. PMID:26213926

Technologies for Single-Cell Isolation

Directory of Open Access Journals (Sweden)

Andre Gross

2015-07-01

Full Text Available The handling of single cells is of great importance in applications such as cell line development or single-cell analysis, e.g., for cancer research or for emerging diagnostic methods. This review provides an overview of technologies that are currently used or in development to isolate single cells for subsequent single-cell analysis. Data from a dedicated online market survey conducted to identify the most relevant technologies, presented here for the first time, shows that FACS (fluorescence activated cell sorting respectively Flow cytometry (33% usage, laser microdissection (17%, manual cell picking (17%, random seeding/dilution (15%, and microfluidics/lab-on-a-chip devices (12% are currently the most frequently used technologies. These most prominent technologies are described in detail and key performance factors are discussed. The survey data indicates a further increasing interest in single-cell isolation tools for the coming years. Additionally, a worldwide patent search was performed to screen for emerging technologies that might become relevant in the future. In total 179 patents were found, out of which 25 were evaluated by screening the title and abstract to be relevant to the field.

Technologies for Single-Cell Isolation.

Science.gov (United States)

Gross, Andre; Schoendube, Jonas; Zimmermann, Stefan; Steeb, Maximilian; Zengerle, Roland; Koltay, Peter

2015-07-24

The handling of single cells is of great importance in applications such as cell line development or single-cell analysis, e.g., for cancer research or for emerging diagnostic methods. This review provides an overview of technologies that are currently used or in development to isolate single cells for subsequent single-cell analysis. Data from a dedicated online market survey conducted to identify the most relevant technologies, presented here for the first time, shows that FACS (fluorescence activated cell sorting) respectively Flow cytometry (33% usage), laser microdissection (17%), manual cell picking (17%), random seeding/dilution (15%), and microfluidics/lab-on-a-chip devices (12%) are currently the most frequently used technologies. These most prominent technologies are described in detail and key performance factors are discussed. The survey data indicates a further increasing interest in single-cell isolation tools for the coming years. Additionally, a worldwide patent search was performed to screen for emerging technologies that might become relevant in the future. In total 179 patents were found, out of which 25 were evaluated by screening the title and abstract to be relevant to the field.

Proinflammatory adipokine leptin mediates disinfection byproduct bromodichloromethane-induced early steatohepatitic injury in obesity

International Nuclear Information System (INIS)

Das, Suvarthi; Kumar, Ashutosh; Seth, Ratanesh Kumar; Tokar, Erik J.; Kadiiska, Maria B.; Waalkes, Michael P.; Mason, Ronald P.; Chatterjee, Saurabh

2013-01-01

Today's developed world faces a major public health challenge in the rise in the obese population and the increased incidence in fatty liver disease. There is a strong association among diet induced obesity, fatty liver disease and development of nonalcoholic steatohepatitis but the environmental link to disease progression remains unclear. Here we demonstrate that in obesity, early steatohepatitic lesions induced by the water disinfection byproduct bromodichloromethane are mediated by increased oxidative stress and leptin which act in synchrony to potentiate disease progression. Low acute exposure to bromodichloromethane (BDCM), in diet-induced obesity produced oxidative stress as shown by increased lipid peroxidation, protein free radical and nitrotyrosine formation and elevated leptin levels. Exposed obese mice showed histopathological signs of early steatohepatitic injury and necrosis. Spontaneous knockout mice for leptin or systemic leptin receptor knockout mice had significantly decreased oxidative stress and TNF-α levels. Co-incubation of leptin and BDCM caused Kupffer cell activation as shown by increased MCP-1 release and NADPH oxidase membrane assembly, a phenomenon that was decreased in Kupffer cells isolated from leptin receptor knockout mice. In obese mice that were BDCM-exposed, livers showed a significant increase in Kupffer cell activation marker CD68 and, increased necrosis as assessed by levels of isocitrate dehydrogenase, events that were decreased in the absence of leptin or its receptor. In conclusion, our results show that exposure to the disinfection byproduct BDCM in diet-induced obesity augments steatohepatitic injury by potentiating the effects of leptin on oxidative stress, Kupffer cell activation and cell death in the liver. - Highlights: ► BDCM acute exposure sensitizes liver to increased free radical stress in obesity. ► BDCM-induced higher leptin contributes to early steatohepatitic lesions. ► Increased leptin mediates protein

Proinflammatory adipokine leptin mediates disinfection byproduct bromodichloromethane-induced early steatohepatitic injury in obesity

Energy Technology Data Exchange (ETDEWEB)

Das, Suvarthi [Environmental Health and Disease Laboratory, Department of Environmental Health Sciences, Arnold School of Public Health, University of South Carolina, Columbia, SC 29208 (United States); Kumar, Ashutosh [Free Radical Metabolism Group, Laboratory of Toxicology and Pharmacology, Research Triangle Park, NC 27709 (United States); Seth, Ratanesh Kumar [Environmental Health and Disease Laboratory, Department of Environmental Health Sciences, Arnold School of Public Health, University of South Carolina, Columbia, SC 29208 (United States); Tokar, Erik J. [Inorganic Toxicology Group, National Toxicology Program Laboratory, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709 (United States); Kadiiska, Maria B. [Free Radical Metabolism Group, Laboratory of Toxicology and Pharmacology, Research Triangle Park, NC 27709 (United States); Waalkes, Michael P. [Inorganic Toxicology Group, National Toxicology Program Laboratory, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709 (United States); Mason, Ronald P. [Free Radical Metabolism Group, Laboratory of Toxicology and Pharmacology, Research Triangle Park, NC 27709 (United States); Chatterjee, Saurabh, E-mail: schatt@mailbox.sc.edu [Environmental Health and Disease Laboratory, Department of Environmental Health Sciences, Arnold School of Public Health, University of South Carolina, Columbia, SC 29208 (United States)

2013-06-15

Today's developed world faces a major public health challenge in the rise in the obese population and the increased incidence in fatty liver disease. There is a strong association among diet induced obesity, fatty liver disease and development of nonalcoholic steatohepatitis but the environmental link to disease progression remains unclear. Here we demonstrate that in obesity, early steatohepatitic lesions induced by the water disinfection byproduct bromodichloromethane are mediated by increased oxidative stress and leptin which act in synchrony to potentiate disease progression. Low acute exposure to bromodichloromethane (BDCM), in diet-induced obesity produced oxidative stress as shown by increased lipid peroxidation, protein free radical and nitrotyrosine formation and elevated leptin levels. Exposed obese mice showed histopathological signs of early steatohepatitic injury and necrosis. Spontaneous knockout mice for leptin or systemic leptin receptor knockout mice had significantly decreased oxidative stress and TNF-α levels. Co-incubation of leptin and BDCM caused Kupffer cell activation as shown by increased MCP-1 release and NADPH oxidase membrane assembly, a phenomenon that was decreased in Kupffer cells isolated from leptin receptor knockout mice. In obese mice that were BDCM-exposed, livers showed a significant increase in Kupffer cell activation marker CD68 and, increased necrosis as assessed by levels of isocitrate dehydrogenase, events that were decreased in the absence of leptin or its receptor. In conclusion, our results show that exposure to the disinfection byproduct BDCM in diet-induced obesity augments steatohepatitic injury by potentiating the effects of leptin on oxidative stress, Kupffer cell activation and cell death in the liver. - Highlights: ► BDCM acute exposure sensitizes liver to increased free radical stress in obesity. ► BDCM-induced higher leptin contributes to early steatohepatitic lesions. ► Increased leptin mediates

Propagation and isolation of ranaviruses in cell culture

DEFF Research Database (Denmark)

Ariel, Ellen; Nicolajsen, Nicole; Christophersen, Maj-Britt

2009-01-01

The optimal in vitro propagation procedure for a panel of ranavirus isolates and the best method for isolation of Epizootic haematopoietic necrosis virus (EHNV) from organ material in cell-culture were investigated. The panel of ranavirus isolates included: Frog virus 3 (FV3), Bohle iridovirus (BIV......), Pike-perch iridovirus (PPIV), European catfish virus (ECV), European sheatfish virus (ESV), EHNV, Doctor fish virus (DFV), Guppy virus 6 (GF6), short-finned eel virus (SERV) and Rana esculenta virus Italy 282/102 (REV 282/102). Each isolate was titrated in five cell lines: bluegill fry (BF-2......), epithelioma papulosum cyprini (EPC), chinook salmon embryo (CHSE-214) rainbow trout gonad (RTG-2) and fathead minnow (FHM), and incubated at 10, 15, 20, 24 and 28 °C for two weeks. BF-2, EPC and CHSE-214 cells performed well and titers obtained in the three cell lines were similar, whereas FHM and RTG-2 cells...

HIV-1 isolation from infected peripheral blood mononuclear cells.

Science.gov (United States)

Dispinseri, Stefania; Saba, Elisa; Vicenzi, Elisa; Kootstra, Neeltje A; Schuitemaker, Hanneke; Scarlatti, Gabriella

2014-01-01

Human immunodeficiency virus 1 (HIV-1) isolation from peripheral blood mononuclear cells (PBMCs) allows retrieval of replication-competent viral variants. In order to impose the smallest possible selective pressure on the viral isolates, isolation must be carried out in primary cultures of cells and not in tumor derived cell lines. The procedure involves culture of PBMCs from an infected patient with phytohemagglutinin (PHA)-stimulated PBMC from seronegative donors, which provide susceptible target cells for HIV replication. HIV can be isolated from the bulk population of PBMCs or after cloning of the cells to obtain viral biological clones. Viral production is determined with p24 antigen (Ag) detection assays or with reverse transcriptase (RT) activity assay. Once isolated, HIV-1 can be propagated by infecting PHA-stimulated PBMCs from healthy donors. Aliquots from culture with a high production of virus are stored for later use.

Isolation of Human Innate Lymphoid Cells.

Science.gov (United States)

Krabbendam, Lisette; Nagasawa, Maho; Spits, Hergen; Bal, Suzanne M

2018-06-29

Innate lymphoid cells (ILCs) are innate immune cells of lymphoid origin that have important effector and regulatory functions in the first line of defense against pathogens, but also regulate tissue homeostasis, remodeling, and repair. Their function mirrors T helper cells and cytotoxic CD8 + T lymphocytes, but they lack expression of rearranged antigen-specific receptors. Distinct ILC subsets are classified in group 1 ILCs (ILC1s), group 2 ILCs (ILC2s), and group 3 ILCs (ILC3s and lymphoid tissue-inducer cells), based on the expression of transcription factors and the cytokines they produce. As the frequency of ILCs is low, their isolation requires extensive depletion of other cell types. The lack of unique cell surface antigens further complicates the identification of these cells. Here, methods for ILC isolation and characterization from human peripheral blood and different tissues are described. © 2018 by John Wiley & Sons, Inc. © 2018 John Wiley & Sons, Inc.

HIV-1 isolation from infected peripheral blood mononuclear cells

NARCIS (Netherlands)

Dispinseri, Stefania; Saba, Elisa; Vicenzi, Elisa; Kootstra, Neeltje A.; Schuitemaker, Hanneke; Scarlatti, Gabriella

2014-01-01

Human immunodeficiency virus 1 (HIV-1) isolation from peripheral blood mononuclear cells (PBMCs) allows retrieval of replication-competent viral variants. In order to impose the smallest possible selective pressure on the viral isolates, isolation must be carried out in primary cultures of cells and

Isolation of plasmodesmata from Arabidopsis suspension culture cells.

Science.gov (United States)

Grison, Magali S; Fernandez-Calvino, Lourdes; Mongrand, Sébastien; Bayer, Emmanuelle M F

2015-01-01

Due to their position firmly anchored within the plant cell wall, plasmodesmata (PD) are notoriously difficult to isolate from plant tissue. Yet, getting access to isolated PD represents the most straightforward strategy for the identification of their molecular components. Proteomic and lipidomic analyses of such PD fractions have provided and will continue to provide critical information on the functional and structural elements that define these membranous nano-pores. Here, we describe a two-step simple purification procedure that allows isolation of pure PD-derived membranes from Arabidopsis suspension cells. The first step of this procedure consists in isolating cell wall fragments containing intact PD while free of contamination from other cellular compartments. The second step relies on an enzymatic degradation of the wall matrix and the subsequent release of "free" PD. Isolated PD membranes provide a suitable starting material for the analysis of PD-associated proteins and lipids.

CLEC4F is an inducible C-type lectin in F4/80-positive cells and is involved in alpha-galactosylceramide presentation in liver.

Directory of Open Access Journals (Sweden)

Chih-Ya Yang

Full Text Available CLEC4F, a member of C-type lectin, was first purified from rat liver extract with high binding affinity to fucose, galactose (Gal, N-acetylgalactosamine (GalNAc, and un-sialylated glucosphingolipids with GalNAc or Gal terminus. However, the biological functions of CLEC4F have not been elucidated. To address this question, we examined the expression and distribution of murine CLEC4F, determined its binding specificity by glycan array, and investigated its function using CLEC4F knockout (Clec4f-/- mice. We found that CLEC4F is a heavily glycosylated membrane protein co-expressed with F4/80 on Kupffer cells. In contrast to F4/80, CLEC4F is detectable in fetal livers at embryonic day 11.5 (E11.5 but not in yolk sac, suggesting the expression of CLEC4F is induced as cells migrate from yolk cells to the liver. Even though CLEC4F is not detectable in tissues outside liver, both residential Kupffer cells and infiltrating mononuclear cells surrounding liver abscesses are CLEC4F-positive upon Listeria monocytogenes (L. monocytogenes infection. While CLEC4F has strong binding to Gal and GalNAc, terminal fucosylation inhibits CLEC4F recognition to several glycans such as Fucosyl GM1, Globo H, Bb3∼4 and other fucosyl-glycans. Moreover, CLEC4F interacts with alpha-galactosylceramide (α-GalCer in a calcium-dependent manner and participates in the presentation of α-GalCer to natural killer T (NKT cells. This suggests that CLEC4F is a C-type lectin with diverse binding specificity expressed on residential Kupffer cells and infiltrating monocytes in the liver, and may play an important role to modulate glycolipids presentation on Kupffer cells.

Isolation of mesenchymal stem cells from equine umbilical cord blood

DEFF Research Database (Denmark)

Koch, Thomas Gadegaard; Heerkens, Tammy; Thomsen, Preben Dybdahl

2007-01-01

. The hypothesis of this study was that equine MSCs could be isolated from fresh whole equine cord blood. Results: Cord blood was collected from 7 foals immediately after foaling. The mononuclear cell fraction was isolated by Ficoll density centrifugation and cultured in a DMEM low glucose based media at 38.5o......Background: There are no published studies on stem cells from equine cord blood although commercial storage of equine cord blood for future autologous stem cell transplantations is available. Mesenchymal stem cells (MSC) have been isolated from fresh umbilical cord blood of humans collected non......-invasively at the time of birth and from sheep cord blood collected invasively by a surgical intrauterine approach. Mesenchymal stem cells isolation percentage from frozen-thawed human cord blood is low and the future isolation percentage of MSCs from cryopreserved equine cord blood is therefore expectedly low...

Susceptibility testing of fish cell lines for virus isolation

DEFF Research Database (Denmark)

Ariel, Ellen; Skall, Helle Frank; Olesen, Niels Jørgen

2009-01-01

and laboratories, but also between lineages of the same cell line. To minimise the occurrence of false negatives in a cell culture based surveillance system, we have investigated methods, to select cell lineages that are relatively superior in their susceptibility to a panel of virus isolates. The procedures...... cell lineages, we increased the number of isolates of each virus, propagated stocks in a given cell line and tested all lineages of that line in use in the laboratory. Testing of relative cell line susceptibility between laboratories is carried out annually via the Inter-laboratory Proficiency Test...... sensitivity for surveillance purposes within a cell line and between laboratories.In terms of economic and practical considerations as well as attempting to approach a realistic test system, we suggest the optimal procedure for susceptibility testing of fish cell lines for virus isolation to be a combination...

Isolation and Characterization of Poliovirus in Cell Culture Systems.

Science.gov (United States)

Thorley, Bruce R; Roberts, Jason A

2016-01-01

The isolation and characterization of enteroviruses by cell culture was accepted as the "gold standard" by clinical virology laboratories. Methods for the direct detection of all enteroviruses by reverse transcription polymerase chain reaction, targeting a conserved region of the genome, have largely supplanted cell culture as the principal diagnostic procedure. However, the World Health Organization's Global Polio Eradication Initiative continues to rely upon cell culture to isolate poliovirus due to the lack of a reliable sensitive genetic test for direct typing of enteroviruses from clinical specimens. Poliovirus is able to infect a wide range of mammalian cell lines, with CD155 identified as the primary human receptor for all three seroytpes, and virus replication leads to an observable cytopathic effect. Inoculation of cell lines with extracts of clinical specimens and subsequent passaging of the cells leads to an increased virus titre. Cultured isolates of poliovirus are suitable for testing by a variety of methods and remain viable for years when stored at low temperature.This chapter describes general procedures for establishing a cell bank and routine passaging of cell lines. While the sections on specimen preparation and virus isolation focus on poliovirus, the protocols are suitable for other enteroviruses.

Lysosomal and endosomal heterogeneity in the liver: A comparison of the intracellular pathways of endocytosis in rat liver cells

International Nuclear Information System (INIS)

Kindberg, G.M.; Tolleshaug, H.; Gjoen, T.; Berg, T.

1991-01-01

Air-filled albumin microspheres, asialoorosomucoid and formaldehyde-treated serum albumin are selectively taken up by endocytosis in rat liver Kupffer cells, parenchymal cells and endothelial cells, respectively. Intracellular transport and degradation of endocytosed material were studied by subcellular fractionation in sucrose and Nycodenz gradients after intravenous injection of the ligand. By using ligands labeled with 125I-tyramine-cellobiose, the subcellular distribution of labeled degradation products can be studied because they are trapped at the site of formation. The results show that the kinetics of intracellular transport are different in hepatic parenchymal, endothelial and Kupffer cells. In endothelial cells, the ligand is associated with two types of endosomes during the first minutes after internalization and then is transferred rapidly to the lysosomes. In parenchymal cells, 125I-tyramine-cellobiose-asialoorosomucoid was located in a relatively slowly sedimenting vesicle during the first minute after internalization and subsequently in denser endosomes. Degradation of 125I-tyramine-cellobiose-asialoorosomucoid in parenchymal cells started later than that of 125I-tyramine-cellobiose-formaldehyde-treated serum albumin in endothelial cells. Furthermore, the ligand seemed to be transferred relatively slowly from endosomes to lysosomes, and most of the undegraded ligand was in the endosomes. The rate-limiting step of proteolysis in parenchymal cells is probably the transport from endosomes to lysosomes. In Kupffer cells, most 125I-tyramine-cellobiose-microspheres are found as undegraded material in very dense endosomes up to 3 hr after injection. After 20 hr, most of the ligand is degraded in lysosomes distributed at a lower density than the endosomes in Nycodenz and sucrose gradients

Cell manipulation tool with combined microwell array and optical tweezers for cell isolation and deposition

International Nuclear Information System (INIS)

Wang, Xiaolin; Gou, Xue; Chen, Shuxun; Yan, Xiao; Sun, Dong

2013-01-01

Isolation from rare cells and deposition of sorted cells with high accuracy for further study are critical to a wide range of biomedical applications. In the current paper, we report an automated cell manipulation tool with combined optical tweezers and a uniquely designed microwell array, which functions for recognition, isolation, assembly, transportation and deposition of the interesting cells. The microwell array allows the passive hydrodynamic docking of cells, while offering the opportunity to inspect the interesting cell phenotypes with high spatio-temporal resolution based on the flexible image processing technique. In addition, dynamic and parallel cell manipulation in three dimensions can realize the target cell levitation from microwell and pattern assembly with multiple optical traps. Integrated with the programmed motorized stage, the optically levitated and assembled cells can be transported and deposited to the predefined microenvironment, so the tool can facilitate the integration of other on-chip functionalities for further study without removing these isolated cells from the chip. Experiments on human embryonic stem cells and yeast cells are performed to demonstrate the effectiveness of the proposed cell manipulation tool. Besides the application to cell isolation and deposition, three other biological applications with this tool are also presented. (paper)

Isolation of Circulating Tumor Cells by Dielectrophoresis

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Peter R. C. Gascoyne

2014-03-01

Full Text Available Dielectrophoresis (DEP is an electrokinetic method that allows intrinsic dielectric properties of suspended cells to be exploited for discrimination and separation. It has emerged as a promising method for isolating circulation tumor cells (CTCs from blood. DEP-isolation of CTCs is independent of cell surface markers. Furthermore, isolated CTCs are viable and can be maintained in culture, suggesting that DEP methods should be more generally applicable than antibody-based approaches. The aim of this article is to review and synthesize for both oncologists and biomedical engineers interested in CTC isolation the pertinent characteristics of DEP and CTCs. The aim is to promote an understanding of the factors involved in realizing DEP-based instruments having both sufficient discrimination and throughput to allow routine analysis of CTCs in clinical practice. The article brings together: (a the principles of DEP; (b the biological basis for the dielectric differences between CTCs and blood cells; (c why such differences are expected to be present for all types of tumors; and (d instrumentation requirements to process 10 mL blood specimens in less than 1 h to enable routine clinical analysis. The force equilibrium method of dielectrophoretic field-flow fractionation (DEP-FFF is shown to offer higher discrimination and throughput than earlier DEP trapping methods and to be applicable to clinical studies.

Isolation of Circulating Tumor Cells by Dielectrophoresis

Energy Technology Data Exchange (ETDEWEB)

Gascoyne, Peter R. C., E-mail: pgascoyn@mdanderson.org [Department of Imaging Physics Research, The University of Texas M.D. Anderson Cancer Center Unit 951, 1515 Holcombe Boulevard, Houston, TX 77030 (United States); Shim, Sangjo [Department of Imaging Physics Research, The University of Texas M.D. Anderson Cancer Center Unit 951, 1515 Holcombe Boulevard, Houston, TX 77030 (United States); Department of Biomedical Engineering, The University of Texas at Austin, 1 University Station, C0800, Austin, TX 78712 (United States); Present address: Micro & Nanotechnology Laboratory, University of Illinois at Urbana-Champaign, Urbana, 208 North Wright Street, Urbana, IL 61801 (United States)

2014-03-12

Dielectrophoresis (DEP) is an electrokinetic method that allows intrinsic dielectric properties of suspended cells to be exploited for discrimination and separation. It has emerged as a promising method for isolating circulation tumor cells (CTCs) from blood. DEP-isolation of CTCs is independent of cell surface markers. Furthermore, isolated CTCs are viable and can be maintained in culture, suggesting that DEP methods should be more generally applicable than antibody-based approaches. The aim of this article is to review and synthesize for both oncologists and biomedical engineers interested in CTC isolation the pertinent characteristics of DEP and CTCs. The aim is to promote an understanding of the factors involved in realizing DEP-based instruments having both sufficient discrimination and throughput to allow routine analysis of CTCs in clinical practice. The article brings together: (a) the principles of DEP; (b) the biological basis for the dielectric differences between CTCs and blood cells; (c) why such differences are expected to be present for all types of tumors; and (d) instrumentation requirements to process 10 mL blood specimens in less than 1 h to enable routine clinical analysis. The force equilibrium method of dielectrophoretic field-flow fractionation (DEP-FFF) is shown to offer higher discrimination and throughput than earlier DEP trapping methods and to be applicable to clinical studies.

Isolation of Circulating Tumor Cells by Dielectrophoresis

International Nuclear Information System (INIS)

Gascoyne, Peter R. C.; Shim, Sangjo

2014-01-01

Dielectrophoresis (DEP) is an electrokinetic method that allows intrinsic dielectric properties of suspended cells to be exploited for discrimination and separation. It has emerged as a promising method for isolating circulation tumor cells (CTCs) from blood. DEP-isolation of CTCs is independent of cell surface markers. Furthermore, isolated CTCs are viable and can be maintained in culture, suggesting that DEP methods should be more generally applicable than antibody-based approaches. The aim of this article is to review and synthesize for both oncologists and biomedical engineers interested in CTC isolation the pertinent characteristics of DEP and CTCs. The aim is to promote an understanding of the factors involved in realizing DEP-based instruments having both sufficient discrimination and throughput to allow routine analysis of CTCs in clinical practice. The article brings together: (a) the principles of DEP; (b) the biological basis for the dielectric differences between CTCs and blood cells; (c) why such differences are expected to be present for all types of tumors; and (d) instrumentation requirements to process 10 mL blood specimens in less than 1 h to enable routine clinical analysis. The force equilibrium method of dielectrophoretic field-flow fractionation (DEP-FFF) is shown to offer higher discrimination and throughput than earlier DEP trapping methods and to be applicable to clinical studies

Efficient Isolation of Cardiac Stem Cells from Brown Adipose

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Zhiqiang Liu

2010-01-01

Full Text Available Cardiac stem cells represent a logical cell type to exploit in cardiac regeneration. The efficient harvest of cardiac stem cells from a suitable source would turn promising in cardiac stem cell therapy. Brown adipose was recently found to be a new source of cardiac stem cells, instrumental to myocardial regeneration. Unfortunately, an efficient method for the cell isolation is unavailable so far. In our study we have developed a new method for the efficient isolation of cardiac stem cells from brown adipose by combining different enzymes. Results showed that the total cell yield dramatically increased (more than 10 times, P<.01 compared with that by previous method. The content of CD133-positive cells (reported to differentiate into cardiomyocytes with a high frequency was much higher than that in the previous report (22.43% versus 3.5%. Moreover, the isolated cells could be the efficiently differentiated into functional cardiomyocytes in optimized conditions. Thus, the new method we established would be of great use in further exploring cardiac stem cell therapy.

Reticuloendothelial hyperphagocytosis occurs in streptozotocin-diabetic rats. Studies with colloidal carbon, albumin microaggregates, and soluble fibrin monomers

International Nuclear Information System (INIS)

Cornell, R.P.

1982-01-01

In contrast to previous studies of diabetic humans and animals, which reported unchanged or depressed function, reticuloendothelial system (RES) hyperphagocytosis of colloidal carbon, 125 I-albumin microaggregates, and 125 I-fibrin monomers were observed in rats as early as 14 days after the induction of diabetes with streptozotocin (STZ). The fact that enhanced phagocytosis by RE macrophages was prevented by chronic insulin replacement therapy indicates that the diabetic internal environment of hyperglycemia and hypoinsulinemia was perhaps responsible for the observed changes. Experiments involving organ localization of intravenously administered particles, perfusion of isolated livers, and microscopic examination of the liver all suggested that increased Kupffer cell activity was the primary event in RES hyperphagocytosis by STZ-diabetic rats. Both hypertrophy and hyperplasia of Kupffer cells were apparent in livers of STZ-diabetic animals as evidenced by photomicrographs and hepatic cell quantification. Plasma fibronectin, which binds fibrin monomers to RE macrophages before phagocytosis, was significantly decreased in the circulation of STZ-diabetic rats, but the level of cell-associated fibronectin was not measured. Renal localization of urea-soluble 125 I-fibrin monomers exceeded splenic and pulmonary uptake in normal control rats and was enhanced in animals with STZ-diabetes. Changes in fibronectin levels, fibrin monomer localization, and Kupffer cell size and numbers in experimental diabetes in rats may have implications for the pathogenesis of vascular disease involving phagocytic mesangial and foam cells in diabetic humans

Reticuloendothelial hyperphagocytosis occurs in streptozotocin-diabetic rats. Studies with colloidal carbon, albumin microaggregates, and soluble fibrin monomers.

Science.gov (United States)

Cornell, R P

1982-02-01

In contrast to previous studies of diabetic humans and animals, which reported unchanged or depressed function, reticuloendothelial system (RES) hyperphagocytosis of colloidal carbon, 125I-albumin microaggregates, and 125I-fibrin monomers were observed in rats as early as 14 days after the induction of diabetes with streptozotocin (STZ). The fact that enhanced phagocytosis by RE macrophages was prevented by chronic insulin replacement therapy indicates that the diabetic internal environment of hyperglycemia and hypoinsulinemia was perhaps responsible for the observed changes. Experiments involving organ localization of intravenously administered particles, perfusion of isolated livers, and microscopic examination of the liver all suggested that increased Kupffer cell activity was the primary event in RES hyperphagocytosis by STZ-diabetic rats. Both hypertrophy and hyperplasia of Kupffer cells were apparent in livers of STZ-diabetic animals as evidenced by photomicrographs and hepatic cell quantification. Plasma fibronectin, which binds fibrin monomers to RE macrophages before phagocytosis, was significantly decreased in the circulation of STZ-diabetic rats, but the level of cell-associated fibronectin was not measured. Renal localization of urea-soluble 125I-fibrin monomers exceeded splenic and pulmonary uptake in normal control rats and was enhanced in animals with STZ-diabetes. Changes in fibronectin levels, fibrin monomer localization, and Kupffer cell size and numbers in experimental diabetes in rats may have implications for the pathogenesis of vascular disease involving phagocytic mesangial and foam cells in diabetic humans.

Novel protocol for persister cells isolation.

Directory of Open Access Journals (Sweden)

Silvia J Cañas-Duarte

Full Text Available Bacterial persistence, where a fraction of a population presents a transient resistance to bactericidal substances, has great medical importance due to its relation with the appearance of antibiotic resistances and untreatable bacterial chronic infections. The mechanisms behind this phenomenon remain largely unknown in spite of recent advances, in great part because of the difficulty in isolating the very small fraction of the population that is in this state at any given time. Current protocols for persister isolation have resulted in possible biases because of the induction of this state by the protocol itself. Here we present a novel protocol that allows rapid isolation of persister cells both from exponential and stationary phase. Moreover, it is capable of differentiating between type I and type II persister cells, which should allow the field to move beyond its current state of studying only one type. While this protocol prompts a revision of many of the current results, it should greatly facilitate further advances in the field.

Optimized exosome isolation protocol for cell culture supernatant and human plasma

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Richard J. Lobb

2015-07-01

Full Text Available Extracellular vesicles represent a rich source of novel biomarkers in the diagnosis and prognosis of disease. However, there is currently limited information elucidating the most efficient methods for obtaining high yields of pure exosomes, a subset of extracellular vesicles, from cell culture supernatant and complex biological fluids such as plasma. To this end, we comprehensively characterize a variety of exosome isolation protocols for their efficiency, yield and purity of isolated exosomes. Repeated ultracentrifugation steps can reduce the quality of exosome preparations leading to lower exosome yield. We show that concentration of cell culture conditioned media using ultrafiltration devices results in increased vesicle isolation when compared to traditional ultracentrifugation protocols. However, our data on using conditioned media isolated from the Non-Small-Cell Lung Cancer (NSCLC SK-MES-1 cell line demonstrates that the choice of concentrating device can greatly impact the yield of isolated exosomes. We find that centrifuge-based concentrating methods are more appropriate than pressure-driven concentrating devices and allow the rapid isolation of exosomes from both NSCLC cell culture conditioned media and complex biological fluids. In fact to date, no protocol detailing exosome isolation utilizing current commercial methods from both cells and patient samples has been described. Utilizing tunable resistive pulse sensing and protein analysis, we provide a comparative analysis of 4 exosome isolation techniques, indicating their efficacy and preparation purity. Our results demonstrate that current precipitation protocols for the isolation of exosomes from cell culture conditioned media and plasma provide the least pure preparations of exosomes, whereas size exclusion isolation is comparable to density gradient purification of exosomes. We have identified current shortcomings in common extracellular vesicle isolation methods and provide a

Assessment of hepatocyte and kupffer cell function using Tc-99m DISIDA/Tc-99m tin colloid in thioacetamide-induced liver injury

International Nuclear Information System (INIS)

Ahn, B. C.; Chun, K. A.; Lee, J.; Lee, K. B.

1997-01-01

Toxic liver injury is not unusual in clinical field and liver biopsy is one of the most accurate method to define the severity of liver injury. But occasionally, it is impossible to obtain liver tissue in patients with acute toxic liver injury. The aim of this study is to evaluate the possibility of liver scintigraphy with Tc-99m DISIDA or Tc-99m tin colloid as a non-invasive tool in predicting functional status of hepatocyte and Kupffer cell and severity of liver injury. Intraperitoneal injection of thioacetamide was performed to make acute liver injury in mice and rats, and liver status was assessed by pathologic specimen and scintigraphic methods. Scintigraphic evaluation were performed by biodistribution of Tc-99m DISIDA or Tc-99m tin colloid in thioacetamide-treated mice. Liver time-activity curves were generated. Comparison between histologic data and scintigraphic data was done with SAS program. Thioacetamide-treated mice demonstrated hepatocyte necrosis in histologic examination and low liver/blood uptake ratios in biodistribution studies using both radiotracers. Biodistribution study using Tc-99m tin colloid revealed increased lung radioactivity in thioacetamide-treated mice. Twenty-four hours after thioacetamide administration, thioacetamide-treated rats demonstrated maximal hepatocyte necrosis and inflammation in histologic finding and delayed maximal uptake time (Tmax) and prolonged half time (T 1/2 ) of liver time-activity curve in liver scintigraphy. Histologic results and scintigraphic data were well correlated, and these two scintigraphic parameters (Tmax T 1/2 ) seemed to be good predictors of histologic change of liver. These data showed that liver injury could be assessed by non-invasive scintigraphic study in rat and mouse. This experimental study might be used as a animal model to evaluate the liver protecting drugs, and this scintigraphic study could be applied to acute toxic hepatitis for assessment of liver status in men

Isolation, Culture, Functional Assays, and Immunofluorescence of Myofiber-Associated Satellite Cells.

Science.gov (United States)

Vogler, Thomas O; Gadek, Katherine E; Cadwallader, Adam B; Elston, Tiffany L; Olwin, Bradley B

2016-01-01

Adult skeletal muscle stem cells, termed satellite cells, regenerate and repair the functional contractile cells in adult skeletal muscle called myofibers. Satellite cells reside in a niche between the basal lamina and sarcolemma of myofibers. Isolating single myofibers and their associated satellite cells provides a culture system that partially mimics the in vivo environment. We describe methods for isolating and culturing intact individual myofibers and their associated satellite cells from the mouse extensor digitorum longus muscle. Following dissection and isolation of individual myofibers we provide protocols for myofiber transplantation, satellite cell transfection, immune detection of satellite cell antigens, and assays to examine satellite cell self-renewal and proliferation.

Simultaneous isolation of vascular endothelial cells and mesenchymal stem cells from the human umbilical cord.

Science.gov (United States)

Kadam, Sachin S; Tiwari, Shubha; Bhonde, Ramesh R

2009-01-01

The umbilical cord represents the link between mother and fetus during pregnancy. This cord is usually discarded as a biological waste after the child's birth; however, its importance as a "store house" of stem cells has been explored recently. We developed a method of simultaneous isolation of endothelial cells (ECs) from the vein and mesenchymal stem cells from umbilical cord Wharton's jelly of the same cord. The isolation protocol has been simplified, modified, and improvised with respect to choice of enzyme and enzyme mixture, digestion time, cell yield, cell growth, and culture medium. Isolated human umbilical vascular ECs (hUVECs) were positive for von-Willibrand factor, a classical endothelial marker, and could form capillary-like structures when seeded on Matrigel, thus proving their functionality. The isolated human umbilical cord mesenchymal stem cells (hUCMSCs) were found positive for CD44, CD90, CD 73, and CD117 and were found negative for CD33, CD34, CD45, and CD105 surface markers; they were also positive for cytoskeleton markers of smooth muscle actin and vimentin. The hUCMSCs showed multilineage differentiation potential and differentiated into adipogenic, chondrogenic, osteogenic, and neuronal lineages under influence of lineage specific differentiation medium. Thus, isolating endothelial cells as well as mesenchymal cells from the same umbilical cord could lead to complete utilization of the available tissue for the tissue engineering and cell therapy.

Single Cell Isolation and Analysis

Directory of Open Access Journals (Sweden)

Ping Hu

2016-10-01

Full Text Available Increasing evidence shows that the heterogeneity of individual cells within a genetically identical population can be critical to their peculiar function and fate. Conventional cell based assays mainly analysis the average responses from a population cells, while the difference within individual cells may often be masked. The cell size, RNA transcripts and protein expression level are quite different within individual cells and these variations are key point to answer the problems in cancer, neurobiology, stem cell biology, immunology and developmental biology. To better understand the cell-to-cell variations, the single cell analysis can provide much more detailed information which may be helpful for therapeutic decisions in an increasingly personalized medicine. In this review, we will focus on the recent development in single cell analysis, including methods used in single cell isolation, analysis and some application examples. The review provides the historical background to single cell analysis, discusses limitations, and current and future possibilities in this exciting field of research.

Human Induced Pluripotent Stem Cell-Derived Macrophages Share Ontogeny with MYB-Independent Tissue-Resident Macrophages

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Julian Buchrieser

2017-02-01

Full Text Available Tissue-resident macrophages, such as microglia, Kupffer cells, and Langerhans cells, derive from Myb-independent yolk sac (YS progenitors generated before the emergence of hematopoietic stem cells (HSCs. Myb-independent YS-derived resident macrophages self-renew locally, independently of circulating monocytes and HSCs. In contrast, adult blood monocytes, as well as infiltrating, gut, and dermal macrophages, derive from Myb-dependent HSCs. These findings are derived from the mouse, using gene knockouts and lineage tracing, but their applicability to human development has not been formally demonstrated. Here, we use human induced pluripotent stem cells (iPSCs as a tool to model human hematopoietic development. By using a CRISPR-Cas9 knockout strategy, we show that human iPSC-derived monocytes/macrophages develop in an MYB-independent, RUNX1-, and SPI1 (PU.1-dependent fashion. This result makes human iPSC-derived macrophages developmentally related to and a good model for MYB-independent tissue-resident macrophages, such as alveolar and kidney macrophages, microglia, Kupffer cells, and Langerhans cells.

Liver and Skin Histopathology in Adults with Acid Sphingomyelinase Deficiency (Niemann-Pick Disease Type B)

Science.gov (United States)

Thurberg, Beth L.; Wasserstein, Melissa P.; Schiano, Thomas; O’Brien, Fanny; Richards, Susan; Cox, Gerald F.; McGovern, Margaret M.

2012-01-01

Acid sphingomyelinase deficiency (ASMD) is a lysosomal storage disorder characterized by the pathologic accumulation of sphingomyelin in multiple cells types, and occurs most prominently within the liver, spleen and lungs, leading to significant clinical disease. Seventeen ASMD patients underwent a liver biopsy during baseline screening for a Phase 1 trial of recombinant human acid sphingomyelinase (rhASM) in adults with Niemann-Pick disease type B. Eleven of the 17 were enrolled in the trial and each received a single dose of rhASM and underwent a repeat liver biopsy on Day 14. Biopsies were evaluated for fibrosis, sphingomyelin accumulation and macrophage infiltration by light and electron microscopy. When present, fibrosis was periportal and pericellular, predominantly surrounding affected Kupffer cells. Two baseline biopsies exhibited frank cirrhosis. Sphingomyelin was localized to isolated Kupffer cells in mildly affected biopsies and was present in both Kupffer cells and hepatocytes in more severely affected cases. Morphometric quantification of sphingomyelin storage in liver biopsies ranged from 4–44% of the microscopic field. Skin biopsies were also performed at baseline and Day 14 in order to compare the sphingomyelin distribution in a peripheral tissue to that of liver. Sphingomyelin storage was present at lower levels in multiple cell types of the skin, including dermal fibroblasts, macrophages, vascular endothelial cells, vascular smooth muscle cells and Schwann cells. This Phase 1 trial of rhASM in adults with ASMD provided a unique opportunity for a prospective assessment of hepatic and skin pathology in this rare disease and their potential usage as pharmacodynamic biomarkers. PMID:22613999

Isolation of Lysosomes from Mammalian Tissues and Cultured Cells.

Science.gov (United States)

Aguado, Carmen; Pérez-Jiménez, Eva; Lahuerta, Marcos; Knecht, Erwin

2016-01-01

Lysosomes participate within the cells in the degradation of organelles, macromolecules, and a wide variety of substrates. In any study on specific roles of lysosomes, both under physiological and pathological conditions, it is advisable to include methods that allow their reproducible and reliable isolation. However, purification of lysosomes is a difficult task, particularly in the case of cultured cells. This is mainly because of the heterogeneity of these organelles, along with their low number and high fragility. Also, isolation methods, while disrupting plasma membranes, have to preserve the integrity of lysosomes, as the breakdown of their membranes releases enzymes that could damage all cell organelles, including themselves. The protocols described below have been routinely used in our laboratory for the specific isolation of lysosomes from rat liver, NIH/3T3, and other cultured cells, but can be adapted to other mammalian tissues or cell lines.

Cell damage caused by vaginal Candida albicans isolates from women with different symptomatologies.

Science.gov (United States)

Faria, Daniella Renata; Sakita, Karina Mayumi; Akimoto-Gunther, Luciene Setsuko; Kioshima, Érika Seki; Svidzinski, Terezinha Inez Estivalet; Bonfim-Mendonça, Patrícia de Souza

2017-08-01

The present study aimed to characterize cell damage caused by vaginal Candida albicans isolates from women with different symptomatologies. It was evaluated 12 clinical isolates of C. albicans from vaginal samples: 4 from asymptomatic women (AS), 4 from women with a single episode of vulvovaginal candidiasis (VVC) and 4 from women with recurrent vulvovaginal candidiasis (RVVC). We evaluated the ability of C. albicans to adhere to human cervical cancer cells (SiHa), the yeast-SiHa cell interactions and cell damage. All of the clinical isolates presented a high adhesion capacity on SiHa cells. However, clinical isolates from symptomatic women (VVC and RVVC) had higher filamentation after contact (24 h) with SiHa cells and a greater capacity to cause cell damage (>80 %). Clinical isolates from symptomatic women had greater potential to invade SiHa cells, suggesting that they are more pathogenic than AS isolates.

Micro+nanotexturing of substrates to enhance ligand-assisted cancer cell isolation

International Nuclear Information System (INIS)

Mahmood, Mohammed Arif I; Islam, Muhymin; Ali, Waqas; Hanif, Madiha; Iqbal, Samir M; Wan, Yuan; Kim, Young-tae

2014-01-01

This paper presents a simple approach to create a two-tiered surface for superior cancer cell isolation. The idea is inspired by the interactions of cells with a nanotextured basement membrane. The texture mimicked the extracellular matrix and basement membrane for superior target cell adhesion. Prepared micro+nanotextured surfaces showed enhanced cell capture. Preparation of the two-tiered surface was done using micro- and nanotexturing and was easily reproducible. It has been shown before that the larger surface area of a nanotextured surface assists the cell’s attachment through surface-anchored ligands. Taking it a step further, ligand functionalized two-level micro+nanotextured surfaces improved the sensitivity of the cancer cell isolation over simple flat nanotexturing. The isolation efficiency increased by 208% compared to the surface with a single-level nanotexture. The two-tiered surface was compatible with previously reported nanotextured devices used for cancer cell isolation. Micro-texture on the glass surface was created using simple sand gritting, followed by reactive ion etching (RIE) of the entire surface. The approach could create large surface areas within a short time while maintaining superior cell isolation efficiency. (paper)

Rapid isolation of bone marrow mesenchymal stromal cells using integrated centrifuge-based technology.

Science.gov (United States)

Meppelink, Amanda M; Wang, Xing-Hua; Bradica, Gino; Barron, Kathryn; Hiltz, Kathleen; Liu, Xiang-Hong; Goldman, Scott M; Vacanti, Joseph P; Keating, Armand; Hoganson, David M

2016-06-01

The use of bone marrow-derived mesenchymal stromal cells (MSCs) in cell-based therapies is currently being developed for a number of diseases. Thus far, the clinical results have been inconclusive and variable, in part because of the variety of cell isolation procedures and culture conditions used in each study. A new isolation technique that streamlines the method of concentration and demands less time and attention could provide clinical and economic advantages compared with current methodologies. In this study, we evaluated the concentrating capability of an integrated centrifuge-based technology compared with standard Ficoll isolation. MSCs were concentrated from bone marrow aspirate using the new device and the Ficoll method. The isolation capabilities of the device and the growth characteristics, secretome production, and differentiation capacity of the derived cells were determined. The new MSC isolation device concentrated the bone marrow in 90 seconds and resulted in a mononuclear cell yield 10-fold higher and with a twofold increase in cell retention compared with Ficoll. The cells isolated using the device were shown to exhibit similar morphology and functional activity as assessed by growth curves and secretome production compared to the Ficoll-isolated cells. The surface marker and trilineage differentiation profile of the device-isolated cells was consistent with the known profile of MSCs. The faster time to isolation and greater cell yield of the integrated centrifuge-based technology may make this an improved approach for MSC isolation from bone marrow aspirates. Copyright © 2016 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

A primary study on the phagocytic activity of Kupffer cells with superparamagnetic iron oxide particles enhanced MR imaging in a rat nonalcoholic steatohepatitis model

International Nuclear Information System (INIS)

Jiao Zhiyun; Li Cheng; Ma Zhanlong; Chen Wenjuan

2010-01-01

Objective: To investigate the feasibility of using superparamgnetic iron oxide (SPIO) as MRI contrast agent to assess rat nonalcoholic steatohepatitis Kupffer cells (KC) function. Methods: Twenty male SD rats were randomly divided into A and B groups, group A (n=10) was the experimental group fed high fat diet, group B (n=10) was the control group fed normal diet. After 8 weeks, plain MR and SPIO enhanced MR were performed in all the rats. Blood lipids were measured, and HE and Perl's blue staining in all livers specimen was done. The related results of the staining were analyzed with t test. Results: Group A TC and TG levels [(6.58 ± 1.25) and (1.53 ± 0.23) mmol/L respectively] were significantly higher than group B[(1.64 ± 0.22) and (0.55 ± 0.14) mmol/L respectively] (t=11.716 and 11.588, P 1 WI, ad statistically significant differences (t=-18.451 and -16.240, P 2 WI, T 2 WI and T 1 WI (t=10.745, 19.800, 39.168 and 92.785, P<0.01). Typical histological hepatic lesions of NASH were observed in group A, Perl's staining-positive particles in group A (2.33 ± 0.50) were fewer than in group B (4) (t=-10.000, P<0.01). Conclusion: The high-fat diet induced model of SD rats was close to the human NASH and was easy to establish. Clinical application of SPIO enhanced MR successfullly assessed the phagocytic activity of KC in the study, and it suggested that the pathogenesis of NASH was related to the decreased phagocytic activity of KC. (authors)

Isolation of hair follicle bulge stem cells from YFP-expressing reporter mice.

Science.gov (United States)

Nakrieko, Kerry-Ann; Irvine, Timothy S; Dagnino, Lina

2013-01-01

In this article we provide a method to isolate hair follicle stem cells that have undergone targeted gene inactivation. The mice from which these cells are isolated are bred into a Rosa26-yellow fluorescent protein (YFP) reporter background, which results in YFP expression in the targeted stem cell population. These cells are isolated and purified by fluorescence-activated cell sorting, using epidermal stem cell-specific markers in conjunction with YFP fluorescence. The purified cells can be used for gene expression studies, clonogenic experiments, and biological assays, such as viability and capacity for directional migration.

Identification of Adenovirus Serotype 5 Hexon Regions That Interact with Scavenger Receptors

Energy Technology Data Exchange (ETDEWEB)

Khare, Reeti; Reddy, Vijay S.; Nemerow, Glen R.; Barry, Michael A. (Scripps); (Mayo)

2012-05-04

Most of an intravenous dose of species C adenovirus serotype 5 (Ad5) is destroyed by liver Kupffer cells. In contrast, another species C virus, Ad6, evades these cells to mediate more efficient liver gene delivery. Given that this difference in Kupffer cell interaction is mediated by the hypervariable (HVR) loops of the virus hexon protein, we genetically modified each of the seven HVRs of Ad5 with a cysteine residue to enable conditional blocking of these sites with polyethylene glycol (PEG). We show that these modifications do not affect in vitro virus transduction. In contrast, after intravenous injection, targeted PEGylation at HVRs 1, 2, 5, and 7 increased viral liver transduction up to 20-fold. Elimination or saturation of liver Kupffer cells did not significantly affect this increase in the liver transduction. In vitro, PEGylation blocked uptake of viruses via the Kupffer cell scavenger receptor SRA-II. These data suggest that HVRs 1, 2, 5, and 7 of Ad5 may be involved in Kupffer cell recognition and subsequent destruction. These data also demonstrate that this conditional genetic-chemical mutation strategy is a useful tool for investigating the interactions of viruses with host tissues.

Isolation and In Vitro Characterization of Epidermal Stem Cells

DEFF Research Database (Denmark)

Moestrup, Kasper S; Andersen, Marianne Stemann; Jensen, Kim Bak

2017-01-01

flow cytometry. Using markers that define the spatial origin of epidermal cells, it is possible to interrogate the specific characteristics of subpopulations of cells based on their in vivo credentials. Here, we describe how to isolate, culture, and characterize keratinocytes from murine back and tail......Colony-forming assays represent prospective methods, where cells isolated from enzymatically dissociated tissues or from tissue cultures are assessed for their proliferative capacity in vitro. Complex tissues such as the epithelial component of the skin (the epidermis) are characterized...

Kupffer's vesicle is a ciliated organ of asymmetry in the zebrafish embryo that initiates left-right development of the brain, heart and gut.

Science.gov (United States)

Essner, Jeffrey J; Amack, Jeffrey D; Nyholm, Molly K; Harris, Erin B; Yost, H Joseph

2005-03-01

Monocilia have been proposed to establish the left-right (LR) body axis in vertebrate embryos by creating a directional fluid flow that triggers asymmetric gene expression. In zebrafish, dorsal forerunner cells (DFCs) express a conserved ciliary dynein gene (left-right dynein-related1, lrdr1) and form a ciliated epithelium inside a fluid-filled organ called Kupffer's vesicle (KV). Here, videomicroscopy demonstrates that cilia inside KV are motile and create a directional fluid flow just prior to the onset of asymmetric gene expression in lateral cells. Laser ablation of DFCs and surgical disruption of KV provide direct evidence that ciliated KV cells are required during early somitogenesis for subsequent LR patterning in the brain, heart and gut. Antisense morpholinos against lrdr1 disrupt KV fluid flow and perturb LR development. Furthermore, lrdr1 morpholinos targeted to DFC/KV cells demonstrate that Lrdr1 functions in these ciliated cells to control LR patterning. This provides the first direct evidence, in any vertebrate, that impairing cilia function in derivatives of the dorsal organizer, and not in other cells that express ciliogenic genes, alters LR development. Finally, genetic analysis reveals novel roles for the T-box transcription factor no tail and the Nodal signaling pathway as upstream regulators of lrdr1 expression and KV morphogenesis. We propose that KV is a transient embryonic 'organ of asymmetry' that directs LR development by establishing a directional fluid flow. These results suggest that cilia are an essential component of a conserved mechanism that controls the transition from bilateral symmetry to LR asymmetry in vertebrates.

Isolation and culture of porcine neural progenitor cells from embryos and pluripotent stem cells

DEFF Research Database (Denmark)

Rasmussen, Mikkel Aabech; Hall, Vanessa Jane; Hyttel, Poul

2013-01-01

from porcine embryos or induced pluripotent stem cells is presented. The neural induction is performed in coculture and the isolation of rosette structures is carried out manually to ensure a homogenous population of NPCs. Using this method, multipotent NPCs can be obtained in approximately 1 month......The isolation and culture of neural progenitor cells (NPCs) from pluripotent stem cells has facilitated in vitro mechanistic studies of diseases related to the nervous system, as well as discovery of new medicine. In addition, NPCs are envisioned to play a crucial role in future cell replacement...... therapy. The pig has become recognized as an important large animal model and establishment of in vitro-derived porcine NPCs would allow for preclinical safety testing by transplantation in a porcine biomedical model. In this chapter, a detailed method for isolation and in vitro culture of porcine NPCs...

Formation of Cell-To-Cell Connection between Bone Marrow Cells and Isolated Rat Cardiomyocytes in a Cocultivation Model

Czech Academy of Sciences Publication Activity Database

Skopalík, J.; Pásek, Michal; Rychtárik, M.; Koristek, Z.; Gabrielová, E.; Sheer, P.; Matejovič, P.; Modrianský, M.; Klabusay, M.

2014-01-01

Roč. 5, č. 5 (2014), s. 1000185 ISSN 2157-7013 Institutional support: RVO:61388998 Keywords : bone marrow * mononuclear cells * isolated cardiomyocytes * cocultivation Subject RIV: BO - Biophysics http://omicsonline.org/ open - access /formation-of-celltocell-connection-between-bone-marrow-cells- and -isolated-rat-cardiomyocytes-2157-7013.1000185.php?aid=33364

Isolation of dendritic-cell-like S100β-positive cells in rat anterior pituitary gland.

Science.gov (United States)

Horiguchi, Kotaro; Fujiwara, Ken; Yoshida, Saishu; Higuchi, Masashi; Tsukada, Takehiro; Kanno, Naoko; Yashiro, Takashi; Tateno, Kozue; Osako, Shunji; Kato, Takako; Kato, Yukio

2014-07-01

S100β-protein-positive cells in the anterior pituitary gland appear to possess multifunctional properties. Because of their pleiotropic features, S100β-positive cells are assumed to be of a heterogeneous or even a non-pituitary origin. The observation of various markers has allowed these cells to be classified into populations such as stem/progenitor cells, epithelial cells, astrocytes and dendritic cells. The isolation and characterization of each heterogeneous population is a prerequisite for clarifying the functional character and origin of the cells. We attempt to isolate two of the subpopulations of S100β-positive cells from the anterior lobe. First, from transgenic rats that express green fluorescent protein (GFP) driven by the S100β protein promoter, we fractionate GFP-positive cells with a cell sorter and culture them so that they can interact with laminin, a component of the extracellular matrix. We observe that one morphological type of GFP-positive cells possesses extended cytoplasmic processes and shows high adhesiveness to laminin (process type), whereas the other is round in shape and exhibits low adherence to laminin (round type). We successfully isolate cells of the round type from the cultured GFP-positive cells by taking advantage of their low affinity to laminin and then measure mRNA levels of the two cell types by real-time polymerase chain reaction. The resultant data show that the process type expresses vimentin (mesenchymal cell marker) and glial fibrillary acidic protein (astrocyte marker). The round type expresses dendritic cell markers, CD11b and interleukin-6. Thus, we found a method for isolating dendritic-cell-like S100β-positive cells by means of their property of adhering to laminin.

Isolation of Autolysosomes from Tobacco BY-2 Cells.

Science.gov (United States)

Takatsuka, Chihiro; Inoue-Aono, Yuko; Moriyasu, Yuji

2017-01-01

Autolysosomes are organelles that sequester and degrade a portion of the cytoplasm during autophagy. Although autophagosomes are short lived compared to other organelles such as mitochondria, plastids, and peroxisomes, many autolysosomes accumulate in tobacco BY-2 cells cultured under sucrose starvation conditions in the presence of a cysteine protease inhibitor. We here describe our methodology for isolating autolysosomes from BY-2 cells by conventional cell fractionation using a Percoll gradient. The autolysosome fraction separates clearly from fractions containing mitochondria and peroxisomes. It contains acid phosphatase, vacuolar H + -ATPase, and protease activity. Electron micrographs show that the fraction contains partially degraded cytoplasm seen in autolysosomes before isolation although an autolysosome structure is only partially preserved.

Isolation of cell cycle-dependent gamma ray-sensitive Chinese hamster ovary cell

International Nuclear Information System (INIS)

Stamato, T.D.; Weinstein, R.; Giaccia, A.; Mackenzie, L.

1983-01-01

A technique for the isolation of gamma ray-sensitive Chinese hamster ovary (CHO) cell mutants is described, which uses nylon cloth replica plating and photography with dark-field illumination to directly monitor colonies for growth after gamma irradiation. Two gamma ray-sensitive mutants were isolated using this method. One of these cells (XR-1) had a two-slope survival curve: an initial steep slope and then a flattening of the curve at about 10% survival. Subsequently, it was found that this cell is sensitive to gamma irradiation in G1, early S, and late G2 phases of the cell cycle, whereas in the resistant phase (late S phase) its survival approaches that of the parental cells. The D37 in the sensitive G1 period is approximately 30 rads, compared with 300 rads of the parental cell. This mutant cell is also sensitive to killing by the DNA breaking agent, bleomycin, but is relatively insensitive to UV light and ethyl methane sulfonate, suggesting that the defect is specific for agents that produce DNA strand breakage

Isolation, Characterization, Cryopreservation of Human Amniotic Stem Cells and Differentiation to Osteogenic and Adipogenic Cells.

Directory of Open Access Journals (Sweden)

Shiva Gholizadeh-Ghaleh Aziz

Full Text Available Human stem cells and progenitor cells can be used to treat cancer and replace dysfunctional cells within a tissue or organ. The objective of this study was to identify the appropriate cells type in regenerative medicine and targeted therapy. As an alternative to embryonic and bone marrow stem cells, we examined human amniotic fluid stem cells (hAFSCs, one of the potential source of multipotent stem cells isolated from both cell pellet (using single-stage method, and supernatant of human amniotic fluid. Source of isolation and unique property of the cells emphasize that these cells are one of the promising new tools in therapeutic field. Double sources for isolation and availability of the left over samples in diagnostic laboratory at the same time have less legal and ethical concerns compared with embryonic stem cell studies. Cells were isolated, cultured for 18th passage for 6 months and characterized using qPCR and flow cytometry. Cells showed good proliferative ability in culture condition. The cells successfully differentiated into the adipogenic and osteogenic lineages. Based on these findings, amniotic fluid can be considered as an appropriate and convenient source of human amniotic fluid stem cells. These cells provide potential tools for therapeutic applications in the field of regenerative medicine. To get a better understanding of crosstalk between Oct4/NANOG with osteogenesis and adipogenesis, we used network analysis based on Common Targets algorithm and Common Regulators algorithm as well as subnetwork discovery based on gene set enrichment. Network analysis highlighted the possible role of MIR 302A and MIR let-7g. We demonstrated the high expression of MIR 302A and low expression of MIR let7g in hAFSCs by qPCR.

Cardiac endothelial cells isolated from mouse heart - a novel model for radiobiology

International Nuclear Information System (INIS)

Jelonek, K.; Walaszczyk, A.; Gabrys, D.; Pietrowska, M.; Widlak, P.; Kanthou, Ch.

2011-01-01

Cardiovascular disease is recognized as an important clinical problem in radiotherapy and radiation protection. However, only few radiobiological models relevant for assessment of cardiotoxic effects of ionizing radiation are available. Here we describe the isolation of mouse primary cardiac endothelial cells, a possible target for cardiotoxic effects of radiation. Cells isolated from hearts of juvenile mice were cultured and irradiated in vitro. In addition, cells isolated from hearts of locally irradiated adult animals (up to 6 days after irradiation) were tested. A dose-dependent formation of histone γH 2 A.X foci was observed after in vitro irradiation of cultured cells. However, such cells were resistant to radiation-induced apoptosis. Increased levels of actin stress fibres were observed in the cytoplasm of cardiac endothelial cells irradiated in vitro or isolated from irradiated animals. A high dose of 16 Gy did not increase permeability to Dextran in monolayers formed by endothelial cells. Up-regulated expression of Vcam1, Sele and Hsp70i genes was detected after irradiation in vitro and in cells isolated few days after irradiation in vivo. The increased level of actin stress fibres and enhanced expression of stress-response genes in irradiated endothelial cells are potentially involved in cardiotoxic effects of ionizing radiation. (authors)

Velocity effect on aptamer-based circulating tumor cell isolation in microfluidic devices.

Science.gov (United States)

Wan, Yuan; Tan, Jifu; Asghar, Waseem; Kim, Young-tae; Liu, Yaling; Iqbal, Samir M

2011-12-01

The isolation and detection of rare circulating tumor cells (CTCs) has been one of the focuses of intense research recently. In a microfluidic device, a number of factors can influence the enrichment capability of surface-bound probe molecules. This article analyzes the important factor of flow velocity in a microfluidic channel. The competition of surface-grafted anti-EGFR aptamers to bind the overexpressed EGFR on cell membranes against the drag force from the fluid flow is an important efficiency determining factor. The flow rate variations are applied both in experiments and in simulation models to study their effects on CTC capture efficiency. A mixture of mononuclear cells and human Glioblastoma cells is used to isolate cancer cells from the cellular flow. The results show interdependence between the adhesion probability, isolation efficiency, and flow rate. This work can help in designing flow-through lab-on-chip devices that use surface-bound probe affinities against overexpressed biomarkers for cell isolation. This work demonstrates that microfluidic based approaches have strong potential applications in CTC detection and isolation. © 2011 American Chemical Society

Chronic inflammation-elicited liver progenitor cell conversion to liver cancer stem cell with clinical significance.

Science.gov (United States)

Li, Xiao-Feng; Chen, Cheng; Xiang, Dai-Min; Qu, Le; Sun, Wen; Lu, Xin-Yuan; Zhou, Teng-Fei; Chen, Shu-Zhen; Ning, Bei-Fang; Cheng, Zhuo; Xia, Ming-Yang; Shen, Wei-Feng; Yang, Wen; Wen, Wen; Lee, Terence Kin Wah; Cong, Wen-Ming; Wang, Hong-Yang; Ding, Jin

2017-12-01

The substantial heterogeneity and hierarchical organization in liver cancer support the theory of liver cancer stem cells (LCSCs). However, the relationship between chronic hepatic inflammation and LCSC generation remains obscure. Here, we observed a close correlation between aggravated inflammation and liver progenitor cell (LPC) propagation in the cirrhotic liver of rats exposed to diethylnitrosamine. LPCs isolated from the rat cirrhotic liver initiated subcutaneous liver cancers in nonobese diabetic/severe combined immunodeficient mice, suggesting the malignant transformation of LPCs toward LCSCs. Interestingly, depletion of Kupffer cells in vivo attenuated the LCSC properties of transformed LPCs and suppressed cytokeratin 19/Oval cell 6-positive tumor occurrence. Conversely, LPCs cocultured with macrophages exhibited enhanced LCSC properties. We further demonstrated that macrophage-secreted tumor necrosis factor-α triggered chromosomal instability in LPCs through the deregulation of ubiquitin D and checkpoint kinase 2 and enhanced the self-renewal of LPCs through the tumor necrosis factor receptor 1/Src/signal transducer and activator of transcription 3 pathway, which synergistically contributed to the conversion of LPCs to LCSCs. Clinical investigation revealed that cytokeratin 19/Oval cell 6-positive liver cancer patients displayed a worse prognosis and exhibited superior response to sorafenib treatment. Our results not only clarify the cellular and molecular mechanisms underlying the inflammation-mediated LCSC generation but also provide a molecular classification for the individualized treatment of liver cancer. (Hepatology 2017;66:1934-1951). © 2017 by the American Association for the Study of Liver Diseases.

Oxidative and ER stress-dependent ASK1 activation in steatotic hepatocytes and Kupffer cells sensitizes mice fatty liver to ischemia/reperfusion injury.

Science.gov (United States)

Imarisio, Chiara; Alchera, Elisa; Bangalore Revanna, Chandrashekar; Valente, Guido; Follenzi, Antonia; Trisolini, Elena; Boldorini, Renzo; Carini, Rita

2017-11-01

Steatosis intensifies hepatic ischemia/reperfusion (I/R) injury increasing hepatocyte damage and hepatic inflammation. This study evaluates if this process is associated to a differential response of steatotic hepatocytes (HP) and Kupffer cells (KC) to I/R injury and investigates the molecular mechanisms involved. Control or steatotic (treated with 50 μmol palmitic acid, PA) mouse HP or KC were exposed to hypoxia/reoxygenation (H/R). C57BL/6 mice fed 9 week with control or High Fat diet underwent to partial hepatic IR. PA increased H/R damage of HP and further activated the ASK1-JNK axis stimulated by ER stress during H/R. PA also induced the production of oxidant species (OS), and OS prevention nullified the capacity of PA to increase H/R damage and ASK1/JNK stimulation. ASK1 inhibition prevented JNK activation and entirely protected HP damage. In KC, PA directly activated ER stress, ASK1 and p38 MAPK and increased H/R damage. However, in contrast to HP, ASK1 inhibition further increased H/R damage by preventing p38 MAPK activation. In mice liver, steatosis induced the expression of activated ASK1 in only KC, whereas I/R exposure of steatotic liver activated ASK1 expression also in HP. "In vivo", ASK1 inhibition prevented ASK1, JNK and p38 MAPK activation and protected I/R damage and expression of inflammatory markers. Lipids-induced ASK1 stimulation differentially affects HP and KC by promoting cytotoxic or protective signals. ASK1 increases H/R damage of HP by stimulating JNK and protects KC activating p38MAPK. These data support the potentiality of the therapeutic employment of ASK1 inhibitors that can antagonize the damaging effects of I/R upon fatty liver surgery by the contextual reduction of HP death and of KC-mediated reactions. Copyright © 2017 Elsevier Inc. All rights reserved.

An optimized method for mouse liver sinusoidal endothelial cell isolation

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Meyer, Jeremy, E-mail: jeremy.meyer@hcuge.ch [Division of Digestive and Transplantation Surgery, University Hospitals of Geneva, Rue Gabrielle-Perret-Gentil 4, 1211 Genève 14 (Switzerland); Unit of Surgical Research, University of Geneva, Rue Michel-Servet 1, 1206 Genève (Switzerland); Lacotte, Stéphanie, E-mail: stephanie.lacotte@unige.ch [Unit of Surgical Research, University of Geneva, Rue Michel-Servet 1, 1206 Genève (Switzerland); Morel, Philippe, E-mail: philippe.morel@hcuge.ch [Division of Digestive and Transplantation Surgery, University Hospitals of Geneva, Rue Gabrielle-Perret-Gentil 4, 1211 Genève 14 (Switzerland); Unit of Surgical Research, University of Geneva, Rue Michel-Servet 1, 1206 Genève (Switzerland); Gonelle-Gispert, Carmen, E-mail: carmen.gonelle@unige.ch [Unit of Surgical Research, University of Geneva, Rue Michel-Servet 1, 1206 Genève (Switzerland); Bühler, Léo, E-mail: leo.buhler@hcuge.ch [Division of Digestive and Transplantation Surgery, University Hospitals of Geneva, Rue Gabrielle-Perret-Gentil 4, 1211 Genève 14 (Switzerland); Unit of Surgical Research, University of Geneva, Rue Michel-Servet 1, 1206 Genève (Switzerland)

2016-12-10

The objective of the present study was to develop an accurate and reproducible method for liver sinusoidal endothelial cell (LSEC) isolation in mice. Non-parenchymal cells were isolated using a modified two-step collagenase digestion combined with Optiprep density gradient centrifugation. LSEC were further purified using two prevalent methods, short-term selective adherence and CD146+ magnetic-activated cell sorting (MACS), and compared in terms of cell yield, viability and purity to our purification technique using CD11b cell depletion combined with long-term selective adherence. LSEC purification using our technique allowed to obtain 7.07±3.80 million LSEC per liver, while CD146+ MACS and short-term selective adherence yielded 2.94±1.28 and 0.99±0.66 million LSEC, respectively. Purity of the final cell preparation reached 95.10±2.58% when using our method. In contrast, CD146+ MACS and short-term selective adherence gave purities of 86.75±3.26% and 47.95±9.82%, respectively. Similarly, contamination by non-LSEC was the lowest when purification was performed using our technique, with a proportion of contaminating macrophages of only 1.87±0.77%. Further, isolated cells analysed by scanning electron microscopy presented typical LSEC fenestrations organized in sieve plates, demonstrating that the technique allowed to isolate bona fide LSEC. In conclusion, we described a reliable and reproducible technique for the isolation of high yields of pure LSEC in mice. This protocol provides an efficient method to prepare LSEC for studying their biological functions. - Highlights: • This protocol provides an efficient method to prepare primary mouse LSEC for studying their biological functions. • The liver cell dispersion step was improved by performing a retrograde cannulation of the liver. • The cell yield and the purity obtained were higher than comparative techniques in mice. • Contaminating macrophages were removed by introducing a CD11b- magnetic �

An optimized method for mouse liver sinusoidal endothelial cell isolation

International Nuclear Information System (INIS)

Meyer, Jeremy; Lacotte, Stéphanie; Morel, Philippe; Gonelle-Gispert, Carmen; Bühler, Léo

2016-01-01

The objective of the present study was to develop an accurate and reproducible method for liver sinusoidal endothelial cell (LSEC) isolation in mice. Non-parenchymal cells were isolated using a modified two-step collagenase digestion combined with Optiprep density gradient centrifugation. LSEC were further purified using two prevalent methods, short-term selective adherence and CD146+ magnetic-activated cell sorting (MACS), and compared in terms of cell yield, viability and purity to our purification technique using CD11b cell depletion combined with long-term selective adherence. LSEC purification using our technique allowed to obtain 7.07±3.80 million LSEC per liver, while CD146+ MACS and short-term selective adherence yielded 2.94±1.28 and 0.99±0.66 million LSEC, respectively. Purity of the final cell preparation reached 95.10±2.58% when using our method. In contrast, CD146+ MACS and short-term selective adherence gave purities of 86.75±3.26% and 47.95±9.82%, respectively. Similarly, contamination by non-LSEC was the lowest when purification was performed using our technique, with a proportion of contaminating macrophages of only 1.87±0.77%. Further, isolated cells analysed by scanning electron microscopy presented typical LSEC fenestrations organized in sieve plates, demonstrating that the technique allowed to isolate bona fide LSEC. In conclusion, we described a reliable and reproducible technique for the isolation of high yields of pure LSEC in mice. This protocol provides an efficient method to prepare LSEC for studying their biological functions. - Highlights: • This protocol provides an efficient method to prepare primary mouse LSEC for studying their biological functions. • The liver cell dispersion step was improved by performing a retrograde cannulation of the liver. • The cell yield and the purity obtained were higher than comparative techniques in mice. • Contaminating macrophages were removed by introducing a CD11b- magnetic �

Silicon Dioxide Thin Film Mediated Single Cell Nucleic Acid Isolation

Science.gov (United States)

Bogdanov, Evgeny; Dominova, Irina; Shusharina, Natalia; Botman, Stepan; Kasymov, Vitaliy; Patrushev, Maksim

2013-01-01

A limited amount of DNA extracted from single cells, and the development of single cell diagnostics make it necessary to create a new highly effective method for the single cells nucleic acids isolation. In this paper, we propose the DNA isolation method from biomaterials with limited DNA quantity in sample, and from samples with degradable DNA based on the use of solid-phase adsorbent silicon dioxide nanofilm deposited on the inner surface of PCR tube. PMID:23874571

A modified efficient method for dental pulp stem cell isolation.

Science.gov (United States)

Raoof, Maryam; Yaghoobi, Mohammad Mehdi; Derakhshani, Ali; Kamal-Abadi, Ali Mohammadi; Ebrahimi, Behnam; Abbasnejad, Mehdi; Shokouhinejad, Noushin

2014-03-01

Dental pulp stem cells can be used in regenerative endodontic therapy. The aim of this study was to introduce an efficient method for dental pulp stem cells isolation. In this in-vitro study, 60 extracted human third molars were split and pulp tissue was extracted. Dental pulp stem cells were isolated by the following three different methods: (1) digestion of pulp by collagenase/dispase enzyme and culture of the released cells; (2) outgrowth of the cells by culture of undigested pulp pieces; (3) digestion of pulp tissue pieces and fixing them. The cells were cultured in minimum essential medium alpha modification (αMEM) medium supplemented with 20% fetal bovine serum(FBS) in humid 37°C incubator with 5% CO 2. The markers of stem cells were studied by reverse transcriptase polymerase chain reaction (PCR). The student t-test was used for comparing the means of independent groups. P third method, we obtained stem cells successfully with about 60% efficiency after 2 days. The results of RT-PCR suggested the expression of Nanog, Oct-4, and Nucleostemin markers in the isolated cells from dental pulps. This study proposes a new method with high efficacy to obtain dental pulp stem cells in a short time.

Biosynthesis and release of proteins by isolated pulmonary Clara cells

International Nuclear Information System (INIS)

Patton, S.E.; Gilmore, L.B.; Jetten, A.M.; Nettesheim, P.; Hook, G.E.

1986-01-01

The major proteins synthesized and released by Clara cells were identified and compared with those synthesized and released by mixed lung cells. Highly purified Clara cells (85.9 +/- 2.4%) and mixed lung cells (Clara cells 4%, Type II cells 33%, granulocytes 18%, macrophages 2.7%, ciliated cells 1.2%) were isolated from rabbit lungs, incubated with Ham's F12 medium in collagen/fibronectin-coated plastic culture dishes in the presence of 35 S-methionine for periods of 4 and 18 hrs. Radiolabelled proteins were isolated from the cells and from the culture medium, electrophoresed on polyacrylamide gels in the presence of SDS under reducing conditions, and then autoradiographed. After 4 and 18 hr of incubation of the Clara cells the major radiolabelled cell-associated proteins were those with molecular weights of 6, 48, and 180 Kd. The major radiolabelled proteins released by Clara cells into the medium after 4 hrs of incubation had molecular weights of 6, 48, and 180 Kd, accounting for 42, 16, and 10%, respectively, of the total extracellular protein-associated radioactivity. After 18 hr of incubation the 6 and 48 Kd proteins represented 30 and 18% of the total released radioactivity, and the relative amount of the 180 Kd protein had decreased to 3%. With the mixed lung cells, the major proteins released into the medium had molecular weights of 6 and 48 Kd. Under nonreducing conditions the 6 Kd protein released by Clara cells had an apparent molecular weight of 12 Kd. Labelling isolated Clara cells with a mixture of 14 C-amino acids also identified this low molecular weight protein as the major secretory product of the Clara cell. The 6 Kd protein did not label when the cells were incubated with 14 C-glucosamine indicating that it was not a glycoprotein. Data demonstrate the release of several proteins from isolated Clara cells but the major protein had a M.W. of 6 Kd

Isolation and functional interrogation of adult human prostate epithelial stem cells at single cell resolution.

Science.gov (United States)

Hu, Wen-Yang; Hu, Dan-Ping; Xie, Lishi; Li, Ye; Majumdar, Shyama; Nonn, Larisa; Hu, Hong; Shioda, Toshi; Prins, Gail S

2017-08-01

Using primary cultures of normal human prostate epithelial cells, we developed a novel prostasphere-based, label-retention assay that permits identification and isolation of stem cells at a single cell level. Their bona fide stem cell nature was corroborated using in vitro and in vivo regenerative assays and documentation of symmetric/asymmetric division. Robust WNT10B and KRT13 levels without E-cadherin or KRT14 staining distinguished individual stem cells from daughter progenitors in spheroids. Following FACS to isolate label-retaining stem cells from label-free progenitors, RNA-seq identified unique gene signatures for the separate populations which may serve as useful biomarkers. Knockdown of KRT13 or PRAC1 reduced sphere formation and symmetric self-renewal highlighting their role in stem cell maintenance. Pathways analysis identified ribosome biogenesis and membrane estrogen-receptor signaling enriched in stem cells with NF-ĸB signaling enriched in progenitors; activities that were biologically confirmed. Further, bioassays identified heightened autophagy flux and reduced metabolism in stem cells relative to progenitors. These approaches similarly identified stem-like cells from prostate cancer specimens and prostate, breast and colon cancer cell lines suggesting wide applicability. Together, the present studies isolate and identify unique characteristics of normal human prostate stem cells and uncover processes that maintain stem cell homeostasis in the prostate gland. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Isolation and functional interrogation of adult human prostate epithelial stem cells at single cell resolution

Directory of Open Access Journals (Sweden)

Wen-Yang Hu

2017-08-01

Full Text Available Using primary cultures of normal human prostate epithelial cells, we developed a novel prostasphere-based, label-retention assay that permits identification and isolation of stem cells at a single cell level. Their bona fide stem cell nature was corroborated using in vitro and in vivo regenerative assays and documentation of symmetric/asymmetric division. Robust WNT10B and KRT13 levels without E-cadherin or KRT14 staining distinguished individual stem cells from daughter progenitors in spheroids. Following FACS to isolate label-retaining stem cells from label-free progenitors, RNA-seq identified unique gene signatures for the separate populations which may serve as useful biomarkers. Knockdown of KRT13 or PRAC1 reduced sphere formation and symmetric self-renewal highlighting their role in stem cell maintenance. Pathways analysis identified ribosome biogenesis and membrane estrogen-receptor signaling enriched in stem cells with NF-ĸB signaling enriched in progenitors; activities that were biologically confirmed. Further, bioassays identified heightened autophagy flux and reduced metabolism in stem cells relative to progenitors. These approaches similarly identified stem-like cells from prostate cancer specimens and prostate, breast and colon cancer cell lines suggesting wide applicability. Together, the present studies isolate and identify unique characteristics of normal human prostate stem cells and uncover processes that maintain stem cell homeostasis in the prostate gland.

A method for isolating identifying and culturing of rat trachea-bronchia epithelial cells

International Nuclear Information System (INIS)

Cui Fengmei; Su Shibiao; Nie Jihua; Li Bingyan; Tong Jian

2005-01-01

Objective: To explore a method for isolating identifying and culturing the rat trachea-bronchia epithelial cells. Methods: The rat trachea-bronchia epithelial cells were isolated by digestion with pronase and brushing with cell brush, identified using confocul and cultured in entire F12 media with no serum. Results: With this method, cells in high purity and high viability could be obtained, and about 10 6 cells per rat. The cells grow well in entire F12 media with no serum. Conclusion: The method is useful for isolating rate trachea-bronchia epithelial cells and the entire F12 media with no serum is effective for culturing. (authors)

Isolation and propagation of colon cancer stem cells

NARCIS (Netherlands)

Prasetyanti, Pramudita R.; Zimberlin, Cheryl; de Sousa E Melo, Felipe; Medema, Jan Paul

2013-01-01

The design of tissue culture conditions that faithfully reproduce the characteristics of cells in their native environment remains one of the main challenges of cancer stem cell (CSC) biology. Here we describe a detailed methodology for the isolation and expansion of both human colon CSCs and mouse

miR-217 regulates ethanol-induced hepatic inflammation by disrupting sirtuin 1-lipin-1 signaling.

Science.gov (United States)

Yin, Huquan; Liang, Xiaomei; Jogasuria, Alvin; Davidson, Nicholas O; You, Min

2015-05-01

Ethanol-mediated injury, combined with gut-derived lipopolysaccharide (LPS), provokes generation of proinflammatory cytokines in Kupffer cells, causing hepatic inflammation. Among the mediators of these effects, miR-217 aggravates ethanol-induced steatosis in hepatocytes. However, the role of miR-217 in ethanol-induced liver inflammation process is unknown. Here, we examined the role of miR-217 in the responses to ethanol, LPS, or a combination of ethanol and LPS in RAW 264.7 macrophages and in primary Kupffer cells. In macrophages, ethanol substantially exacerbated LPS-mediated induction of miR-217 and production of proinflammatory cytokines compared with LPS or ethanol alone. Consistently, ethanol administration to mice led to increases in miR-217 abundance and increased production of inflammatory cytokines in isolated primary Kupffer cells exposed to the combination of ethanol and LPS. miR-217 promoted combined ethanol and LPS-mediated inhibition of sirtuin 1 expression and activity in macrophages. Moreover, miR-217-mediated sirtuin 1 inhibition was accompanied by increased activities of two vital inflammatory regulators, NF-κB and the nuclear factor of activated T cells c4. Finally, adenovirus-mediated overexpression of miR-217 led to steatosis and inflammation in mice. These findings suggest that miR-217 is a pivotal regulator involved in ethanol-induced hepatic inflammation. Strategies to inhibit hepatic miR-217 could be a viable approach in attenuating alcoholic hepatitis. Copyright © 2015 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Use of Multicolor Flow Cytometry for Isolation of Specific Cell Populations Deriving from Differentiated Human Embryonic Stem Cells

NARCIS (Netherlands)

Mengarelli, Isabella; Fryga, Andrew; Barberi, Tiziano

2016-01-01

Flow Cytometry-Sorting (FCM-Sorting) is a technique commonly used to identify and isolate specific types of cells from a heterogeneous population of live cells. Here we describe a multicolor flow cytometry technique that uses five distinct cell surface antigens to isolate four live populations with

Ex vivo hyperpolarized MR spectroscopy on isolated renal tubular cells: A novel technique for cell energy phenotyping.

Science.gov (United States)

Juul, Troels; Palm, Fredrik; Nielsen, Per Mose; Bertelsen, Lotte Bonde; Laustsen, Christoffer

2017-08-01

It has been demonstrated that hyperpolarized 13 C MR is a useful tool to study cultured cells. However, cells in culture can alter phenotype, which raises concerns regarding the in vivo significance of such findings. Here we investigate if metabolic phenotyping using hyperpolarized 13 C MR is suitable for cells isolated from kidney tissue, without prior cell culture. Isolation of tubular cells from freshly excised kidney tissue and treatment with either ouabain or antimycin A was investigated with hyperpolarized MR spectroscopy on a 9.4 Tesla preclinical imaging system. Isolation of tubular cells from less than 2 g of kidney tissue generally resulted in more than 10 million live tubular cells. This amount of cells was enough to yield robust signals from the conversion of 13 C-pyruvate to lactate, bicarbonate and alanine, demonstrating that metabolic flux by means of both anaerobic and aerobic pathways can be quantified using this technique. Ex vivo metabolic phenotyping using hyperpolarized 13 C MR in a preclinical system is a useful technique to study energy metabolism in freshly isolated renal tubular cells. This technique has the potential to advance our understanding of both normal cell physiology as well as pathological processes contributing to kidney disease. Magn Reson Med 78:457-461, 2017. © 2016 International Society for Magnetic Resonance in Medicine. © 2016 International Society for Magnetic Resonance in Medicine.

Isolation, separation, and characterization of epithelial and connective cells from rat palate

Energy Technology Data Exchange (ETDEWEB)

Terranova, Victor Paul [Univ. of Rochester, NY (United States)

1979-01-01

Epithelial and connective tissue cells were isolated from rat palate by sequential collagenase, hyaluronidase and trypsin digestion of the extracellular matrix. Differences between the two populations were noted with respect to total cell protein, total cell water, proline uptake and incorporation, percent collagen synthesized, effects of parathyroid hormone, metabolism of D-valine and cell density. Basal epithelial cells were subsequently separated from the heterogeneous epithelial cell population on shallow linear density gradients by velocity centrifugation. The type of collagen synthesized by the basal epithelial cells was compared to the type of collagen synthesized by the connective tissue cells by means of labeled amino acid incorporation ratios. Cells isolated from the epithelial and connective tissue were compared. From these studies it can be concluded that epithelial and connective tissue cells can be isolated from rat palate as viable and distinct populations with respect to the biochemical parameters examined. Furthermore, subpopulations can be separated and biochemically characterized.

Reproducible isolation of lymph node stromal cells reveals site-dependent differences in fibroblastic reticular cells.

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Fletcher, Anne L; Malhotra, Deepali; Acton, Sophie E; Lukacs-Kornek, Veronika; Bellemare-Pelletier, Angelique; Curry, Mark; Armant, Myriam; Turley, Shannon J

2011-01-01

Within lymph nodes, non-hematopoietic stromal cells organize and interact with leukocytes in an immunologically important manner. In addition to organizing T and B cell segregation and expressing lymphocyte survival factors, several recent studies have shown that lymph node stromal cells shape the naïve T cell repertoire, expressing self-antigens which delete self-reactive T cells in a unique and non-redundant fashion. A fundamental role in peripheral tolerance, in addition to an otherwise extensive functional portfolio, necessitates closer study of lymph node stromal cell subsets using modern immunological techniques; however this has not routinely been possible in the field, due to difficulties reproducibly isolating these rare subsets. Techniques were therefore developed for successful ex vivo and in vitro manipulation and characterization of lymph node stroma. Here we discuss and validate these techniques in mice and humans, and apply them to address several unanswered questions regarding lymph node composition. We explored the steady-state stromal composition of lymph nodes isolated from mice and humans, and found that marginal reticular cells and lymphatic endothelial cells required lymphocytes for their normal maturation in mice. We also report alterations in the proportion and number of fibroblastic reticular cells (FRCs) between skin-draining and mesenteric lymph nodes. Similarly, transcriptional profiling of FRCs revealed changes in cytokine production from these sites. Together, these methods permit highly reproducible stromal cell isolation, sorting, and culture.

Cell fusion in tumor progression: the isolation of cell fusion products by physical methods

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Vincitorio Massimo

2011-09-01

Full Text Available Abstract Background Cell fusion induced by polyethylene glycol (PEG is an efficient but poorly controlled procedure for obtaining somatic cell hybrids used in gene mapping, monoclonal antibody production, and tumour immunotherapy. Genetic selection techniques and fluorescent cell sorting are usually employed to isolate cell fusion products, but both procedures have several drawbacks. Results Here we describe a simple improvement in PEG-mediated cell fusion that was obtained by modifying the standard single-step procedure. We found that the use of two PEG undertreatments obtains a better yield of cell fusion products than the standard method, and most of these products are bi- or trinucleated polykaryocytes. Fusion rate was quantified using fluorescent cell staining microscopy. We used this improved cell fusion and cell isolation method to compare giant cells obtained in vitro and giant cells obtained in vivo from patients with Hodgkin's disease and erythroleukemia. Conclusions In the present study we show how to improve PEG-mediated cell fusion and that cell separation by velocity sedimentation offers a simple alternative for the efficient purification of cell fusion products and to investigate giant cell formation in tumor development.

Kupffer cells ameliorate hepatic insulin resistance induced by high-fat diet rich in monounsaturated fatty acids: the evidence for the involvement of alternatively activated macrophages

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Papackova Zuzana

2012-03-01

Full Text Available Abstract Background Resident macrophages (Kupffer cells, KCs in the liver can undergo both pro- or anti-inflammatory activation pathway and exert either beneficiary or detrimental effects on liver metabolism. Until now, their role in the metabolically dysfunctional state of steatosis remains enigmatic. Aim of our study was to characterize the role of KCs in relation to the onset of hepatic insulin resistance induced by a high-fat (HF diet rich in monounsaturated fatty acids. Methods Male Wistar rats were fed either standard (SD or high-fat (HF diet for 4 weeks. Half of the animals were subjected to the acute GdCl3 treatment 24 and 72 hrs prior to the end of the experiment in order to induce the reduction of KCs population. We determined the effect of HF diet on activation status of liver macrophages and on the changes in hepatic insulin sensitivity and triacylglycerol metabolism imposed by acute KCs depletion by GdCl3. Results We found that a HF diet rich in MUFA itself triggers an alternative but not the classical activation program in KCs. In a steatotic, but not in normal liver, a reduction of the KCs population was associated with a decrease of alternative activation and with a shift towards the expression of pro-inflammatory activation markers, with the increased autophagy, elevated lysosomal lipolysis, increased formation of DAG, PKCε activation and marked exacerbation of HF diet-induced hepatic insulin resistance. Conclusions We propose that in the presence of a high MUFA content the population of alternatively activated resident liver macrophages may mediate beneficial effects on liver insulin sensitivity and alleviate the metabolic disturbances imposed by HF diet feeding and steatosis. Our data indicate that macrophage polarization towards an alternative state might be a useful strategy for treating type 2 diabetes.

Isolation of chlamydia in irradiated and non-irradiated McCoy cells

International Nuclear Information System (INIS)

Johnson, L.; Harper, I.A.

1975-01-01

Specimens from eye and genital tract were cultured in parallel in irradiated and non-irradiated McCoy cells and the frequency of isolation of chlamydia using these culture methods was compared. There was a significant difference between the frequencies of isolation; irradiated McCoy cells produced a greater number of positive results. (author)

Cortactin and phagocytosis in isolated Sertoli cells

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Wolski Katja M

2005-12-01

Full Text Available Abstract Background Cortactin, an actin binding protein, has been associated with Sertoli cell ectoplasmic specializations in vivo, based on its immunolocalization around the heads of elongated spermatids, but not previously identified in isolated Sertoli cells. In an in vitro model of Sertoli cell-spermatid binding, cortactin was identified around debris and dead germ cells. Based on this observation, we hypothesized that this actin binding protein may be associated with a non-junction-related physiological function, such as phagocytosis. The purpose of this study was to identify the presence and distribution of cortactin in isolated rat Sertoli cells active in phagocytic activity following the addition of 0.8 μm latex beads. Results Sertoli cell monocultures were incubated with or without follicle stimulating hormone (FSH; 0.1 μg/ml in the presence or absence of cytochalasin D (2 μM, as an actin disrupter. Cortactin was identified by standard immunostaining with anti-cortactin, clone 4F11 (Upstate after incubation times of 15 min, 2 hr, and 24 hr with or without beads. Cells exposed to no hormone and no beads appeared to have a ubiquitous distribution of cortactin throughout the cytoplasm. In the presence of cytochalasin D, cortactin immunostaining was punctate and distributed in a pattern similar to that reported for actin in cells exposed to cytochalasin D. Sertoli cells not exposed to FSH, but activated with beads, did not show cortactin immunostaining around the phagocytized beads at any of the time periods. FSH exposure did not alter the distribution of cortactin within Sertoli cells, even when phagocytic activity was upregulated by the presence of beads. Conclusion Results of this study suggest cortactin is not associated with peripheralized actin at junctional or phagocytic sites. Further studies are necessary to clarify the role of cortactin in Sertoli cells.

Isolation and Characterization of Cancer Stem Cells of the Non-Small-Cell Lung Cancer (A549) Cell Line.

Science.gov (United States)

Halim, Noor Hanis Abu; Zakaria, Norashikin; Satar, Nazilah Abdul; Yahaya, Badrul Hisham

2016-01-01

Cancer is a major health problem worldwide. The failure of current treatments to completely eradicate cancer cells often leads to cancer recurrence and dissemination. Studies have suggested that tumor growth and spread are driven by a minority of cancer cells that exhibit characteristics similar to those of normal stem cells, thus these cells are called cancer stem cells (CSCs). CSCs are believed to play an important role in initiating and promoting cancer. CSCs are resistant to currently available cancer therapies, and understanding the mechanisms that control the growth of CSCs might have great implications for cancer therapy. Cancer cells are consist of heterogeneous population of cells, thus methods of identification, isolation, and characterisation of CSCs are fundamental to obtain a pure CSC populations. Therefore, this chapter describes in detail a method for isolating and characterizing a pure population of CSCs from heterogeneous population of cancer cells and CSCs based on specific cell surface markers.

Glutathione synthesis and homeostasis in isolated type II alveolar cells

International Nuclear Information System (INIS)

Saito, K.; Warshaw, J.B.; Prough, R.A.

1986-01-01

After isolation of Type II cells from neonatal rat lung, the glutathione (GSH) levels in these cells were greatly depressed. The total glutathione content could be increased 5-fold within 12-24 h by incubating the cells in media containing sulfur amino acids. Similarly, the activity of γ-glutamyltranspeptidase was low immediately after isolation, but was increased 2-fold during the first 24 h culture. Addition of either GSH or GSSG to the culture media increased the GSH content of Type II cells 2-2.5-fold. Buthionine sulfoximine and NaF prevented this replenishment of GSH during 24 h culture. When the rates of de novo synthesis of GSH and GSSG from 35 S-cysteine were measured, the amounts of newly formed GSH decreased to 80% in the presence of GSH or GSSG. This suggests that exogenous GSH/GSSG can be taken up by the Type II cells to replenish the intracellular pool of GSH. Methionine was not as effective as cysteine in the synthesis of GSH. These results suggest that GSH levels in the isolated Type II cell can be maintained by de novo synthesis or uptake of exogenous GSH. Most of the GSH synthesized from cysteine, however, was excreted into the media of the cultured cells indicative of a potential role for the type II cell in export of the non-protein thiol

Propagation of Asian isolates of canine distemper virus (CDV in hamster cell lines

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Yamaguchi Ryoji

2009-10-01

Full Text Available Abstract Backgrounds The aim of this study was to confirm the propagation of various canine distemper viruses (CDV in hamster cell lines of HmLu and BHK, since only a little is known about the possibility of propagation of CDV in rodent cells irrespective of their epidemiological importance. Methods The growth of CDV in hamster cell lines was monitored by titration using Vero.dogSLAMtag (Vero-DST cells that had been proven to be susceptible to almost all field isolates of CDV, with the preparations of cell-free and cell-associated virus from the cultures infected with recent Asian isolates of CDV (13 strains and by observing the development of cytopathic effect (CPE in infected cultures of hamster cell lines. Results Eleven of 13 strains grew in HmLu cells, and 12 of 13 strains grew in BHK cells with apparent CPE of cell fusion in the late stage of infection. Two strains and a strain of Asia 1 group could not grow in HmLu cells and BHK cells, respectively. Conclusion The present study demonstrates at the first time that hamster cell lines can propagate the majority of Asian field isolates of CDV. The usage of two hamster cell lines suggested to be useful to characterize the field isolates biologically.

Isolation and expansion of adipose-derived stem cells for tissue engineering

DEFF Research Database (Denmark)

Fink, Trine; Rasmussen, Jeppe Grøndahl; Lund, Pia

2011-01-01

For treatment of cardiac failure with bone marrow-derived mesenchymal stem cells, several clinical trials are ongoing. However, more attention is gathering on the use of adipose tissue-derived stem cells (ASCs). This paper describes the optimization of isolation and propagation of ASCs for subseq......For treatment of cardiac failure with bone marrow-derived mesenchymal stem cells, several clinical trials are ongoing. However, more attention is gathering on the use of adipose tissue-derived stem cells (ASCs). This paper describes the optimization of isolation and propagation of ASCs...

Isolation of nucleoli from Ehrlich ascites tumor cells and dynamics of nascent RNA within isolated nucleoli.

Science.gov (United States)

Thiry, Marc; Ploton, Dominique

2008-01-01

Here we describe a new, rapid method for isolating nucleoli from Ehrlich tumor cells that preserves their morphological integrity and high transcriptional activity. Until now, methods for isolation of nucleoli were generally assumed to empty one of their three main compartments, the fibrillar center, of its contents. This new method consists of sonicating cells in an isotonic medium containing MgSO(4), spermidine, and spermine, followed by separation of nucleoli through a Percoll density gradient. Using the nonisotopic approach of labelling with BrUTP, we have further investigated the dynamics of nascent ribosomal RNAs (rRNAs) within morphologically intact isolated nucleoli at the electron microscope level. We show that ribosomal transcripts are elongated in the cortex of the fibrillar center and then enter the surrounding dense fibrillar component.

Reproducible isolation of lymph node stromal cells reveals site-dependent differences in fibroblastic reticular cells

Directory of Open Access Journals (Sweden)

Anne L Fletcher

2011-09-01

Full Text Available Within lymph nodes, non-hematopoietic stromal cells organize and interact with leukocytes in an immunologically important manner. In addition to organizing T and B cell segregation and expressing lymphocyte survival factors, several recent studies have shown that lymph node stromal cells shape the naïve T cell repertoire, expressing self-antigens which delete self-reactive T cells in a unique and non-redundant fashion. A fundamental role in peripheral tolerance, in addition to an otherwise extensive functional portfolio, necessitates closer study of lymph node stromal cell subsets using modern immunological techniques; however this has not routinely been possible in the field, due to difficulties reproducibly isolating these rare subsets. Techniques were therefore developed for successful ex vivo and in vitro manipulation and characterization of lymph node stroma. Here we discuss and validate these techniques in mice and humans, and apply them to address several unanswered questions regarding lymph node composition. We explored the steady-state stromal composition of lymph nodes isolated from mice and humans, and found that marginal reticular cells and lymphatic endothelial cells required lymphocytes for their normal maturation in mice. We also report alterations in the proportion and number of fibroblastic reticular cells (FRCs between skin-draining and mesenteric lymph nodes. Similarly, transcriptional profiling of FRCs revealed changes in cytokine production from these sites. Together, these methods permit highly reproducible stromal cell isolation, sorting, and culture.

Soft switching PWM isolated boost converter for fuel cell application

Energy Technology Data Exchange (ETDEWEB)

Rezaei, M.; Adib, E. [Isfahan Univ. of Technology, Isfahan (Iran, Islamic Republic of)

2009-07-01

This presentation introduced a newly developed soft switching, isolated boost type converter for fuel cell applications. With a simple PWM control circuit, the converter achieves zero voltage switching the main switch. Since the auxiliary circuit is soft switched, the converter can operate at high powers which make it suitable for fuel cell applications. In particular, the converter is suitable for the interface of fuel cell and inverters because of its high voltage gain and isolation between input and output sources. In addition, the input current of the converter (current drained from the fuel cell) is almost constant since it is a boost type converter. The converter was analyzed and the simulation results validate the theoretical analysis.

Isolating peripheral lymphocytes by density gradient centrifugation and magnetic cell sorting.

Science.gov (United States)

Brosseron, Frederic; Marcus, Katrin; May, Caroline

2015-01-01

Combining density gradient centrifugation with magnetic cell sorting provides a powerful tool to isolate blood cells with high reproducibility, yield, and purity. It also allows for subsequent separation of multiple cell types, resulting in the possibility to analyze different purified fractions from one donor's sample. The centrifugation step divides whole blood into peripheral blood mononuclear cells (PBMC), erythrocytes, and platelet-rich plasma. In the following, lymphocyte subtypes can be consecutively isolated from the PBMC fraction. This chapter describes enrichment of erythrocytes, CD14-positive monocytes and CD3-positive T lymphocytes. Alternatively, other cell types can be targeted by using magnetic beads specific for the desired subpopulation.

Isolation and establishment of radiotolerant hepatoma cell subline

International Nuclear Information System (INIS)

Jin Wensen; Kong Zhaolu; Zhang Jianghong; Shen Zhifen; Tong Shungao; Ji Huajun; Jin Yizun

2009-01-01

Objective: To induce and isolate the monoclonal cell subline, in order to establish the experimental model for further investigating biologic characteristics in radiotolerant hepatoma cells. Methods: HepG2 cells were irradiated by γ-rays at the dose of 2 Gy each time with the total absorbed dose of 60 Gy. After monoclonal cell being selected and extensively cultured, the cell subline was named as HepG2/R60. Furthermore, HepG2/R60 cells were identified by observing the changes of morphology, ultrastructure, growth characteristics and radiosensitivity. The levels of radioresistant correlative gene mRNA in HepG2/R60 cells after exposure to 2 Gy irradiation, were also detected by RT-PCR, and then compared with parental HepG2 cells. Results: HepG2/R60 cell subline was successfully established by fractionated irradiation at 2 Gy. HepG2/R60 cells displayed higher irregularity, the clearer appearance and dissociation of cell junctions compared with parental HepG2 cells. Ultrastnictural investigations through transmission electron microscopy (TEM) showed that there was an increase of microvillus on the surfaces of HepG2/R60 cells with plenty of rough endo-plasmic reticulum, abundance of mitochondria and viable Golgi complex. Further observation found that the growth of HepG2/R60 cells was slower and its population doubling time (PDT) prolonged arrived at 34.9 h. Moreover, the radiosensitivity of HepG2/R60 cells was lower than that of parental HepG2 cells. Additionally, the levels of radioresistance correlative genes were increased in HepG2/R60 cells by 2 Gy irradiaiton Conclusions: Radiotolerant cell subline - HepG2/R60 was successfully isolated and established by fractionated irradiation. (authors)

Isolation and characterization of equine peripheral blood-derived multipotent mesenchymal stromal cells

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Armando de M. Carvalho

2013-09-01

Full Text Available The objective of the study was to isolate, cultivate and characterize equine peripheral blood-derived multipotent mesenchymal stromal cells (PbMSCs. Peripheral blood was collected, followed by the isolation of mononuclear cells using density gradient reagents, and the cultivation of adherent cells. Monoclonal mouse anti-horse CD13, mouse anti-horse CD44, and mouse anti-rat CD90 antibodies were used for the immunophenotypic characterization of the surface of the PbMSCs. These cells were also cultured in specific media for adipogenic and chondrogenic differentiation. There was no expression of the CD13 marker, but CD44 and CD90 were expressed in all of the passages tested. After 14 days of cell differentiation into adipocytes, lipid droplets were observed upon Oil Red O (ORO staining. Twenty-one days after chondrogenic differentiation, the cells were stained with Alcian Blue. Although the technique for the isolation of these cells requires improvement, the present study demonstrates the partial characterization of PbMSCs, classifying them as a promising type of progenitor cells for use in equine cell therapy.

Isolation of Primary Murine Retinal Ganglion Cells (RGCs) by Flow Cytometry.

Science.gov (United States)

Chintalapudi, Sumana R; Patel, Need N; Goldsmith, Zachary K; Djenderedjian, Levon; Wang, Xiang Di; Marion, Tony N; Jablonski, Monica M; Morales-Tirado, Vanessa M

2017-07-05

Neurodegenerative diseases often have a devastating impact on those affected. Retinal ganglion cell (RGC) loss is implicated in an array of diseases, including diabetic retinopathy and glaucoma, in addition to normal aging. Despite their importance, RGCs have been extremely difficult to study until now due in part to the fact that they comprise only a small percentage of the wide variety of cells in the retina. In addition, current isolation methods use intracellular markers to identify RGCs, which produce non-viable cells. These techniques also involve lengthy isolation protocols, so there is a lack of practical, standardized, and dependable methods to obtain and isolate RGCs. This work describes an efficient, comprehensive, and reliable method to isolate primary RGCs from mice retinae using a protocol based on both positive and negative selection criteria. The presented methods allow for the future study of RGCs, with the goal of better understanding the major decline in visual acuity that results from the loss of functional RGCs in neurodegenerative diseases.

A modified efficient method for dental pulp stem cell isolation

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Maryam Raoof

2014-01-01

Full Text Available Background: Dental pulp stem cells can be used in regenerative endodontic therapy. The aim of this study was to introduce an efficient method for dental pulp stem cells isolation. Materials and Methods: In this in-vitro study, 60 extracted human third molars were split and pulp tissue was extracted. Dental pulp stem cells were isolated by the following three different methods: (1 digestion of pulp by collagenase/dispase enzyme and culture of the released cells; (2 outgrowth of the cells by culture of undigested pulp pieces; (3 digestion of pulp tissue pieces and fixing them. The cells were cultured in minimum essential medium alpha modification (αMEM medium supplemented with 20% fetal bovine serum(FBS in humid 37°C incubator with 5% CO 2 . The markers of stem cells were studied by reverse transcriptase polymerase chain reaction (PCR. The student t-test was used for comparing the means of independent groups. P <0.05 was considered as significant. Results: The results indicated that by the first method a few cell colonies with homogenous morphology were detectable after 4 days, while in the outgrowth method more time was needed (10-12 days to allow sufficient numbers of heterogeneous phenotype stem cells to migrate out of tissue. Interestingly, with the improved third method, we obtained stem cells successfully with about 60% efficiency after 2 days. The results of RT-PCR suggested the expression of Nanog, Oct-4, and Nucleostemin markers in the isolated cells from dental pulps. Conclusion: This study proposes a new method with high efficacy to obtain dental pulp stem cells in a short time.

Specific single-cell isolation and genomic amplification of uncultured microorganisms

DEFF Research Database (Denmark)

Kvist, Thomas; Ahring, Birgitte Kiær; Lasken, R.S.

2007-01-01

We in this study describe a new method for genomic studies of individual uncultured prokaryotic organisms, which was used for the isolation and partial genome sequencing of a soil archaeon. The diversity of Archaea in a soil sample was mapped by generating a clone library using group-specific pri......We in this study describe a new method for genomic studies of individual uncultured prokaryotic organisms, which was used for the isolation and partial genome sequencing of a soil archaeon. The diversity of Archaea in a soil sample was mapped by generating a clone library using group......-specific primers in combination with a terminal restriction fragment length polymorphism profile. Intact cells were extracted from the environmental sample, and fluorescent in situ hybridization probing with Cy3-labeled probes designed from the clone library was subsequently used to detect the organisms...... of interest. Single cells with a bright fluorescent signal were isolated using a micromanipulator and the genome of the single isolated cells served as a template for multiple displacement amplification (MDA) using the Phi29 DNA polymerase. The generated MDA product was afterwards used for 16S rRNA gene...

Large-scale Isolation of Highly Pure "Untouched" Regulatory T Cells in a GMP Environment for Adoptive Cell Therapy.

Science.gov (United States)

Haase, Doreen; Puan, Kia Joo; Starke, Mireille; Lai, Tuck Siong; Soh, Melissa Yan Ling; Karunanithi, Iyswariya; San Luis, Boris; Poh, Tuang Yeow; Yusof, Nurhashikin; Yeap, Chun Hsien; Phang, Chew Yen; Chye, Willis Soon Yuan; Chan, Marieta; Koh, Mickey Boon Chai; Goh, Yeow Tee; Bertin-Maghit, Sebastien; Nardin, Alessandra; Ho, Liam Pock; Rotzschke, Olaf

2015-01-01

Adoptive cell therapy is an emerging treatment strategy for a number of serious diseases. Regulatory T (Treg) cells represent 1 cell type of particular interest for therapy of inflammatory conditions, as they are responsible for controlling unwanted immune responses. Initial clinical trials of adoptive transfer of Treg cells in patients with graft-versus-host disease were shown to be safe. However, obtaining sufficient numbers of highly pure and functional Treg cells with minimal contamination remains a challenge. We developed a novel approach to isolate "untouched" human Treg cells from healthy donors on the basis of negative selection using the surface markers CD49d and CD127. This procedure, which uses an antibody cocktail and magnetic beads for separation in an automated system (RoboSep), was scaled up and adapted to be compatible with good manufacturing practice conditions. With this setup we performed 9 Treg isolations from large-scale leukapheresis samples in a good manufacturing practice facility. These runs yielded sufficient numbers of "untouched" Treg cells for immediate use in clinical applications. The cell preparations consisted of viable highly pure FoxP3-positive Treg cells that were functional in suppressing the proliferation of effector T cells. Contamination with CD4 effector T cells was cell types did not exceed 2% in the final product. Remaining isolation reagents were reduced to levels that are considered safe. Treg cells isolated with this procedure will be used in a phase I clinical trial of adoptive transfer into leukemia patients developing graft-versus-host disease after stem cell transplantation.

Identification of different subsets of lung cells using Raman microspectroscopy and whole cell nucleus isolation.

Science.gov (United States)

Pijanka, Jacek K; Stone, Nicholas; Rutter, Abigail V; Forsyth, Nicholas; Sockalingum, Ganesh D; Yang, Ying; Sulé-Suso, Josep

2013-09-07

Raman spectroscopy has been widely used to study its possible clinical application in cancer diagnosis. However, in order to make it into clinical practice, it is important that this technique is able not only to identify cancer cells from their normal counterparts, but also from the array of cells present in human tissues. To this purpose, we used Raman spectroscopy to assess whether this technique was able to differentiate not only between lung cancer cells and lung epithelial cells but also from lung fibroblasts. Furthermore, we studied whether the differences were due to cell lineage (epithelial versus fibroblast) or to different proliferative characteristics of cells, and where in the cell compartment these differences might reside. To answer these questions we studied cell cytoplasm, cell nucleus and isolated whole cell nuclei. Our data suggests that Raman spectroscopy can differentiate between lung cancer, lung epithelial cells and lung fibroblasts. More important, it can also differentiate between 2 cells from the same lineage (fibroblast) but with one of them rendered immortal and with an increased proliferative activity. Finally, it seems that the main spectral differences reside in the cell nucleus and that the study of isolated nuclei strengthens the differences between cells.

Isolation of pronephros cells which endocytose chemically modified proteins in the rainbow trout

International Nuclear Information System (INIS)

Dannevig, B.H.; Berg, T.

1986-01-01

Modified serum albumin is cleared from the blood by kidney cells in salmonid fishes. The present study deals with isolation of cells from pronephros which endocytose formaldehyde-treated human serum albumin (fHSA). Radioactively labelled fHSA or dinitrophenyl-conjugated albumin (DNP-HSA) were injected intravenously into rainbow trouts. Pronephros cells, containing the endocytosed protein, were isolated and further separated by centrifugal elutriation and density-gradient centrifugation. Most of the radioactive protein was elutriated together with small cells. After centrifuging the cells through a Percoll density gradient, radioactive protein was located in cells recovered in the upper part of the gradient. In mammals, fHSA and other modified proteins are mainly taken up by sinusoidal endothelial cells in the liver via a scavenger receptor 0. Our results suggest that a comparable function in salmonids is located in a subpopulation of relatively small cells in kidney tissue, possibly sinusoidal lining cells. The separation techniques used seemed to be suitable for isolation of different populations of pronephros cells

Etanercept blocks inflammatory responses orchestrated by TNF-α to promote transplanted cell engraftment and proliferation in rat liver

Science.gov (United States)

Viswanathan, Preeti; Kapoor, Sorabh; Kumaran, Vinay; Joseph, Brigid; Gupta, Sanjeev

2014-01-01

Engraftment of transplanted cells is critical for liver-directed cell therapy but most transplanted cells are rapidly cleared from liver sinusoids by proinflammatory cytokines/chemokines/receptors after activation of neutrophils or Kupffer cells. To define whether TNF-α served roles in cell-transplantation-induced hepatic inflammation, we used TNF-α antagonist, etanercept, for studies in syngeneic rat hepatocyte transplantation systems. After cell transplantation, multiple cytokines/chemokines/receptors were overexpressed, whereas etanercept prior to cell transplantation essentially normalized these responses. Moreover, ETN downregulated cell transplantation-induced intrahepatic release of secretory cytokines, such as high mobility group box 1. These effects of etanercept decreased cell transplantation-induced activation of neutrophils but not of Kupffer cells. Transplanted cell engraftment improved by several-fold in etanercept-treated animals. These gains in cell engraftment were repeatedly realized after pretreatment of animals with etanercept before multiple cell transplantation sessions. Transplanted cell numbers did not change over time indicating absence of cell proliferation after etanercept alone. By contrast, in animals preconditioned with retrorsine and partial hepatectomy, cell transplantation after etanercept pretreatment significantly accelerated liver repopulation compared with control rats. We concluded that TNF-α played a major role in orchestrating cell transplantation-induced inflammation through regulation of multiple cytokines/chemokines/receptor expression. As TNF-α antagonism by etanercept decreased transplanted cell clearance, improved cell engraftment and accelerated liver repopulation, this pharmacological approach to control hepatic inflammation will help optimize clinical strategies for liver cell therapy. PMID:24844924

Isolation, culture and adenoviral transduction of parietal cells from mouse gastric mucosa

International Nuclear Information System (INIS)

Gliddon, Briony L; Nguyen, Nhung V; Gunn, Priscilla A; Gleeson, Paul A; Driel, Ian R van

2008-01-01

Here we describe a method for the isolation of intact gastric glands from mice and primary culture and transfection of mouse gastric epithelial cells. Collagenase digestion of PBS-perfused mouse stomachs released large intact gastric glands that were plated on a basement membrane matrix. The heterogeneous gland cell cultures typically contain ∼60% parietal cells. Isolated mouse parietal cells remain viable in culture for up to 5 days and react strongly with an antibody specific to the gastric H + /K + ATPase. Isolated intact mouse gastric glands and primary cultures of mouse parietal cells respond to the secretagogue, histamine. Typical morphological changes from a resting to an acid-secreting active parietal cell were observed. In resting cultures of mouse parietal cells, the H + /K + ATPase displayed a cytoplasmic punctate staining pattern consistent with tubulovesicle element structures. Following histamine stimulation, an expansion of internal apical vacuole structures was observed together with a pronounced redistribution of the H + /K + ATPase from the cytoplasm to the apical vacuoles. A reproducible procedure to express genes of interest exogenously in these cultures of mouse parietal cells was also established. This method combines recombinant adenoviral transduction with magnetic field-assisted transfection resulting in ∼30% transduced parietal cells. Adenoviral-transduced parietal cells maintain their ability to undergo agonist-induced activation. This protocol will be useful for the isolation, culture and expression of genes in parietal cells from genetically modified mice and as such will be an invaluable tool for studying the complex exocytic and endocytic trafficking events of the H + /K + ATPase which underpin the regulation of acid secretion

Isolation and differentiation of stromal vascular cells to beige/brite cells

DEFF Research Database (Denmark)

Aune, Ulrike Liisberg; Ruiz, Lauren; Kajimura, Shingo

2013-01-01

cold exposure or by PPARγ agonists, therefore, this cell type has potential as a therapeutic target for obesity treatment. Although most immortalized adipocyte lines cannot recapitulate the process of "browning" of white fat in culture, primary adipocytes isolated from stromal vascular fraction...

Cell Culture Isolation of Piscine Nodavirus (Betanodavirus) in Fish-Rearing Seawater.

Science.gov (United States)

Nishi, Shinnosuke; Yamashita, Hirofumi; Kawato, Yasuhiko; Nakai, Toshihiro

2016-04-01

Piscine nodavirus (betanodavirus) is the causative agent of viral nervous necrosis (VNN) in a variety of cultured fish species, particularly marine fish. In the present study, we developed a sensitive method for cell culture isolation of the virus from seawater and applied the method to a spontaneous fish-rearing environment. The virus in seawater was concentrated by an iron-based flocculation method and subjected to isolation with E-11 cells. A real-time reverse transcriptase PCR (RT-PCR) assay was used to quantify the virus in water. After spiking into seawater was performed, a betanodavirus strain (red spotted grouper nervous necrosis virus [RGNNV] genotype) was effectively recovered in the E-11 cells at a detection limit of approximately 10(5)copies (equivalent to 10(2)50% tissue culture infective doses [TCID50])/liter seawater. In an experimental infection of juvenile sevenband grouper (Epinephelus septemfasciatus) with the virus, the virus was isolated from the drainage of a fish-rearing tank when the virus level in water was at least approximately 10(5)copies/liter. The application of this method to seven band grouper-rearing floating net pens, where VNN prevailed, resulted in the successful isolation of the virus from seawater. No differences were found in the partial sequences of the coat protein gene (RNA2) between the clinical virus isolates of dead fish and the cell-cultured virus isolates from seawater, and the viruses were identified as RGNNV. The infection experiment showed that the virus isolates from seawater were virulent to seven band grouper. These results showed direct evidence of the horizontal transmission of betanodavirus via rearing water in marine aquaculture. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Cell-cell interactions of isolated and cultured oligodendrocytes: formation of linear occluding junctions and expression of peculiar intramembrane particles.

Science.gov (United States)

Massa, P T; Szuchet, S; Mugnaini, E

1984-12-01

Oligodendrocytes were isolated from lamb brain. Freshly isolated cells and cultured cells, either 1- to 4-day-old unattached or 1- to 5-week-old attached, were examined by thin section and freeze-fracture electron microscopy. Freeze-fracture of freshly isolated oligodendrocytes showed globular and elongated intramembrane particles similar to those previously described in oligodendrocytes in situ. Enrichment of these particles was seen at sites of inter-oligodendrocyte contact. Numerous gap junctions and scattered linear tight junctional arrays were apparent. Gap junctions were connected to blebs of astrocytic plasma membrane sheared off during isolation, whereas tight junctions were facing extracellular space or blebs of oligodendrocytic plasma membrane. Thin sections of cultured, unattached oligodendrocytes showed rounded cell bodies touching one another at points without forming specialized cell junctions. Cells plated on polylysine-coated aclar dishes attached, emanated numerous, pleomorphic processes, and expressed galactocerebroside and myelin basic protein, characteristic markers for oligodendrocytes. Thin sections showed typical oligodendrocyte ultrastructure but also intermediate filaments not present in unattached cultures. Freeze-fracture showed intramembrane particles similar to but more numerous, and with a different fracture face repartition, than those seen in oligodendrocytes, freshly isolated or in situ. Gap junctions were small and rare. Apposed oligodendrocyte plasma membrane formed linear tight junctions which became more numerous with time in culture. Thus, cultured oligodendrocytes isolated from ovine brains develop and maintain features characteristic of mature oligodendrocytes in situ and can be used to explore formation and maintenance of tight junctions and possibly other classes of cell-cell interactions important in the process of myelination.

Isolation and in vitro expansion of human colonic stem cells

NARCIS (Netherlands)

Jung, P.; Sato, T.; Merlos-Suarez, A.; Barriga, F.M.; Iglesias, M.; Rossell, D.; Auer, H.; Gallardo, M.; Blasco, M.A.; Sancho, E.; Clevers, H.; Batlle, E.

2011-01-01

Here we describe the isolation of stem cells of the human colonic epithelium. Differential cell surface abundance of ephrin type-B receptor 2 (EPHB2) allows the purification of different cell types from human colon mucosa biopsies. The highest EPHB2 surface levels correspond to epithelial colonic

Isolation of intact RNA from murine CD4+ T cells after intracellular cytokine staining and fluorescence-activated cell sorting.

Science.gov (United States)

Kunnath-Velayudhan, Shajo; Porcelli, Steven A

2018-05-01

Intracellular cytokine staining (ICS) is a powerful method for identifying functionally distinct lymphocyte subsets, and for isolating these by fluorescence activated cell sorting (FACS). Although transcriptomic analysis of cells sorted on the basis of ICS has many potential applications, this is rarely performed because of the difficulty in isolating intact RNA from cells processed using standard fixation and permeabilization buffers for ICS. To address this issue, we compared three buffers shown previously to preserve RNA in nonhematopoietic cells subjected to intracellular staining for their effects on RNA isolated from T lymphocytes processed for ICS. Our results showed that buffers containing the recombinant ribonuclease inhibitor RNasin or high molar concentrations of salt yielded intact RNA from fixed and permeabilized T cells. As proof of principle, we successfully used the buffer containing RNasin to isolate intact RNA from CD4 + T cells that were sorted by FACS on the basis of specific cytokine production, thus demonstrating the potential of this approach for coupling ICS with transcriptomic analysis. Copyright © 2018 Elsevier B.V. All rights reserved.

Cellular location of rat muscle ferritins and their preferential loss during cell isolation.

Science.gov (United States)

Linder, M C; Roboz, M; McKown, M J; Pardridge, W M; Zak, R

1984-04-10

Heart and other muscles of the rat contain two forms of ferritin separable in polyacrylamide gel electrophoresis. The cellular location of the fast- and slow-migrating ferritins was investigated using primary cultures of hindlimb skeletal muscle, and isolated myocardial cell populations. Muscle and non-muscle cells were isolated in good yield from hearts of adult rats pretreated with large doses of iron to increase their ferritin content. In virtually all cases, the isolated muscle cells contained traces only of the fast-migrating species and the non-muscle cells contained small amounts of the slow-migrating ferritin. During cell isolation, 90-100% of both ferritins was lost and could be recovered in the perfusates and solutions employed, while one third of the total tissue protein, and a larger percentage of creatine phosphokinase, was recovered in the isolated cells. Primary cultures of thigh muscle from adult rats which had differentiated into multi-nucleated myotubes, were incubated for 1-3 days with chelated iron. These cells contained substantial amounts of the electrophoretically fast migrating ferritin, with its characteristic larger Stokes' radius (determined by quantitative polyacrylamide gel electrophoresis). None of the slow-migrating ferritin species was detected, although hindlimb muscle from iron-treated rats contained both forms. It is concluded that the fast-migrating ferritin of muscle, which is much larger and more asymmetric than other ferritins, is confined to the muscle cell population, while the other form is predominantly or exclusively in the non-muscle cells. Both ferritins are lost preferentially over other proteins during procedures which injure muscle tissue.

Effects of methyl palmitate on cytokine release, liver injury and survival in mice with sepsis.

Science.gov (United States)

Villa, P; Demitri, M T; Meazza, C; Sironi, M; Gnocchi, P; Ghezzi, P

1996-12-01

The effects of methyl palmitate (MP), a known inhibitor of Kupffer cells, were studied in a model of polymicrobial sepsis induced in CD-1 mice by cecal ligation and puncture (CLP). The inhibition of Kupffer cells by pretreatment with MP was shown by the reduced phagocytosis, the production of tumor necrosis factor (TNF) and interleukin-6 (IL-6) after lipopolysaccharide (LPS) challenge. The reduced activation of Kupffer cells resulted in lower levels of inflammatory products after CLP. TNF and IL-6 were significantly reduced in serum 2 h and 24 h respectively after CLP, interleukin-1 beta (IL-1 beta) was reduced in liver 4 h after CLP, nitric oxide (NO) and serum amyloid A (SAA) were significantly reduced 8 and 24 h respectively after CLP. Liver toxicity was significantly reduced in MP-treated mice and survival was significantly prolonged at all intervals, reaching 45% after six to ten days compared with 3% in control mice. These findings suggest that Kupffer cells play an important role in liver damage and survival in sepsis.

Host-pathogen interactions in bovine mammary epithelial cells and HeLa cells by Staphylococcus aureus isolated from subclinical bovine mastitis.

Science.gov (United States)

Castilho, Ivana G; Dantas, Stéfani Thais Alves; Langoni, Hélio; Araújo, João P; Fernandes, Ary; Alvarenga, Fernanda C L; Maia, Leandro; Cagnini, Didier Q; Rall, Vera L M

2017-08-01

Staphylococcus aureus is a common pathogen that causes subclinical bovine mastitis due to several virulence factors. In this study, we analyzed S. aureus isolates collected from the milk of cows with subclinical mastitis that had 8 possible combinations of bap, icaA, and icaD genes, to determine their capacity to produce biofilm on biotic (bovine primary mammary epithelial cells and HeLa cells) and abiotic (polystyrene microplates) surfaces, and their ability to adhere to and invade these cells. We also characterized isolates for microbial surface components recognizing adhesive matrix molecules (MSCRAMM) and agr genes, and for their susceptibility to cefquinome sulfate in the presence of biofilm. All isolates adhered to and invaded both cell types, but invasion indexes were higher in bovine primary mammary epithelial cells. Using tryptic soy broth + 1% glucose on abiotic surfaces, 5 out of 8 isolates were biofilm producers, but only the bap + icaA + icaD + isolate was positive in Dulbecco's Modified Eagle's medium. The production of biofilm on biotic surfaces occurred only with this isolate and only on HeLa cells, because the invasion index for bovine primary mammary epithelial cells was too high, making it impossible to use these cells in this assay. Of the 5 biofilm producers in tryptic soy broth + 1% glucose, 4 presented with the bap/fnbA/clfA/clfB/eno/fib/ebpS combination, and all were protected from cefquinome sulfate. We found no predominance of any agr group. The high invasive potential of S. aureus made it impossible to observe biofilm in bovine primary mammary epithelial cells, and we concluded that cells with lower invasion rates, such as HeLa cells, were more appropriate for this assay. Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Culture conditions for bovine embryonic stem cell-like cells isolated from blastocysts after external fertilization

OpenAIRE

Jin, Muzi; Wu, Asga; Dorzhin, Sergei; Yue, Qunhua; Ma, Yuzhen; Liu, Dongjun

2012-01-01

Although isolation and characterization of embryonic stem cells have been successful in cattle, maintenance of bovine embryonic stem cells in culture remains difficult. In this study, we compared different methods of cell passaging, feeder cell layers and medium conditions for bovine embryonic stem cell-like cells. We found that a murine embryonic fibroblast feeder layer is more suitable for embryonic stem cell-like cells than bovine embryonic fibroblasts. When murine embryonic fibroblasts we...

Isolation, culture expansion and characterization of canine bone marrow derived mesenchymal stem cells

Directory of Open Access Journals (Sweden)

D Kazemi

2016-07-01

Full Text Available The purpose of the present study was to isolate, culture expand and characterize canine bone marrow derived mesenchymal stem cells. Bone marrow aspirates of 15 adult male dogs were collected to this end and their mononuclear cells isolated by centrifugation and cultured in standard media. The adherent cells were isolated and their mesenchymal origin was confirmed at 3rd passage by cellular morphology, expression of surface antigens and differentiation to osteogenic and adipogenic lineage. After 4 days, spindle shaped fibroblast like cells which were apparently bone marrow derived mesenchymal stem cells appeared in culture medium and their numbers increased over time. The cells reached 3rd passage with over 75% confluent after a mean of 22.89±5.75 days. Flow cytometric analysis revealed that the cells negatively expressed CD34 and CD45 antigens while positively expressing CD44 and CD105 antigens. Differentiation into osteogenic and adipogenic lineage had taken place after one month culture in induction medium. VDR, COL1A1, BGLAP and SPARC gene expression indicated that mesenchymal stem cells isolated from canine bone marrow had differentiated into osteogenic lineage. These findings can form the basis of any forthcoming clinical studies involving the use of canine mesenchymal stem cells particularly in the field of bone and cartilage regeneration.

Cell pairing ratio controlled micro-environment with valve-less electrolytic isolation

KAUST Repository

Chen, Yu-Chih; Lou, Xia; Ingram, Patrick; Yoon, Euisik

2012-01-01

We present a ratio controlled cell-to-cell interaction chip using valve-less isolation. We incorporated electrolysis in a microfluidic channel. In each microfluidic chamber, we loaded two types of different cells at various pairing ratios. More than

Isolated tumor endothelial cells maintain specific character during long-term culture

International Nuclear Information System (INIS)

Matsuda, Kohei; Ohga, Noritaka; Hida, Yasuhiro; Muraki, Chikara; Tsuchiya, Kunihiko; Kurosu, Takuro; Akino, Tomoshige; Shih, Shou-Ching

2010-01-01

Tumor angiogenesis is necessary for solid tumor progression and metastasis. Increasing evidence indicates that tumor endothelial cells (TECs) are more relevant to the study of tumor angiogenesis than normal endothelial cells (NECs) because their morphologies and gene expression are different from NECs. However, it is challenging to isolate and culture large numbers of pure ECs from tumor tissue since the percentage of ECs is only about 1-2% and tumor cells and fibroblasts easily overgrow them. In addition, there has been concern that isolated TECs may lose their special phenotype once they are dissociated from tumor cells. In this study, we have successfully purified murine TECs from four different human tumor xenografts and NECs from murine dermal tissue. Isolated ECs expressed endothelial markers, such as CD31, VE-cadherin (CD144), and endoglin (CD105), for more than 3 months after isolation. TECs maintained tumor endothelial-specific markers, such as tumor endothelial marker 8 (TEM8) and aminopeptidase N (APN), as in tumor blood vessels in vivo. In addition, TECs were more proliferative and motile than NECs. TECs showed a higher response to VEGF and higher expression of VEGF receptors-1 and -2 than NECs did. Stem cell antigen-1 was up-regulated in all four TECs, suggesting that they have a kind of stemness. Cultured TECs maintain distinct biological differences from NECs as in vivo. In conclusion, it was suggested that TECs are relevant material for tumor angiogenesis research.

Effects of defibrotide, a novel oligodeoxyribonucleotide, on ischaemia and reperfusion injury of the rat liver.

Science.gov (United States)

Kim, Kwang Joon; Shin, Yong Kyoo; Song, Jin Ho; Oh, Byung Kwon; Choi, Myung Sup; Sohn, Uy Dong

2002-02-01

1. The purpose of this study was to investigate the protective effects of defibrotide, a single-stranded polydeoxyribonucleotide, on ischaemia-reperfusion injury to the liver using a rat model. 2. Ischaemia of the left and median lobes was created by total inflow occlusion for 30 min followed by 60 min of reperfusion. Hepatic injury was assessed by the release of liver enzymes (alanine transferase, ALT and lactic dehydrogenase, LDH). Hepatic oxidant stress was measured by superoxide production, lipid peroxidation and nitrite/nitrate formation. Leukocyte-endothelium interaction and Kupffer cell mobilization were quantified by measuring hepatic myeloperoxidase (MPO), polymorphonuclear leukocyte adherence to superior mesenteric artery (SMA) and immunostaining of Kupffer cell. 3. Defibrotide treatment resulted in a significant inhibition of postreperfusion superoxide generation, lipid peroxidation, serum ALT activity, serum LDH activity, MPO activity, serum nitrite/nitrate level, leukocyte adherence to SMA, and Kupffer cell mobilization, indicating a significant attenuation of hepatic dysfunction. 4. A significant correlation existed between liver ischaemia/reperfusion and hepatic injury, suggesting that liver ischaemia/reperfusion injury is mediated predominantly by generation of oxygen free radicals and mobilization of Kupffer cells. 5. We conclude that defibrotide significantly protects the liver against liver ischaemia/reperfusion injury by interfering with Kupffer cell mobilization and formation of oxygen free radicals. This study provides strong evidence that defibrotide has important beneficial effects on acute inflammatory tissue injury such as that occurring in the reperfusion of the ischaemic liver.

Isolation of sphere-forming stem cells from the mouse inner ear.

Science.gov (United States)

Oshima, Kazuo; Senn, Pascal; Heller, Stefan

2009-01-01

The mammalian inner ear has very limited ability to regenerate lost sensory hair cells. This deficiency becomes apparent when hair cell loss leads to hearing loss as a result of either ototoxic insult or the aging process. Coincidently, with this inability to regenerate lost hair cells, the adult cochlea does not appear to harbor cells with a proliferative capacity that could serve as progenitor cells for lost cells. In contrast, adult mammalian vestibular sensory epithelia display a limited ability for hair cell regeneration, and sphere-forming cells with stem cell features can be isolated from the adult murine vestibular system. The neonatal inner ear, however, does harbor sphere-forming stem cells residing in cochlear and vestibular tissues. Here, we provide protocols to isolate sphere-forming stem cells from neonatal vestibular and cochlear sensory epithelia as well as from the spiral ganglion. We further describe procedures for sphere propagation, cell differentiation, and characterization of inner ear cell types derived from spheres. Sphere-forming stem cells from the mouse inner ear are an important tool for the development of cellular replacement strategies of damaged inner ears and are a bona fide progenitor cell source for transplantation studies.

Transplantation of an alginate-matrigel matrix containing isolated ovarian cells: first step in developing a biodegradable scaffold to transplant isolated preantral follicles and ovarian cells.

Science.gov (United States)

Vanacker, Julie; Luyckx, Valérie; Dolmans, Marie-Madeleine; Des Rieux, Anne; Jaeger, Jonathan; Van Langendonckt, Anne; Donnez, Jacques; Amorim, Christiani A

2012-09-01

For women diagnosed with leukemia, transplantation of cryopreserved ovarian tissue after disease remission is not advisable. Therefore, to restore fertility in these patients, we aim to develop a biodegradable artificial ovary that offers an environment where isolated follicles and ovarian cells (OCs) can survive and grow. Four NMRI mice were ovariectomized and their ovaries used to isolate OCs. Groups of 50,000 OCs were embedded in an alginate-matrigel matrix for further fixation (fresh controls), one week of in vitro culture (IVC) or heterotopic autografting. OC proliferation (Ki67), apoptosis (TUNEL), scaffold degradation, vessel formation (CD34) and inflammation (CD45) were analyzed. Ki67-positive OCs were found in 2.3%, 9.0% and 15.5% cells of cases in fresh, IVC and grafted beads respectively, while cells were TUNEL-positive in 0%, 1.5% and 6.9% of cases. After IVC or grafting, the beads degraded, losing their original round aspect, and infiltrating blood capillaries could be observed in the grafted beads. CD34-positive cells and 22% CD45-positive cells were found around and inside the matrix. In conclusion, our results demonstrate that an alginate-based matrix is a promising proposition to graft isolated OCs. After transplantation, this matrix was able to degrade, allowed vascularization and elicited a low inflammatory response. Copyright © 2012 Elsevier Ltd. All rights reserved.

Isolation and culture of neural crest cells from embryonic murine neural tube.

Science.gov (United States)

Pfaltzgraff, Elise R; Mundell, Nathan A; Labosky, Patricia A

2012-06-02

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types. NC also has the unique ability to influence the differentiation and maturation of target organs. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. NC multipotency was first described from explants of the avian neural tube. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter. The method presented here is adapted from

Live Cell Genomics: RNA Exon-Specific RNA-Binding Protein Isolation.

Science.gov (United States)

Bell, Thomas J; Eberwine, James

2015-01-01

RNA-binding proteins (RBPs) are essential regulatory proteins that control all modes of RNA processing and regulation. New experimental approaches to isolate these indispensable proteins under in vivo conditions are needed to advance the field of RBP biology. Historically, in vitro biochemical approaches to isolate RBP complexes have been useful and productive, but biological relevance of the identified RBP complexes can be imprecise or erroneous. Here we review an inventive experimental to isolate RBPs under the in vivo conditions. The method is called peptide nucleic acid (PNA)-assisted identification of RBP (PAIR) technology and it uses cell-penetrating peptides (CPPs) to deliver photo-activatible RBP-capture molecule to the cytoplasm of the live cells. The PAIR methodology provides two significant advantages over the most commonly used approaches: (1) it overcomes the in vitro limitation of standard biochemical approaches and (2) the PAIR RBP-capture molecule is highly selective and adaptable which allows investigators to isolate exon-specific RBP complexes. Most importantly, the in vivo capture conditions and selectivity of the RBP-capture molecule yield biologically accurate and relevant RBP data.

Isolation and evaluation of dental pulp stem cells from teeth with advanced periodontal disease.

Science.gov (United States)

Derakhshani, Ali; Raoof, Maryam; Dabiri, Shahriar; Farsinejad, Ali Reza; Gorjestani, Hedayat; Yaghoobi, Mohammad Mehdi; Shokouhinejad, Noushin; Ehsani, Maryam

2015-04-01

Successful isolation of mesenchymal stem cells from waste tissues might be extremely promising for developing stem cell-based therapies. This study aimed to explore whether cells retrieved from teeth extracted due to advanced periodontal disease present mesenchymal stem cell-like properties. Pulp cells were isolated from 15 intact molars and 15 teeth with advanced periodontal disease. Cell proliferation and markers of mesenchymal stem cells were evaluated. Based on the RT-PCR and agarose gel electrophoresis, nucleostemin, Oct-4 and jmj2c, but not Nanog, were expressed in undifferentiated mesenchymal stem cells of both groups. Interestingly, diseased pulp exhibited higher gene expressions although it was not statistically significant. The average percentage of BrdU positive cells in the diseased group (84.4%, n = 5) was significantly higher than that of the control group (65.4%, n = 5) (t-test, P = 0.001). Our results indicate the successful isolation of mesenchymal stem cells from the pulp tissue of hopeless periodontally involved teeth.

Isolation of Mesenchymal Stromal Cells (MSCs from Human Adenoid Tissue

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Yoon Se Lee

2013-04-01

Full Text Available Background: Mesenchymal stromal cells (MSCs are multipotent progenitor cells that originally derived from bone marrow. Clinical use of bone marrow-derived MSC is difficult due to morbidity and low MSC abundance and isolation efficiency. Recently, MSCs have been isolated from various adult tissues. Here we report the isolation of adenoid tissue-derived MSCs (A-MSCs and their characteristics. Methods: We compared the surface markers, morphologies, and differentiation and proliferation capacities of previously established tonsil-derived MSCs (T-MSCs and bone marrow-derived MSCs (BM-MSCs with cells isolated from adenoid tissue. The immunophenotype of A-MSCs was investigated upon interferon (IFN-γ stimulation. Results: A-MSCs, T-MSCs, and BM-MSCs showed negative CD45, CD31 HLA-DR, CD34, CD14, CD19 and positive CD 90, CD44, CD73, CD105 expression. A-MSCs were fibroblast-like, spindle-shaped non-adherent cells, similar to T-MSCs and BM-MSCs. Adipogenesis was observed in A-MSCs by the formation of lipid droplets after Oil Red O staining. Osteogenesis was observed by the formation of the matrix mineralization in Alizarin Red staining. Chondrogenesis was observed by the accumulation of sulfated glycosaminoglycan-rich matrix in collagen type II staining. These data were similar to those of T-MSCs and BM-MSCs. Expression of marker genes (i.e., adipogenesis; lipoprotein lipase, proliferator-activator receptor-gamma, osteogenesis; osteocalcin, alkaline phasphatase, chondrogenesis; aggrecan, collagen type II α1 in A-MSCs were not different from those in T-MSCs and BM-MSCs. Conclusions: A-MSCs possess the characteristics of MSCs in terms of morphology, multipotent differentiation capacity, cell surface markers, and immunogeneity. Therefore, A-MSCs fulfill the definition of MSCs and represent an alternate source of MSCs.

Methods Development for the Isolation and Culture of Primary Corneal Endothelial Cells

Science.gov (United States)

2017-02-01

a cell population particularly suitable for low serum propagation, provided that appropriate growth factors are available. A low serum medium...of MGK. 15. SUBJECT TERMS Cornea, chemical warfare agent, corneal endothelial cell, endothelium, growth , isolation, mouse, rabbit, porcine, in...with corneal SM exposure.2 A primary requirement in achieving this goal is to develop methods that enable the isolation of a pure CEC population and

Effect of neurturin on multipotent cells isolated from the adult skeletal muscle

International Nuclear Information System (INIS)

Vourc'h, Patrick; Lacar, Benjamin; Mignon, Laurence; Lucas, Paul A.; Young, Henry E.; Chesselet, Marie-Francoise

2005-01-01

Ligands of the glial cell line-derived neurotrophic factors (GDNF)-family are trophic factors for the development and survival of multiple cell types, however their effects on non-neuronal stem cells are unknown. We examined the action of neurturin on a candidate stem cell population isolated from adult skeletal muscles. When grown as spheres, these cells expressed mRNAs for GDNF, persephin, GFR-α2, GFR-α4 (neurturin receptor), and Ret. Exposure of these cells to neurturin significantly augmented cell numbers via increased cell proliferation. After addition of retinoic acid, the cells exited the cell cycle, developed thin processes, and became immunoreactive for βIII-tubulin, while Ret mRNA expression decreased, without changes in the level of GFR-α2 mRNA. Neurturin induced an outgrowth of processes on these βIII-tubulin positive cells. Neurturin may therefore be beneficial in the use of these multipotent cells isolated from adult muscles for autologous transplants in neurological applications

Isolation and genetic characterization of mother-of-snow-white, a maternal effect allele affecting laterality and lateralized behaviors in zebrafish.

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Alice Domenichini

Full Text Available In the present work we report evidence compatible with a maternal effect allele affecting left-right development and functional lateralization in vertebrates. Our study demonstrates that the increased frequency of reversed brain asymmetries in a zebrafish line isolated through a behavioral assay is due to selection of mother-of-snow-white (msw, a maternal effect allele involved in early stages of left-right development in zebrafish. msw homozygous females could be identified by screening of their progeny for the position of the parapineal organ because in about 50% of their offspring we found an altered, either bilateral or right-sided, expression of lefty1 and spaw. Deeper investigations at earlier stages of development revealed that msw is involved in the specification and differentiation of precursors of the Kupffer's vesicle, a structure homologous to the mammalian node. To test the hypothesis that msw, by controlling Kupffer's vesicle morphogenesis, controls lateralized behaviors related to diencephalic asymmetries, we analyzed left- and right-parapineal offspring in a "viewing test". As a result, left- and right-parapineal individuals showed opposite and complementary eye preference when scrutinizing a model predator, and a different degree of lateralization when scrutinizing a virtual companion. As maternal effect genes are expected to evolve more rapidly when compared to zygotic ones, our results highlight the driving force of maternal effect alleles in the evolution of vertebrates behaviors.

FXR agonist obeticholic acid reduces hepatic inflammation and fibrosis in a rat model of toxic cirrhosis

Science.gov (United States)

Verbeke, Len; Mannaerts, Inge; Schierwagen, Robert; Govaere, Olivier; Klein, Sabine; Vander Elst, Ingrid; Windmolders, Petra; Farre, Ricard; Wenes, Mathias; Mazzone, Massimiliano; Nevens, Frederik; van Grunsven, Leo A.; Trebicka, Jonel; Laleman, Wim

2016-01-01

Hepatic inflammation drives hepatic stellate cells (HSC), resulting in liver fibrosis. The Farnesoid-X receptor (FXR) antagonizes inflammation through NF-κB inhibition. We investigated preventive and therapeutic effects of FXR agonist obeticholic acid (OCA) on hepatic inflammation and fibrosis in toxic cirrhotic rats. Cirrhosis was induced by thioacetamide (TAA) intoxication. OCA was given during or after intoxication with vehicle-treated rats as controls. At sacrifice, fibrosis, hemodynamic and biochemical parameters were assessed. HSC activation, cell turn-over, hepatic NF-κB activation, pro-inflammatory and pro-fibrotic cytokines were determined. The effect of OCA was further evaluated in isolated HSC, Kupffer cells, hepatocytes and liver sinusoidal endothelial cells (LSEC). OCA decreased hepatic inflammation and fibrogenesis during TAA-administration and reversed fibrosis in established cirrhosis. Portal pressure decreased through reduced intrahepatic vascular resistance. This was paralleled by decreased expression of pro-fibrotic cytokines (transforming growth-factor β, connective tissue growth factor, platelet-derived growth factor β-receptor) as well as markers of hepatic cell turn-over, by blunting effects of pro-inflammatory cytokines (e.g. monocyte chemo-attractant protein-1). In vitro, OCA inhibited both LSEC and Kupffer cell activation; while HSC remained unaffected. This related to NF-κB inhibition via up-regulated IκBα. In conclusion, OCA inhibits hepatic inflammation in toxic cirrhotic rats resulting in decreased HSC activation and fibrosis. PMID:27634375

Edaravone Decreases Paraquat Toxicity in A549 Cells and Lung Isolated Mitochondria

OpenAIRE

Shokrzadeh, Mohammad; Shaki, Fatemeh; Mohammadi, Ebrahim; Rezagholizadeh, Neda; Ebrahimi, Fatemeh

2014-01-01

Edaravone, an antioxidant and radical scavenger, showed protective effects against oxidative stress-like condition. Paraquat (PQ) is toxic herbicide considerable evidence suggests that oxidative stress and mitochondrial dysfunction contribute to PQ toxicity. In this study, protective effect of edaravone against PQ induced toxicity and reactive oxygen species (ROS) generation in A549 cells and lung isolated mitochondria were evaluated. A549 cells and lung isolated mitochondria were divided int...

THE PROGRAMED CELL DEATH REGULATORS OF ISOLATED MODEL SYSTEMS

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D. V. Vatlitsov

2016-06-01

Full Text Available The technology evolution creates the prerequisites for the emergence of new informational concept and approaches to the formation of a fundamentally new principles of biological objects understanding. The aim was to study the activators of the programmed cell death in an isolated system model. Cell culture aging parameters were performed on flow cytometer. It had formed the theory that the changes in the concentrations of metal ions and increase their extracellular concentration had formed a negative gradient into the cells.regulation of cell death. It was shown that the metals ions concentrations.

Capture, isolation and release of cancer cells with aptamer-functionalized glass bead array.

Science.gov (United States)

Wan, Yuan; Liu, Yaling; Allen, Peter B; Asghar, Waseem; Mahmood, M Arif Iftakher; Tan, Jifu; Duhon, Holli; Kim, Young-tae; Ellington, Andrew D; Iqbal, Samir M

2012-11-21

Early detection and isolation of circulating tumor cells (CTC) can enable better prognosis for cancer patients. A Hele-Shaw device with aptamer functionalized glass beads is designed, modeled, and fabricated to efficiently isolate cancer cells from a cellular mixture. The glass beads are functionalized with anti-epidermal growth factor receptor (EGFR) aptamer and sit in ordered array of pits in polydimethylsiloxane (PDMS) channel. A PDMS encapsulation is then used to cover the channel and to flow through cell solution. The beads capture cancer cells from flowing solution depicting high selectivity. The cell-bound glass beads are then re-suspended from the device surface followed by the release of 92% cells from glass beads using combination of soft shaking and anti-sense RNA. This approach ensures that the cells remain in native state and undisturbed during capture, isolation and elution for post-analysis. The use of highly selective anti-EGFR aptamer with the glass beads in an array and subsequent release of cells with antisense molecules provide multiple levels of binding and release opportunities that can help in defining new classes of CTC enumeration devices.

Anti-inflammatory liposomes have no impact on liver regeneration in rats

DEFF Research Database (Denmark)

Jepsen, Betina Norman; Andersen, Kasper Jarlhelt; Knudsen, Anders Riegels

2015-01-01

Introduction: Surgical resection is the gold standard in treatment of hepatic malignancies, giving the patient the best chance to be cured. The liver has a unique capacity to regenerate. However, an inflammatory response occurs during resection, in part mediated by Kupffer cells, that influences...... the speed of regeneration. The aim of this study was to investigate the effect of a Kupffer cell targeted anti-inflammatory treatment on liver regeneration in rats. Methods: Two sets of animals, each including four groups of eight rats, were included. Paired groups from each set received treatment......-6. Conclusion: Low dose dexamethasone targeted to Kupffer cells does not affect histological liver cell regeneration after 70% hepatectomy in rats, but reduces the inflammatory response judged by circulating markers of inflammation. (C) 2015 The Authors. Published by Elsevier Ltd on behalf of IJS...

Administration of progesterone after trauma and hemorrhagic shock prevents hepatocellular injury.

Science.gov (United States)

Kuebler, Joachim F; Yokoyama, Yukihiro; Jarrar, Doraid; Toth, Balazs; Rue, Loring W; Bland, Kirby I; Wang, Ping; Chaudry, Irshad H

2003-07-01

Administration of a single dose of progesterone following trauma and hemorrhage in progesterone-deficient rats would ameliorate the inflammatory response and hepatocellular damage. A university laboratory. Ovariectomized female Sprague-Dawley rats (250-350 g; Charles River Laboratories, Wilmington, Mass) underwent a 5-cm midline laparotomy (ie, induction of soft tissue trauma), were bled to a mean arterial blood pressure of 35 mm Hg for about 90 minutes, and then were resuscitated using Ringer lactate solution. Progesterone (25 mg/kg of body weight) or vehicle was administered subcutaneously at the end of resuscitation. In additional animals, Kupffer cells were isolated following trauma, hemorrhage, and resuscitation and treated in vitro with progesterone, lipopolysaccharide, or both. Six hours following resuscitation, plasma tumor necrosis factor alpha (TNF-alpha) and interleukin 6 (IL-6) levels and liver myeloperoxidase activity were determined. Hepatocellular function (maximum velocity of indocyanine green clearance [Vmax] and the efficiency of the active transport or Michaelis-Menten constant [Km]) and plasma levels of transaminases were measured 20 hours after resuscitation. Kupffer cell IL-6 and TNF-alpha production were assessed. Plasma levels of TNF-alpha, IL-6, aspartate aminotransferase, and alanine aminotransferase, as well as hepatic myeloperoxidase activity were increased, whereas indocyanine green clearance was depressed in vehicle-treated rats following trauma-hemorrhage. Animals treated with progesterone showed significantly reduced levels of the TNF-alpha, IL-6, and transaminases as well as reduced myeloperoxidase activity in the liver. Progesterone-treated animals showed increased Vmax and Kmax values for indocyanine green. In vitro treatment of Kupffer cells with progesterone decreased TNF-alpha production but did not affect the production of IL-6. Progesterone administration following trauma-hemorrhage ameliorates the proinflammatory response

Isolation of Individual Egg Cells and Zygotes in Alstroemeria Followed by Manual Selection with a Microcapillary-connected Micropump

Science.gov (United States)

HOSHINO, YOICHIRO; MURATA, NAHO; SHINODA, KOICHI

2006-01-01

• Aims To develop a procedure for isolating living egg cells and zygotes from Alstroemeria ovules. • Scope An attempt was made to isolate egg cells and zygotes from the ovules of Alstroemeria aurea. The ovules were histologically observed using a clearing procedure which revealed the localization and sizes of the embryo sacs and egg apparatus within the ovules. For the isolation of egg cells, ovules were cut into sections with a surgical blade and treated with an enzyme solution. Subsequently, these ovule sections were dissected using a glass needle under an inverted microscope. Egg cells successfully isolated by this procedure were collected using microcapillaries connected to a micropump. For zygote isolation, ovules were excised from ovaries 24 h after self-pollination. By treating excised ovules with an enzyme solution and subsequently dissecting them using a glass needle, zygotes were successfully isolated from the ovules and collected with a microcapillary. The isolated zygotes were associated with pollen tubes and one of the synergids. Egg cells and zygotes were viable for up to 2 h following isolation, as determined by fluorescein diacetate staining. • Conclusions The procedures for isolating egg cells and zygotes in Alstroemeria were established, and each egg cell and zygote was captured with a microcapillary. PMID:16621859

Establishment of an agamid cell line and isolation of adenoviruses from central bearded dragons (Pogona vitticeps).

Science.gov (United States)

Ball, Inna; Hoferer, Marc; Marschang, Rachel E

2014-03-01

A cell line was established from whole 6-8-week-old central bearded dragon (Pogona vitticeps) embryos. Cells were mid-sized and showed an elongated and polymorphic form. The cell line grew in a monolayer and has been serially passaged for 17 passages at time of publication. This cell line has been used with samples from adenovirus polymerase chain reaction (PCR)-positive bearded dragons, and 2 virus isolates have been obtained so far. The isolates show a clear cytopathic effect in inoculated cells. Both virus isolates have been serially passaged on this cell line, and have been identified by PCR amplification and sequencing of a portion of the DNA-dependent DNA polymerase gene and show 100% nucleotide identity to the corresponding region of an agamid adenovirus. Electron microscopic examination of supernatant from infected cells demonstrated the presence of nonenveloped particles, with a diameter of approximately 80 nm in both virus isolates.

Efficient Generation of Glucose-Responsive Beta Cells from Isolated GP2+ Human Pancreatic Progenitors

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Jacqueline Ameri

2017-04-01

Full Text Available Stem cell-based therapy for type 1 diabetes would benefit from implementation of a cell purification step at the pancreatic endoderm stage. This would increase the safety of the final cell product, allow the establishment of an intermediate-stage stem cell bank, and provide a means for upscaling β cell manufacturing. Comparative gene expression analysis revealed glycoprotein 2 (GP2 as a specific cell surface marker for isolating pancreatic endoderm cells (PECs from differentiated hESCs and human fetal pancreas. Isolated GP2+ PECs efficiently differentiated into glucose responsive insulin-producing cells in vitro. We found that in vitro PEC proliferation declines due to enhanced expression of the cyclin-dependent kinase (CDK inhibitors CDKN1A and CDKN2A. However, we identified a time window when reducing CDKN1A or CDKN2A expression increased proliferation and yield of GP2+ PECs. Altogether, our results contribute tools and concepts toward the isolation and use of PECs as a source for the safe production of hPSC-derived β cells.

Isolation of Human Skin Dendritic Cell Subsets.

Science.gov (United States)

Gunawan, Merry; Jardine, Laura; Haniffa, Muzlifah

2016-01-01

Dendritic cells (DCs) are specialized leukocytes with antigen-processing and antigen-presenting functions. DCs can be divided into distinct subsets by anatomical location, phenotype and function. In human, the two most accessible tissues to study leukocytes are peripheral blood and skin. DCs are rare in human peripheral blood (skin covering an average total surface area of 1.8 m(2) has approximately tenfold more DCs than the average 5 L of total blood volume (Wang et al., J Invest Dermatol 134:965-974, 2014). DCs migrate spontaneously from skin explants cultured ex vivo, which provide an easy method of cell isolation (Larsen et al., J Exp Med 172:1483-1493, 1990; Lenz et al., J Clin Invest 92:2587-2596, 1993; Nestle et al., J Immunol 151:6535-6545, 1993). These factors led to the extensive use of skin DCs as the "prototype" migratory DCs in human studies. In this chapter, we detail the protocols to isolate DCs and resident macrophages from human skin. We also provide a multiparameter flow cytometry gating strategy to identify human skin DCs and to distinguish them from macrophages.

Differences in cell morphometry, cell wall topography and gp70 expression correlate with the virulence of Sporothrix brasiliensis clinical isolates.

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Rafaela A Castro

Full Text Available Sporotrichosis is a chronic infectious disease affecting both humans and animals. For many years, this subcutaneous mycosis had been attributed to a single etiological agent; however, it is now known that this taxon consists of a complex of at least four pathogenic species, including Sporothrix schenckii and Sporothrix brasiliensis. Gp70 was previously shown to be an important antigen and adhesin expressed on the fungal cell surface and may have a key role in immunomodulation and host response. The aim of this work was to study the virulence, morphometry, cell surface topology and gp70 expression of clinical isolates of S. brasiliensis compared with two reference strains of S. schenckii. Several clinical isolates related to severe human cases or associated with the Brazilian zoonotic outbreak of sporotrichosis were genotyped and clustered as S. brasiliensis. Interestingly, in a murine subcutaneous model of sporotrichosis, these isolates showed a higher virulence profile compared with S. schenckii. A single S. brasiliensis isolate from an HIV-positive patient not only showed lower virulence but also presented differences in cell morphometry, cell wall topography and abundant gp70 expression compared with the virulent isolates. In contrast, the highly virulent S. brasiliensis isolates showed reduced levels of cell wall gp70. These observations were confirmed by the topographical location of the gp70 antigen using immunoelectromicroscopy in both species. In addition, the gp70 molecule was sequenced and identified using mass spectrometry, and the sequenced peptides were aligned into predicted proteins using Blastp with the S. schenckii and S. brasiliensis genomes.

Tula hantavirus isolate with the full-length ORF for nonstructural protein NSs survives for more consequent passages in interferon-competent cells than the isolate having truncated NSs ORF.

Science.gov (United States)

Jääskeläinen, Kirsi M; Plyusnina, Angelina; Lundkvist, Ake; Vaheri, Antti; Plyusnin, Alexander

2008-01-11

The competitiveness of two Tula hantavirus (TULV) isolates, TULV/Lodz and TULV/Moravia, was evaluated in interferon (IFN) -competent and IFN-deficient cells. The two isolates differ in the length of the open reading frame (ORF) encoding the nonstructural protein NSs, which has previously been shown to inhibit IFN response in infected cells. In IFN-deficient Vero E6 cells both TULV isolates survived equally well. In contrast, in IFN-competent MRC5 cells TULV/Lodz isolate, that possesses the NSs ORF for the full-length protein of 90 aa, survived for more consequent passages than TULV/Moravia isolate, which contains the ORF for truncated NSs protein (66-67 aa). Our data show that expression of a full-length NSs protein is beneficial for the virus survival and competitiveness in IFN-competent cells and not essential in IFN-deficient cells. These results suggest that the N-terminal aa residues are important for the full activity of the NSs protein.

Chemical analysis of isolated cell walls of Gram-positive bacteria and the determination of the cell wall to cell mass ratio.

NARCIS (Netherlands)

Wal, van der A.; Norde, W.; Bendinger, B.; Zehnder, A.J.B.; Lyklema, J.

1997-01-01

Cell walls of five Gram-positive bacterial strains, including four coryneforms and a Bacillus brevis strain were isolated and subsequently chemically analysed. The wall contribution to the total cell mass is calculated from a comparison of D-Lactate concentrations in hydrolysates of whole cells and

Isolation and Culture of Postnatal Stem Cells from Deciduous Teeth

OpenAIRE

Olávez, Daniela; Facultad de Odontología Universidad de Los Andes; Salmen, Siham; Instituto de Inmunología Clínica, Universidad de Los Andes.; Padrón, Karla; Facultad de Odontología. Univerisdad de Los Andes.; Lobo, Carmine; Facultad de Odontología. Univerisdad de Los Andes.; Díaz, Nancy; Facultad de Odontología, Universidad de Los Andes.; Berrueta, Lisbeth; Doctora en Inmunología por Instituto Venezolano de Investigaciones Científicas (IVIC). Instituto de Inmunología Clínica, Facultad de Medicina, Universidad de Los Andes, Venezuela.; Solorzanio, Eduvigis; Facultad de Odontología, Universidad de Los Andes.

2014-01-01

Background: Currently, degenerative diseases represent a public health problem; therefore, the development and implementation of strategies to fully or partially recover of damaged tissues has a special interest in the biomedical field. Therapeutic strategies based on mesenchymal stem cells transplantation from dental pulp have been proposed as an alternative. Purpose: To develop a mesenchymal stem cells culture isolated from dental pulp of deciduous teeth. Methods: The mesenchymal stem cells...

Isolation, culture and biological characteristics of multipotent porcine skeletal muscle satellite cells.

Science.gov (United States)

Yang, Jinjuan; Liu, Hao; Wang, Kunfu; Li, Lu; Yuan, Hongyi; Liu, Xueting; Liu, Yingjie; Guan, Weijun

2017-12-01

Skeletal muscle has a huge regenerative potential for postnatal muscle growth and repair, which mainly depends on a kind of muscle progenitor cell population, called satellite cell. Nowadays, the majority of satellite cells were obtained from human, mouse, rat and other animals but rarely from pig. In this article, the porcine skeletal muscle satellite cells were isolated and cultured in vitro. The expression of surface markers of satellite cells was detected by immunofluorescence and RT-PCR assays. The differentiation capacity was assessed by inducing satellite cells into adipocytes, myoblasts and osteoblasts. The results showed that satellite cells isolated from porcine tibialis anterior were subcultured up to 12 passages and were positive for Pax7, Myod, c-Met, desmin, PCNA and NANOG but were negative for Myogenin. Satellite cells were also induced to differentiate into adipocytes, osteoblasts and myoblasts, respectively. These findings indicated that porcine satellite cells possess similar biological characteristics of stem cells, which may provide theoretical basis and experimental evidence for potential therapeutic application in the treatment of dystrophic muscle and other muscle injuries.

A novel method for isolation of epithelial cells from ovine esophagus for tissue engineering.

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Macheiner, Tanja; Kuess, Anna; Dye, Julian; Saxena, Amulya K

2014-01-01

The yield of a critical number of basal epithelial cells with high mitotic rates from native tissue is a challenge in the field of tissue engineering. There are many protocols that use enzymatic methods for isolation of epithelial cells with unsatisfactory results for tissue engineering. This study aimed to develop a protocol for isolating a sufficient number of epithelial cells with a high Proliferating Index from ovine esophagus for tissue engineering applications. Esophageal mucosa was pretreated with dispase-collagenase solution and plated on collagen-coated culture dishes. Distinction of the various types of epithelial cells and developmental stages was done with specific primary antibodies to Cytokeratins and to Proliferating Cell Nuclear Antigen (PCNA). Up to approximately 8100 epithelial cells/mm2 of mucosa tissue were found after one week of migration. Cytokeratin 14 (CK 14) was positive identified in cells even after 83 days. At the same time the Proliferating Index was 71%. Our protocol for isolation of basal epithelial cells was successful to yield sufficient numbers of cells predominantly with proliferative character and without noteworthy negative enzymatic affection. The results at this study offer the possibility of generation critical cell numbers for tissue engineering applications.

Isolation and characterization of node/notochord-like cells from mouse embryonic stem cells

DEFF Research Database (Denmark)

Winzi, Maria K.; Hyttel, Poul; Dale, Jacqueline Kim

2011-01-01

The homeobox gene Noto is expressed in the node and its derivative the notochord. Here we use a targeted Noto-GFP reporter to isolate and characterize node/notochord-like cells derived from mouse embryonic stem cells. We find very few Noto-expressing cells after spontaneous differentiation. However......, the number of Noto-expressing cells was increased when using Activin A to induce a Foxa2- and Brachyury-expressing progenitor population, whose further differentiation into Noto-expressing cells was improved by simultaneous inhibition of BMP, Wnt, and retinoic acid signaling. Noto-GFP(+) cells expressed...... the node/notochord markers Noto, Foxa2, Shh, Noggin, Chordin, Foxj1, and Brachyury; showed a vacuolarization characteristic of notochord cells; and can integrate into midline structures when grafted into Hensen's node of gastrulating chicken embryos. The ability to generate node/notochord-like cells...

Cytotopographical specialization of enzymatically isolated rabbit retinal Müller (glial) cells: K+ conductivity of the cell membrane.

Science.gov (United States)

Reichenbach, A; Eberhardt, W

1988-01-01

Müller (radial glial) cells were isolated from rabbit retinae by means of papaine and mechanical dissociation. Regional membrane properties of these cells were studied by intracellular microelectrode recordings of potential responses to local application of high K+ solutions. When different parts of the cell membrane were exposed to high K+, the amplitude of the depolarizing responses varied greatly, indicating a strong regional specialization of the membrane properties. Using morphometrical data of isolated rabbit Müller cells, and a simple circuit model, we calculated the endfoot membrane to constitute more than 80% of the total K+ conductance of the cell; the specific resistivity of the endfoot membrane was about 400 omega cm2, i.e., more than 40 times less than that of the membrane of the vitread process, which is immediately adjacent. This kind of regional membrane specialization seems to be optimized in respect to the Müller cells' ability to carry spatial buffering K+ currents.

Improved isolation protocol for equine cord blood-derived mesenchymal stromal cells

DEFF Research Database (Denmark)

Koch, Thomas Gadegaard; Thomsen, Preben Dybdahl; Betts, Dean H.

2009-01-01

  BACKGROUND AIMS: A robust methodology for the isolation of cord blood-derived multipotent mesenchymal stromal cells (CB-MSCs) from fresh umbilical cord blood has not been reported in any species. The objective of this study was to improve the isolation procedure for equine CB-MSCs. METHODS: Pre......-culture separation of red and white blood cells was done using either PrepaCyte?-EQ medium or Ficoll-Paque? PREMIUM density medium. Regular FBS and MSC-qualified FBS were compared for their ability to support the establishment of putative primary MSC colonies. RESULTS AND CONCLUSIONS: Our results indicate that Prepa...

A Novel Method for Isolation of Pluripotent Stem Cells from Human Umbilical Cord Blood.

Science.gov (United States)

Monti, Manuela; Imberti, Barbara; Bianchi, Niccolò; Pezzotta, Anna; Morigi, Marina; Del Fante, Claudia; Redi, Carlo Alberto; Perotti, Cesare

2017-09-01

Very small embryonic-like cells (VSELs) are a population of very rare pluripotent stem cells isolated in adult murine bone marrow and many other tissues and organs, including umbilical cord blood (UCB). VSEL existence is still not universally accepted by the scientific community, so for this purpose, we sought to investigate whether presumptive VSELs (pVSELs) could be isolated from human UCB with an improved protocol based on the isolation of enriched progenitor cells by depletion of nonprogenitor cells with magnetic separation. Progenitor cells, likely including VSELs, cultured with retinoic acid were able to form dense colonies and cystic embryoid bodies and to differentiate toward the ecto-meso-endoderm lineages as shown by the positivity to specific markers. VSEL differentiative potential toward mesodermal lineage was further demonstrated in vitro upon exposure to an established inductive protocol, which induced the acquisition of renal progenitor cell phenotype. VSEL-derived renal progenitors showed regenerative potential in a cisplatin model of acute kidney injury by restoring renal function and tubular structure through induction of proliferation of endogenous renal cells. The data presented here foster the great debate that surrounds VSELs and, more in general, the existence of cells endowed with pluripotent features in adult tissues. In fact, the possibility to find and isolate subpopulations of cells that fully fit all the criteria utilized to define pluripotency remains, nowadays, almost unproven. Thus, efforts to better characterize the phenotype of these intriguing cells are crucial to understand their possible applications for regenerative and precision medicine purposes.

Simultaneous isolation of mRNA and native protein from minute samples of cells

DEFF Research Database (Denmark)

Petersen, Tonny Studsgaard; Andersen, Claus Yding

2014-01-01

Precious biological samples often lack a sufficient number of cells for multiple procedures, such as extraction of mRNA while maintaining protein in a non-denatured state suitable for subsequent characterization. Here we present a new method for the simultaneous purification of mRNA and native...... in their native state for traditional protein assays. We validated the procedure using neonatal rat ovaries and small numbers of human granulosa cells, demonstrating the extraction of mRNA suitable for gene expression analysis with simultaneous isolation of native proteins suitable for downstream characterization...... proteins from samples containing small numbers of cells. Our approach utilizes oligodeoxythymidylate [oligo(dT)25]-coated paramagnetic beads in an optimized reaction buffer to isolate mRNA comparable in quantity and quality to mRNA isolated with existing methods, while maintaining the proteins...

Bovine annulus fibrosus cell lines isolated from intervertebral discs

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Petra Kraus

2016-12-01

Full Text Available The adult bovine (Bos taurus intervertebral disc is primarily comprised of two major tissue types: The outer annulus fibrosus (AF and the central nucleus pulposus (NP. We isolated several primary cell lineages of passage (P 0 cells from the AF tissue omitting typically used enzymatic tissue digestion protocols. The cells grow past p10 without signs of senescence in DMEM + 10% FCS on 0.1% gelatin coated/uncoated surfaces of standard cell culture plates and survive freeze-thawing. Preliminary analysis of the AF derived cells for expression of the two structural genes Col1a1 and Col2a1 was performed by PISH recapitulating the expression observed in vivo.

Isolation of cell-free bacterial inclusion bodies.

Science.gov (United States)

Rodríguez-Carmona, Escarlata; Cano-Garrido, Olivia; Seras-Franzoso, Joaquin; Villaverde, Antonio; García-Fruitós, Elena

2010-09-17

Bacterial inclusion bodies are submicron protein clusters usually found in recombinant bacteria that have been traditionally considered as undesirable products from protein production processes. However, being fully biocompatible, they have been recently characterized as nanoparticulate inert materials useful as scaffolds for tissue engineering, with potentially wider applicability in biomedicine and material sciences. Current protocols for inclusion body isolation from Escherichia coli usually offer between 95 to 99% of protein recovery, what in practical terms, might imply extensive bacterial cell contamination, not compatible with the use of inclusion bodies in biological interfaces. Using an appropriate combination of chemical and mechanical cell disruption methods we have established a convenient procedure for the recovery of bacterial inclusion bodies with undetectable levels of viable cell contamination, below 10⁻¹ cfu/ml, keeping the particulate organization of these aggregates regarding size and protein folding features. The application of the developed protocol allows obtaining bacterial free inclusion bodies suitable for use in mammalian cell cultures and other biological interfaces.

Isolation and characterization of string-forming female germline stem cells from ovaries of neonatal mice.

Science.gov (United States)

Liu, Jing; Shang, Dantong; Xiao, Yao; Zhong, Pei; Cheng, Hanhua; Zhou, Rongjia

2017-09-29

Germline stem cells are essential in the generation of both male and female gametes. In mammals, the male testis produces sperm throughout the entire lifetime, facilitated by testicular germline stem cells. Oocyte renewal ceases in postnatal or adult life in mammalian females, suggesting that germline stem cells are absent from the mammalian ovary. However, studies in mice, rats, and humans have recently provided evidence for ovarian female germline stem cells (FGSCs). A better understanding of the role of FGSCs in ovaries could help improve fertility treatments. Here, we developed a rapid and efficient method for isolating FGSCs from ovaries of neonatal mice. Notably, our FGSC isolation method could efficiently isolate on average 15 cell "strings" per ovary from mice at 1-3 days postpartum. FGSCs isolated from neonatal mice displayed the string-forming cell configuration at mitosis ( i.e. a "stringing" FGSC (sFGSC) phenotype) and a disperse phenotype in postnatal mice. We also found that sFGSCs undergo vigorous mitosis especially at 1-3 days postpartum. After cell division, the sFGSC membranes tended to be connected to form sFGSCs. Moreover, F-actin filaments exhibited a cell-cortex distribution in sFGSCs, and E-cadherin converged in cell-cell connection regions, resulting in the string-forming morphology. Our new method provides a platform for isolating FGSCs from the neonatal ovary, and our findings indicate that FGCSs exhibit string-forming features in neonatal mice. The sFGSCs represent a valuable resource for analysis of ovary function and an in vitro model for future clinical use to address ovarian dysfunction. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Isolation and characterization of node/notochord-like cells from mouse embryonic stem cells.

Science.gov (United States)

Winzi, Maria K; Hyttel, Poul; Dale, Jacqueline Kim; Serup, Palle

2011-11-01

The homeobox gene Noto is expressed in the node and its derivative the notochord. Here we use a targeted Noto-GFP reporter to isolate and characterize node/notochord-like cells derived from mouse embryonic stem cells. We find very few Noto-expressing cells after spontaneous differentiation. However, the number of Noto-expressing cells was increased when using Activin A to induce a Foxa2- and Brachyury-expressing progenitor population, whose further differentiation into Noto-expressing cells was improved by simultaneous inhibition of BMP, Wnt, and retinoic acid signaling. Noto-GFP(+) cells expressed the node/notochord markers Noto, Foxa2, Shh, Noggin, Chordin, Foxj1, and Brachyury; showed a vacuolarization characteristic of notochord cells; and can integrate into midline structures when grafted into Hensen's node of gastrulating chicken embryos. The ability to generate node/notochord-like cells in vitro will aid the biochemical characterization of these developmentally important structures.

Novel method for isolation of murine clara cell secretory protein-expressing cells with traces of stemness.

Directory of Open Access Journals (Sweden)

Xiao-Yang Wang

Full Text Available Clara cells are non-ciliated, secretory bronchiolar epithelial cells that serve to detoxify harmful inhaled substances. Clara cells also function as stem/progenitor cells for repair in the bronchioles. Clara cell secretory protein (CCSP is specifically expressed in pulmonary Clara cells and is widely used as a Clara cell marker. In addition CCSP promoter is commonly used to direct gene expression into the lung in transgenic models. The discovery of CCSP immunoreactivity in plasma membranes of airway lining cells prompted us to explore the possibility of enriching Clara cells by flow cytometry. We established a novel and simple method for the isolation of CCSP-expressing cell Clara cells using a combination of mechanical and enzymatic dissociation followed by flow cytometry sorting technology. We showed that ∼25% of dissociated cells from whole lung expressed CCSP. In the resulting preparation, up to 98% of cells expressed CCSP. Notably, we found that several common stem cell markers including CD44, CD133, Sca-1 and Sox2 were expressed in CCSP(+ cells. Moreover, CCSP(+ cells were able to form spheroid colonies in vitro with 0.97‰ efficiency. Parallel studies in vivo confirmed that a small population of CCSP(-expressing cells in mouse airways also demonstrates stem cell-like properties such as label retention and harboring rare bronchioalveolar stem cells (BASCs in terminal bronchioles (TBs. We conclude that CCSP(+ cells exhibit a number of stem cell-like features including stem cell marker expression, bronchosphere colony formation and self-renewal ability. Clara cell isolation by flow cytometry sorting is a useful method for investigating the function of primary Clara cells in stem cell research and mouse models.

Isolation of cells for selective treatment and analysis using a magnetic microfluidic chip

KAUST Repository

Yassine, Omar

2014-05-01

This study describes the development and testing of a magnetic microfluidic chip (MMC) for trapping and isolating cells tagged with superparamagnetic beads (SPBs) in a microfluidic environment for selective treatment and analysis. The trapping and isolation are done in two separate steps; first, the trapping of the tagged cells in a main channel is achieved by soft ferromagnetic disks and second, the transportation of the cells into side chambers for isolation is executed by tapered conductive paths made of Gold (Au). Numerical simulations were performed to analyze the magnetic flux and force distributions of the disks and conducting paths, for trapping and transporting SPBs. The MMC was fabricated using standard microfabrication processes. Experiments were performed with E. coli (K12 strand) tagged with 2.8 μm SPBs. The results showed that E. coli can be separated from a sample solution by trapping them at the disk sites, and then isolated into chambers by transporting them along the tapered conducting paths. Once the E. coli was trapped inside the side chambers, two selective treatments were performed. In one chamber, a solution with minimal nutrition content was added and, in another chamber, a solution with essential nutrition was added. The results showed that the growth of bacteria cultured in the second chamber containing nutrient was significantly higher, demonstrating that the E. coli was not affected by the magnetically driven transportation and the feasibility of performing different treatments on selectively isolated cells on a single microfluidic platform.

Isolation of Human Colon Stem Cells Using Surface Expression of PTK7.

Science.gov (United States)

Jung, Peter; Sommer, Christian; Barriga, Francisco M; Buczacki, Simon J; Hernando-Momblona, Xavier; Sevillano, Marta; Duran-Frigola, Miquel; Aloy, Patrick; Selbach, Matthias; Winton, Douglas J; Batlle, Eduard

2015-12-08

Insertion of reporter cassettes into the Lgr5 locus has enabled the characterization of mouse intestinal stem cells (ISCs). However, low cell surface abundance of LGR5 protein and lack of high-affinity anti-LGR5 antibodies represent a roadblock to efficiently isolate human colonic stem cells (hCoSCs). We set out to identify stem cell markers that would allow for purification of hCoSCs. In an unbiased approach, membrane-enriched protein fractions derived from in vitro human colonic organoids were analyzed by quantitative mass spectrometry. Protein tyrosine pseudokinase PTK7 specified a cell population within human colonic organoids characterized by highest self-renewal and re-seeding capacity. Antibodies recognizing the extracellular domain of PTK7 allowed us to isolate and expand hCoSCs directly from patient-derived mucosa samples. Human PTK7+ cells display features of canonical Lgr5+ ISCs and include a fraction of cells that undergo differentiation toward enteroendocrine lineage that resemble crypt label retaining cells (LRCs). Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

A SYSTEM AND A DEVICE FOR ISOLATING CIRCULATING TUMOR CELLS FROM THE PERIPHERAL BLOOD IN VIVO

Directory of Open Access Journals (Sweden)

Michal Mego

2015-08-01

Full Text Available Circulating tumor cells (CTC play a crucial role in disseminating tumors and in the metastatic cascade. CTCs are found only in small numbers, and the limited amount of isolated CTCs makes it impossible to characterize them closely. This paper presents a proposal for a new system for isolating CTCs from the peripheral blood in vivo. The system enables CTCs to be isolated from the whole blood volume for further research and applications. The proposed system consists of magnetic nanoparticles covered by monoclonal antibodies against a common epithelial antigen, large supermagnets, which are used to control the position of the nanoparticles within the human body, and a special wire made of a magnetic core wrapped in a non-magnetic shell. The system could be used not only for isolating CTCs, but also for in vivo isolation of other rare cells from the peripheral blood, including hematopoietic and/or mesenchymal stem cells, with applications in regenerative medicine and/or in stem cell transplantation.

Immunohistochemical characterisation of the hepatic stem cell niche in feline hepatic lipidosis: a preliminary morphological study.

Science.gov (United States)

Valtolina, Chiara; Robben, Joris H; Favier, Robert P; Rothuizen, Jan; Grinwis, Guy Cm; Schotanus, Baukje A; Penning, Louis C

2018-05-01

Objectives The aim of this study was to describe the cellular and stromal components of the hepatic progenitor cell niche in feline hepatic lipidosis (FHL). Methods Immunohistochemical staining for the progenitor/bile duct marker (K19), activated Kupffer cells (MAC387), myofibroblasts (alpha-smooth muscle actin [α-SMA]) and the extracellular matrix component laminin were used on seven liver biopsies of cats with FHL and three healthy cats. Double immunofluorescence stainings were performed to investigate co-localisation of different cell types in the hepatic progenitor cell (HPC) niche. Results HPCs, Kupffer cells, myofibroblasts and laminin deposition were observed in the liver samples of FHL, although with variability in the expression and positivity of the different immunostainings between different samples. When compared with the unaffected cats where K19 positivity and minimal α-SMA and laminin positivity were seen mainly in the portal area, in the majority of FHL samples K19 and α-SMA-positive cells and laminin positivity were seen also in the periportal and parenchymatous area. MAC387-positive cells were present throughout the parenchyma. Conclusions and relevance This is a preliminary morphological study to describe the activation and co-localisation of components of the HPC niche in FHL. Although the HPC niche in FHL resembles that described in hepatopathies in dogs and in feline lymphocytic cholangitis, the expression of K19, α-SMA, MAC387 and lamin is more variable in FHL, and a common pattern of activation could not be established. Nevertheless, when HPCs were activated, a spatial association between HPCs and their niche could be demonstrated.

Data on isolating mesenchymal stromal cells from human adipose tissue using a collagenase-free method

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Wassim Shebaby

2016-03-01

Full Text Available The present dataset describes a detailed protocol to isolate mesenchymal cells from human fat without the use of collagenase. Human fat specimen, surgically cleaned from non-fat tissues (e.g., blood vessels and reduced into smaller fat pieces of around 1–3 mm size, is incubated in complete culture media for five to seven days. Then, cells started to spread out from the fat explants and to grow in cultures according to an exponential pattern. Our data showed that primary mesenchymal cells presenting heterogeneous morphology start to acquire more homogenous fibroblastic-like shape when cultured for longer duration or when subcultured into new flasks. Cell isolation efficiency as well as cell doubling time were also calculated throughout the culturing experimentations and illustrated in a separate figure thereafter. This paper contains data previously considered as an alternative protocol to isolate adipose-derived mesenchymal stem cell published in “Proliferation and differentiation of human adipose-derived mesenchymal stem cells (ASCs into osteoblastic lineage are passage dependent” [1]. Keywords: Adipose tissue, mesenchymal stromal cell, cell culture, doubling time

Saikosaponin D Isolated from Bupleurum falcatum Inhibits Selectin-Mediated Cell Adhesion

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Myoung-Jun Jang

2014-12-01

Full Text Available Three saikosaponins were isolated from the MeOH extract of the roots of Bupleurum falcatum L.: saikosaponins B3 (1; B4 (2; and D (3. Of the three, compound 3 inhibited the interaction of selectins (E, L, and P and THP-1 cells with IC50 values of 1.8, 3.0 and 4.3 µM, respectively. Also, the aglycone structure 4 of compound 3 showed moderate inhibitory activity on L-selectin-mediated cell adhesion. From these results, we suspect that compound 3 isolated from Bupleurum falcatum roots would be a good candidate for therapeutic strategies to treat inflammation.

Isolated cutaneous involvement in a child with nodal anaplastic large cell lymphoma

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Vibhu Mendiratta

2016-01-01

Full Text Available Non-Hodgkin lymphoma is a common childhood T-cell and B-cell neoplasm that originates primarily from lymphoid tissue. Cutaneous involvement can be in the form of a primary extranodal lymphoma, or secondary to metastasis from a non-cutaneous location. The latter is uncommon, and isolated cutaneous involvement is rarely reported. We report a case of isolated secondary cutaneous involvement from nodal anaplastic large cell lymphoma (CD30 + and ALK + in a 7-year-old boy who was on chemotherapy. This case is reported for its unusual clinical presentation as an acute febrile, generalized papulonodular eruption that mimicked deep fungal infection, with the absence of other foci of systemic metastasis.

Isolation of parafluorophenylalanine-resistant mutants from HeLa cell cultures

International Nuclear Information System (INIS)

Yim, L.K.; Stuart, W.D.

1983-01-01

This report describes a method to isolate temperature-conditional phenylalanine transport mutants from the transformed human cell line HeLa. Using ultraviolet light as a mutagenic agent and DL-parafluorophenylalanine (PFPA), a poisonous analogue of L-phenylalanine, as a selective agent, mutagenized cells were selected for survival in the presence of PFPA at a temperature of 39 degrees C. Survivors of the mutagenesis and selection procedures were removed from the culture dishes by trypsin and cloned at a temperature of 35 degrees C. Seven of these lines isolated demonstrated continued resistance to PFPA at 39 degrees C. These lines were tested for uptake of L-phenylalanine at an external concentration of 100 microM and for continued resistance to PFPA at two concentrations. Cells were tested at 35 and at 39 degrees C. The data were compared to those obtained for the parental HeLa cell line under identical conditions. The seven mutant cell lines demonstrated varying resistances to PFPA and varying levels of accumulation of L-phenylalanine when tested at 35 and 39 degrees C. Three mutant lines were additionally tested for L-phenylalanine tRNA charging levels and for transport of L-arginine. The lines had parental cell levels of tRNA charging and L-arginine transport which suggest that the induced genetic defect affects a specific L-phenylalanine transport system

Aptamer-based isolation and subsequent imaging of mesenchymal stem cells in ischemic myocard by magnetic resonance imaging.

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Schäfer, R; Wiskirchen, J; Guo, K; Neumann, B; Kehlbach, R; Pintaske, J; Voth, V; Walker, T; Scheule, A M; Greiner, T O; Hermanutz-Klein, U; Claussen, C D; Northoff, H; Ziemer, G; Wendel, H P

2007-10-01

Mesenchymal stem cells (MSC) seem to be a promising cell source for cellular cardiomyoplasty. We recently developed a new aptamer-based specific selection of MSC to provide "ready to transplant" cells directly after isolation. We evaluated MRI tracking of newly isolated and freshly transplanted MSC in the heart using one short ex vivo selection step combining specific aptamer-based isolation and labeling of the cells. Bone marrow (BM) was collected from healthy pigs. The animals were euthanized and the heart was placed in a perfusion model. During cold ischemia, immunomagnetic isolation of MSC from the BM by MSC-specific aptamers labeled with Dynabeads was performed within 2 h. For histological identification the cells were additionally stained with PKH26. Approx. 3 x 10(6) of the freshly aptamer-isolated cells were injected into the ramus interventricularis anterior (RIVA) and 5 x 10(5) cells were injected directly into myocardial tissue after damaging the respective area by freezing (cryo-scar). 3 x 10(6) of the aptamer-isolated cells were kept for further characterization (FACS and differentiation assays). 20 h after cell transplantation, MRI of the heart using a clinical 3.0 Tesla whole body scanner (Magnetom Trio, Siemens, Germany) was performed followed by histological examinations. The average yield of sorted cells from 120 ml BM was 7 x 10(6) cells. The cells were cultured and showed MSC-like properties. MRI showed reproducible artifacts within the RIVA-perfusion area and the cryo-scar with surprisingly excellent quality. The histological examination of the biopsies showed PKH26-positive cells within the areas which were positive in the MRI in contrast to the control biopsies. Immunomagnetic separation of MSC by specific aptamers linked to magnetic particles is feasible, effective and combines a specific separation and labeling technique to a "one stop shop" strategy.

Cell pairing ratio controlled micro-environment with valve-less electrolytic isolation

KAUST Repository

Chen, Yu-Chih

2012-01-01

We present a ratio controlled cell-to-cell interaction chip using valve-less isolation. We incorporated electrolysis in a microfluidic channel. In each microfluidic chamber, we loaded two types of different cells at various pairing ratios. More than 80% of the microchambers were successfully loaded with a specific target pairing ratio. For the proof of concept, we have demonstrated the cell-to-cell interaction between prostate cancer cells and muscle stem cells can be controlled by cell pairing ratios through growth factor secretion. The experimental data shows that sealing of microenvironment by air generated from electrolysis does not affect cell viability and cell interaction assay results. © 2012 IEEE.

Comparative study on the effect of radiation on whole blood and isolate red blood cells

International Nuclear Information System (INIS)

Selim, N.S.

2009-01-01

Assessment of the dielectric properties of red blood cells requires several steps for preparation and isolation from whole blood. These steps may results in changes in the cells properties, and they are time consuming . The present study aims to compare the properties of both whole blood and isolated red blood cells and the effect of gamma radiation on these properties. Adult male rats were exposed to 1, 3.5 and 7 Gy as single dose, from Cs-137 source.The studies dielectric properties, in the frequency range 40 k Hz to 5 MHz, and light scattering studies for suspensions of whole blood and isolated red blood cells from the same groups were measured. The obtained results showed that whole blood and red blood cells suspensions followed the same trend in their response to radiation, which suggests the possibility of using whole blood suspension for the evaluation of the red blood cells properties

Comparative performance of fetal goat tongue cell line ZZ-R 127 and fetal porcine kidney cell line LFBK-αvβ6 for Foot-and-mouth disease virus isolation.

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Fukai, Katsuhiko; Morioka, Kazuki; Yamada, Manabu; Nishi, Tatsuya; Yoshida, Kazuo; Kitano, Rie; Yamazoe, Reiko; Kanno, Toru

2015-07-01

The fetal goat tongue cell line ZZ-R 127 and the fetal porcine kidney cell line LFBK-α(v)β(6) have been reported to have high sensitivity to various Foot-and-mouth disease virus (FMDV) strains. The suitability of ZZ-R 127 cells for FMDV isolation not only from epithelial suspensions but also from other clinical samples has already been confirmed in a previous study. However, to our knowledge, the suitability of LFBK-α(v)β(6) cells has not been evaluated using clinical samples other than epithelial materials. In addition, both cell lines have never been compared, in terms of use for FMDV isolation, under the same conditions. Therefore, in the current study, the virus isolation rates of both cell lines were compared using clinical samples collected from animals infected experimentally with FMDV. Viruses were successfully isolated from clinical samples other than epithelial suspensions for both cell lines. The virus isolation rates for the 2 cell lines were not significantly different. The Cohen kappa coefficients between the virus isolation results for both cell lines were significantly high. Taken together, these results confirmed the suitability of LFBK-α(v)β(6) cells for FMDV isolation from clinical samples other than epithelial suspensions. The levels of susceptibility of both cell lines to FMDV isolation were also confirmed to be almost the same. © 2015 The Author(s).

Congestive heart failure effects on atrial fibroblast phenotype: differences between freshly-isolated and cultured cells.

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Kristin Dawson

Full Text Available Fibroblasts are important in the atrial fibrillation (AF substrate resulting from congestive heart failure (CHF. We previously noted changes in in vivo indices of fibroblast function in a CHF dog model, but could not detect changes in isolated cells. This study assessed CHF-induced changes in the phenotype of fibroblasts freshly isolated from control versus CHF dogs, and examined effects of cell culture on these differences.Left-atrial fibroblasts were isolated from control and CHF dogs (ventricular tachypacing 240 bpm × 2 weeks. Freshly-isolated fibroblasts were compared to fibroblasts in primary culture. Extracellular-matrix (ECM gene-expression was assessed by qPCR, protein by Western blot, fibroblast morphology with immunocytochemistry, and K(+-current with patch-clamp. Freshly-isolated CHF fibroblasts had increased expression-levels of collagen-1 (10-fold, collagen-3 (5-fold, and fibronectin-1 (3-fold vs. control, along with increased cell diameter (13.4 ± 0.4 µm vs control 8.4 ± 0.3 µm and cell spreading (shape factor 0.81 ± 0.02 vs. control 0.87 ± 0.02, consistent with an activated phenotype. Freshly-isolated control fibroblasts displayed robust tetraethylammonium (TEA-sensitive K(+-currents that were strongly downregulated in CHF. The TEA-sensitive K(+-current differences between control and CHF fibroblasts were attenuated after 2-day culture and eliminated after 7 days. Similarly, cell-culture eliminated the ECM protein-expression and shape differences between control and CHF fibroblasts.Freshly-isolated CHF and control atrial fibroblasts display distinct ECM-gene and morphological differences consistent with in vivo pathology. Culture for as little as 48 hours activates fibroblasts and obscures the effects of CHF. These results demonstrate potentially-important atrial-fibroblast phenotype changes in CHF and emphasize the need for caution in relating properties of cultured fibroblasts to in vivo systems.

[Effects and mechanisms of the inflammatory reaction related to NASH and induced by activation of the cholinergic anti-inflammatory pathway].

Science.gov (United States)

Zhou, Zhou; Chen, Xiaomei; Li, Fuqiang; Tang, Cuilan

2015-01-01

To investigate the effects and mechanisms of the inflammatory reaction related to nonalcoholic steatohepatitis (NASH) and induced by activation of the cholinergic anti-inflammatory pathway. A mouse model of NASH was established by feeding a high-fat and high-sugar diet.Activation of the cholinergic anti-inflammatory pathway was achieved by nicotine administration to the NASH modeled mice and normal controls. Liver biopsies were taken and the concentrations of cytokines were measured. Isolated liver primary Kupffer cells and RAw264.7 cells were cultured, pre-treated or not with lipopolysaccharide (LPS) and exposed to nicotine, after which the supernatant concentrations of IL-6 and TNFa were determined by ELISA. The protein expression levels of phosphorylated (p)-NF-kB and I k B were detected in primary cultured Kupffer cells by western blotting. The mouse model of NASH was successfully established, as evidenced by findings from liver biopsy and serum liver function tests. The degree of liver inflammation in the NASH mice decreased after nicotine administration, and the level of serum TNFa also significantly decreased. The levels of serum TNFa were 21.95+/-0.8 pg/mL in nicotine-treated mice and 38.07+/-1.7 pg/mL in the non-nicotine-treated NASH mice (P less than 0.05). The nicotine treatment also significantly reduced the concentration of TNFa in the culture supernatants of Kupffer cells after LPS stimulation; moreover, the supernatant level of TNFa decreased significantly after the nicotine treatment (Pless than 0.05). LPS stimulation of the RAw264.7 cells led to an increased level ofp-NF-kB and a reduced level ofI-kB, suggesting that the NF-kB pathway had been activated; different doses of nicotine pre-treatment led to down-regulation of the p-NF-kB level and up-regulation of the I-kB level, both in dose-dependent manners. Activating the cholinergic anti-inflammatory pathway inhibits the NASH-related inflammatory reaction, and the mechanism for this inhibition

Isolated Post-Transplantation Lymphoproliferative Disease Involving the Breast and Axilla as Peripheral T-cell Lymphoma

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Hwang, Ji-Young [Department of Radiology, Kangnam Sacred Heart Hospital, Hallym University College of Medicine, Seoul 150-950 (Korea, Republic of); Cha, Eun Suk; Lee, Jee Eun [Department of Radiology, Ewha Womans University School of Medicine, Seoul 158-710 (Korea, Republic of); Sung, Sun Hee [Department of Pathology, Ewha Womans University School of Medicine, Seoul 158-710 (Korea, Republic of)

2013-07-01

Post-transplantation lymphoproliferative disorders (PTLDs) are a heterogeneous group of diseases that represent serious complications following immunosuppressive therapy for solid organ or hematopoietic-cell recipients. In contrast to B-cell PTLD, T-cell PTLD is less frequent and is not usually associated with Epstein Barr Virus infection. Moreover, to our knowledge, isolated T-cell PTLD involving the breast is extremely rare and this condition has never been reported previously in the literature. Herein, we report a rare case of isolated T-cell PTLD of the breast that occurred after a patient had been treated for allogeneic peripheral blood stem cell transplantation due to acute myeloblastic leukemia.

Isolated Post-Transplantation Lymphoproliferative Disease Involving the Breast and Axilla as Peripheral T-cell Lymphoma

International Nuclear Information System (INIS)

Hwang, Ji-Young; Cha, Eun Suk; Lee, Jee Eun; Sung, Sun Hee

2013-01-01

Post-transplantation lymphoproliferative disorders (PTLDs) are a heterogeneous group of diseases that represent serious complications following immunosuppressive therapy for solid organ or hematopoietic-cell recipients. In contrast to B-cell PTLD, T-cell PTLD is less frequent and is not usually associated with Epstein Barr Virus infection. Moreover, to our knowledge, isolated T-cell PTLD involving the breast is extremely rare and this condition has never been reported previously in the literature. Herein, we report a rare case of isolated T-cell PTLD of the breast that occurred after a patient had been treated for allogeneic peripheral blood stem cell transplantation due to acute myeloblastic leukemia

PRODUCTION OF EMBBRYONIC STEM CELLS FROM INNER CELL MASS OF BLASTOCYST ISOLATED BY ENZYMATIC AND IMMUNOSURGERY METHODS

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Thomas Mata Hine

2008-03-01

Full Text Available The objective of the research is determining the ICM isolation method to produce ESC. Blastocyst stage of DDy mice embryos were used in this study. Zona pellucida of blastocysts were removed by 0.25% pronase, the ICM isolation were done by enzimatic or immunosurgery method, and then they were cultured in DMEM-high glucose supplemented with mercaptoethanol, gentamycin, fetal bovine serum, and cumulus cells as feeder layer. The result of the research indicated that immunosurgery method yielding attachment rate and number ESC colony 93.85% and 43.08%, respectively, higher (P<0.05 than enzimatic method that weree 79.63% and 18.52%, respectively, but the viability of ICM cells were equal (P >0.05 that are 93.59% in enzymatic method and 98.56% in immunosurgery method. This research concluded that immunosurgery more effective method for isolation of ICM and ESC production than enzymatic method.

Clinically applicable bulk isolation of blood CD34+ cells for autografting in children.

Science.gov (United States)

Kawano, Y; Takaue, Y; Law, P; Watanabe, T; Abe, T; Okamoto, Y; Makimoto, A; Sato, J; Nakagawa, R; Kajiume, T; Hirao, A; Watanabe, A; Kuroda, Y

1998-11-01

CD34+ cells were purified in bulk from apheresis-collected cells of children with cancer using monoclonal antibody (MoAb) and magnetic beads (Baxter ISOLEX system). To improve the purity of the final product for possibly better tumor cell purging and to make the manufacturer's original procedure more cost-effective, we incubated the cells for 30 min with l-phenylalanine methylester hydrochloride (PME) to reduce the cell number by removing contaminating granulocytes and monocytes in the initial step before incubation with MoAb. Our modification prevented nonspecific interactions between MoAb and magnetic beads, and thereby saved expensive materials for purification. A total of 40 purifications were performed with samples containing a mean of 3.1 x 10(9) blood cells mobilized from 15 children by chemotherapy plus granulocyte colony-stimulating factor (G-CSF). The entire purification procedure, from the end of apheresis to storage, was completed within 5h. After incubation with PME and double-layered (40/60%) Percoll separation, the number of CD34+ cells was reduced to 48+/-29%, which suggests the possibility that half of the CD34+ cells in the inoculum were nonclonogenic in the hematopoietic progenitor assay. PME/Percoll-treated cells were then subjected to a final isolation procedure with MoAb according to the manufacturer's suggestions, and 52+/-42% and 32+/-22%, respectively, of the CFU-GM and CD34+ cells present in the initial bag inoculums were recovered. The recovery rates were, respectively, 54% and 67%, when the calculation was limited to the isolation procedure with MNoAb. The purity of isolated CD34+ cells and the plating efficiency in methylcellulose culture were, respectively, 77+/-24% and 33+/-13%. Fourteen children were subsequently autografted with purified CD34+ cells after marrow ablative chemotherapy. The median number of days to achieve an ANC of 0.5 x 10(9)/l was 12 and that to achieve a platelet count of 50 x 10(9)/l was 22.5, which were

Isolation and characterization of human salivary gland cells for stem cell transplantation to reduce radiation-induced hyposalivation

International Nuclear Information System (INIS)

Feng Jielin; Zwaag, Marianne van der; Stokman, Monique A.; Os, Ronald van; Coppes, Robert P.

2009-01-01

Background: Recently, we showed that transplantation of 100-300 c-Kit + stem cells isolated from cultured salispheres ameliorates radiation-damage in murine salivary glands. The aim of this study is to optimize and translate these findings from mice to man. Methods: Mouse and human non-malignant parotid and submandibular salivary gland tissue was collected and enzymatically digested. The remaining cell suspension was cultured according to our salisphere culture method optimized for murine salispheres. Salisphere cells were tested using 3D matrix culturing for their in vitro stem cell characteristics such as the potential to differentiate into tissue specific cell types. Several potential mouse and human salivary gland stem cells were selected using FACS. Results: In human salivary gland, c-Kit + cells were only detected in excretory ducts as shown previously in mice. From both human parotid and submandibular gland cell suspensions salispheres could be grown, which when placed in 3D culture developed ductal structures and mucin-expressing acinar-like cells. Moreover, cells dispersed from primary salispheres were able to form secondary spheres in matrigel, a procedure that could be repeated for at least seven passages. Approximately 3000 c-Kit + cells could be isolated from primary human salispheres per biopsy. Conclusion: Human salivary glands contain a similar 'putative' stem cell population as rodents, expressing c-kit and capable of in vitro differentiation and self-renewal. In the future, these cells may have the potential to reduce radiotherapy-induced salivary gland dysfunction in patients.

Stainable hepatic iron in 341 African American adults at coroner/medical examiner autopsy

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Acton Ronald T

2005-01-01

Full Text Available Abstract Background Results of previous autopsy studies indicate that increased hepatic iron stores or hepatic iron overload is common in African Americans dying in hospitals, but there are no reports of hepatic iron content in other cohorts of African Americans. Methods We investigated the prevalence of heavy liver iron deposition in African American adults. Using established histochemical criteria, we graded Perls' acid ferrocyanide-reactive iron in the hepatocytes and Kupffer cells of 341 consecutive African American adults who were autopsied in the coroner/medical examiner office. Heavy staining was defined as grade 3 or 4 hepatocyte iron or grade 3 Kupffer cell iron. Results There were 254 men and 85 women (mean age ± 1 SD: 44 ± 13 y vs. 48 ± 14 y, respectively; p = 0.0255; gender was unstated or unknown in two subjects. Approximately one-third of subjects died of natural causes. Heavy staining was observed in 10.2% of men and 4.7% of women. 23 subjects had heavy hepatocyte staining only, six had heavy Kupffer cell staining only, and one had a mixed pattern of heavy staining. 15 subjects had histories of chronic alcoholism; three had heavy staining confined to hepatocytes. We analyzed the relationships of three continuous variables (age at death in years, hepatocyte iron grade, Kupffer cell iron grade and two categorical variables (sex, cause of death (natural and non-natural causes in all 341 subjects using a correlation matrix with Bonferroni correction. This revealed two positive correlations: hepatocyte with Kupffer cell iron grades (p Conclusions The present results confirm and extend previous observations that heavy liver iron staining is relatively common in African Americans. The pertinence of these observations to genetic and acquired causes of iron overload in African Americans is discussed.

In vitro evaluation of isolation possibility of stem cells from intra oral soft tissue and comparison of them with bone mar-row stem cells

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P. Torkzaban

2012-01-01

Full Text Available Objective: Stem cells are of great interest for regenerating disturbed tissues and organs. These cells are commonly isolated from the bone marrow, but there has been interest in other tissues in the recent years. In this study, we evaluated the possibility of isolation of stem cells from oral connective tissue and investigated their characteristics.Materials and Methods: In this experimental study, sampling from the bone marrow and oral connective tissue of a beagle dog was performed under general anesthesia. Bone marrow stem cell isolation was performed according to the established protocols. The samples obtained from oral soft tissue were broken to small pieces and after adding collagenase I, the samples were incubated for 45 minutes in 37°C. Other processes were similar to the processes which were carried out on bone marrow cells. Then cell properties were compared to evaluate if the cells from the connective tissue were stem cells.Results: The cells from the bone marrow and connective tissue had the same morphology. The result of colony forming unit assay was relatively similar. Population doubling time was similar too. In addition, both cell groups differentiated to osteoblasts in osteogenic media.Conclusion: The cells isolated from the oral connective tissue had the characteristics of stem cells, including fibroblastoid morphology, self renewal properties, high proliferation rate and differentiation potential.

Isolation of circulating tumor cells by a magnesium-embedded filter

International Nuclear Information System (INIS)

Liu, Yang; Kang, Dongyang; Xu, Lei; Park, Jungwook; Zhang, Xiaoxiao; Tai, Yu-Chong; Xu, Tong; Xu, Yucheng; Chang, Jay Han-Chieh; Goldkorn, Amir

2015-01-01

Circulating tumor cells (CTCs) are rare cancer cells that are shed by tumors into the bloodstream and that can be valuable biomarkers for various types of cancers. However, CTCs captured on the filter could not be released easily using the existing CTC analysis platforms based on size. To address this limitation, we have developed a novel magnesium (Mg)-embedded cell filter for capture, release and isolation of CTCs. The CTC-filter consists of a thin Ebeam-deposited Mg layer embedded between two parylene-C (PA-C) layers with designed slots for filtration and CTC capture. Thin Mg film has proved highly biocompatible and can be etched in saline, PBS and Dulbecco’s modified eagle medium (DMEM) etc, properties that are of great benefit to help dissociate the filter and thus release the cells. The finite element method (FEM) analysis was performed on the Mg etching process in DMEM for the structure design. After the filtration process, the filter was submerged in DMEM to facilitate Mg etching. The top PA-C filter pieces break apart from the bottom after Mg completely dissolves, enabling captured CTCs to detach. The released CTC can be easily aspirated into a micropipette for further analysis. Thus, the Mg-embedded cell filter provides a new and effective approach for CTCs isolation from the filter, making this a promising new strategy for cancer detection. (paper)

CPM Is a Useful Cell Surface Marker to Isolate Expandable Bi-Potential Liver Progenitor Cells Derived from Human iPS Cells.

Science.gov (United States)

Kido, Taketomo; Koui, Yuta; Suzuki, Kaori; Kobayashi, Ayaka; Miura, Yasushi; Chern, Edward Y; Tanaka, Minoru; Miyajima, Atsushi

2015-10-13

To develop a culture system for large-scale production of mature hepatocytes, liver progenitor cells (LPCs) with a high proliferation potential would be advantageous. We have found that carboxypeptidase M (CPM) is highly expressed in embryonic LPCs, hepatoblasts, while its expression is decreased along with hepatic maturation. Consistently, CPM expression was transiently induced during hepatic specification from human-induced pluripotent stem cells (hiPSCs). CPM(+) cells isolated from differentiated hiPSCs at the immature hepatocyte stage proliferated extensively in vitro and expressed a set of genes that were typical of hepatoblasts. Moreover, the CPM(+) cells exhibited a mature hepatocyte phenotype after induction of hepatic maturation and also underwent cholangiocytic differentiation in a three-dimensional culture system. These results indicated that hiPSC-derived CPM(+) cells share the characteristics of LPCs, with the potential to proliferate and differentiate bi-directionally. Thus, CPM is a useful marker for isolating hiPSC-derived LPCs, which allows development of a large-scale culture system for producing hepatocytes and cholangiocytes. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Isolation and characterization of human umbilical cord-derived endothelial colony-forming cells

Science.gov (United States)

Zhang, Hao; Tao, Yanling; Ren, Saisai; Liu, Haihui; Zhou, Hui; Hu, Jiangwei; Tang, Yongyong; Zhang, Bin; Chen, Hu

2017-01-01

Endothelial colony-forming cells (ECFCs) are a population of endothelial progenitor cells (EPCs) that display robust proliferative potential and vessel-forming capability. Previous studies have demonstrated that a limited number of ECFCs may be obtained from adult bone marrow, peripheral blood and umbilical cord (UC) blood. The present study describes an effective method for isolating ECFCs from human UC. The ECFCs derived from human UC displayed the full properties of EPCs. Analysis of the growth kinetics, cell cycle and colony-forming ability of the isolated human UC-ECFCs indicated that the cells demonstrated properties of stem cells, including relative stability and rapid proliferation in vitro. Gene expression of Fms related tyrosine kinase 1, kinase insert domain receptor, vascular endothelial cadherin, cluster of differentiation (CD)31, CD34, epidermal growth factor homology domains-2, von Willebrand factor and endothelial nitric oxide synthase was assessed by reverse transcription-polymerase chain reaction. The cells were positive for CD34, CD31, CD73, CD105 and vascular endothelial growth factor receptor-2, and negative for CD45, CD90 and human leukocyte antigen-antigen D related protein according to flow cytometry. 1,1′-dioctadecyl-3,3,3′,3′-tetra-methyl-indocarbocyanine perchlorate-labeled acetylated low-density lipoprotein and fluorescein isothiocyanate-Ulex europaeus-l were used to verify the identity of the UC-ECFCs. Matrigel was used to investigate tube formation capability. The results demonstrated that the reported technique is a valuable method for isolating human UC-ECFCs, which have potential for use in vascular regeneration. PMID:29067104

A Method to Identify and Isolate Pluripotent Human Stem Cells and Mouse Epiblast Stem Cells Using Lipid Body-Associated Retinyl Ester Fluorescence

OpenAIRE

Thangaselvam Muthusamy; Odity Mukherjee; Radhika Menon; Megha Prakash Bangalore; Mitradas M. Panicker

2014-01-01

Summary We describe the use of a characteristic blue fluorescence to identify and isolate pluripotent human embryonic stem cells and human-induced pluripotent stem cells. The blue fluorescence emission (450–500 nm) is readily observed by fluorescence microscopy and correlates with the expression of pluripotency markers (OCT4, SOX2, and NANOG). It allows easy identification and isolation of undifferentiated human pluripotent stem cells, high-throughput fluorescence sorting and subsequent propa...

Aptamer-based isolation and subsequent imaging of mesenchymal stem cells in ischemic myocard by magnetic resonance imaging

Energy Technology Data Exchange (ETDEWEB)

Schaefer, R.; Hermanutz-Klein, U.; Northoff, H. [Universitaetsklinikum Tuebingen (Germany). Inst. fuer Klinische und Experimentelle Transfusionsmedizin; Wiskirchen, J.; Kehlbach, R.; Pintaske, J. [Universitaetsklinikum Tuebingen (Germany). Abt. fuer Radiologische Diagnostik; Guo, K.; Neumann, B.; Voth, V.; Walker, T.; Scheule, A.M.; Greiner, T.O.; Ziemer, G.; Wendel, H.P. [Universitaetsklinikum Tuebingen (Germany). Abt. fuer Thorax-, Herz- und Gefaesschirurgie; Claussen, C.D. [Universitaetsklinikum Tuebingen (Germany). Radiologische Universitaetsklinik

2007-10-15

Purpose: Mesenchymal stem cells (MSC) seem to be a promising cell source for cellular cardiomyoplasty. We recently developed a new aptamer-based specific selection of MSC to provide ''ready to transplant'' cells directly after isolation. We evaluated MRI tracking of newly isolated and freshly transplanted MSC in the heart using one short ex vivo selection step combining specific aptamer-based isolation and labeling of the cells. Materials and Methods: Bone marrow (BM) was collected from healthy pigs. The animals were euthanized and the heart was placed in a perfusion model. During cold ischemia, immunomagnetic isolation of MSC from the BM by MSC-specific aptamers labeled with Dynabeads {sup registered} was performed within 2 h. For histological identification the cells were additionally stained with PKH26. Approx. 3 x 10{sup 6} of the freshly aptamer-isolated cells were injected into the ramus interventricularis anterior (RIVA) and 5 x 10{sup 5} cells were injected directly into myocardial tissue after damaging the respective area by freezing (cryo-scar). 3 x 10{sup 6} of the aptamer-isolated cells were kept for further characterization (FACS and differentiation assays). 20 h after cell transplantation, MRI of the heart using a clinical 3.0 Tesla whole body scanner (Magnetom Trio, Siemens, Germany) was performed followed by histological examinations. Results: The average yield of sorted cells from 120 ml BM was 7 x 10{sup 6} cells. The cells were cultured and showed MSC-like properties. MRI showed reproducible artifacts within the RIVA-perfusion area and the cryo-scar with surprisingly excellent quality. The histological examination of the biopsies showed PKH26-positive cells within the areas which were positive in the MRI in contrast to the control biopsies. Conclusion: Immunomagnetic separation of MSC by specific aptamers linked to magnetic particles is feasible, effective and combines a specific separation and labeling technique to a &apos

Isolation and identification of gene mediating radiation-induced apoptosis in human leukemia U937 cells

International Nuclear Information System (INIS)

Tong Xin; Luo Ying; Dong Yan; Sun Zhixian

1998-01-01

Objective: Increasing evidences suggest that Caspase family proteases play an important role in the effector mechanism of apoptotic cell death. Radiation (IR) can induce apoptosis in tumor cells, so it is very important to isolate and identify the member of the Caspase family proteases involved in IR-induced apoptosis, and this would contribute to the understanding of the mechanism responsible for apoptosis execution. Methods: A PCR approach to isolate genes for IR-induced apoptosis was developed. The approach used degenerated oligonucleotide encoding the highly conserved peptides that were present in all known Caspases. Results: Protease inhibitors special for Caspases could block the apoptotic cell death caused by IR, and Caspase-3 was isolated from irradiated human leukemia U937 cells. Conclusion: Caspases involve in IR-induced apoptosis, and Caspase-3 is the pivotal element of IR-induced apoptosis

Recurrence of isolated transitional cell carcinoma in an orthotopic ...

African Journals Online (AJOL)

A.M. Moeen

2015-11-10

Nov 10, 2015 ... rare with less than 10 cases reported to date [2,3]. We present the case of a female patient with isolated recurrent transitional cell carcinoma (TCC) in an ileal neobladder, diagnosed 18 months after radical cystectomy and modified Hautmann ileal bladder substitution. E-mail address: moeen3@yahoo.com.

Isolation, Culture, and Differentiation of Bone Marrow Stromal Cells and Osteoclast Progenitors from Mice.

Science.gov (United States)

Maridas, David E; Rendina-Ruedy, Elizabeth; Le, Phuong T; Rosen, Clifford J

2018-01-06

Bone marrow stromal cells (BMSCs) constitute a cell population routinely used as a representation of mesenchymal stem cells in vitro. They reside within the bone marrow cavity alongside hematopoietic stem cells (HSCs), which can give rise to red blood cells, immune progenitors, and osteoclasts. Thus, extractions of cell populations from the bone marrow results in a very heterogeneous mix of various cell populations, which can present challenges in experimental design and confound data interpretation. Several isolation and culture techniques have been developed in laboratories in order to obtain more or less homogeneous populations of BMSCs and HSCs invitro. Here, we present two methods for isolation of BMSCs and HSCs from mouse long bones: one method that yields a mixed population of BMSCs and HSCs and one method that attempts to separate the two cell populations based on adherence. Both methods provide cells suitable for osteogenic and adipogenic differentiation experiments as well as functional assays.

Embryonic Stem Cell-Derived Cardiomyocyte Heterogeneity and the Isolation of Immature and Committed Cells for Cardiac Remodeling and Regeneration

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Kenneth R. Boheler

2011-01-01

Full Text Available Pluripotent stem cells represent one promising source for cell replacement therapy in heart, but differentiating embryonic stem cell-derived cardiomyocytes (ESC-CMs are highly heterogeneous and show a variety of maturation states. In this study, we employed an ESC clonal line that contains a cardiac-restricted ncx1 promoter-driven puromycin resistance cassette together with a mass culture system to isolate ESC-CMs that display traits characteristic of very immature CMs. The cells display properties of proliferation, CM-restricted markers, reduced mitochondrial mass, and hypoxia-resistance. Following transplantation into rodent hearts, bioluminescence imaging revealed that immature cells, but not more mature CMs, survived for at least one month following injection. These data and comparisons with more mature cells lead us to conclude that immature hypoxia resistant ESC-CMs can be isolated in mass in vitro and, following injection into heart, form grafts that may mediate long-term recovery of global and regional myocardial contractile function following infarction.

Cells isolated from human periapical cysts express mesenchymal stem cell-like properties.

Science.gov (United States)

Marrelli, Massimo; Paduano, Francesco; Tatullo, Marco

2013-01-01

We provide a detailed description of mesenchymal stem cells (MSCs) isolated from human periapical cysts, which we have termed hPCy-MSCs. These cells have a fibroblast-like shape and adhere to tissue culture plastic surfaces. hPCy-MSCs possess high proliferative potential and self-renewal capacity properties. We characterised the immunophenotype of hPCy-MSCs (CD73(+), CD90(+), CD105(+), CD13(+), CD29(+), CD44(+), CD45(-), STRO-1(+), CD146(+)) by flow cytometry and immunofluorescence. hPCy-MSCs possess the potential to differentiate into osteoblast- and adipocyte-like cells in vitro. Multi-potentiality was evaluated with culture-specific staining and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis for osteo/odontogenic and adipogenic markers. This is the first report to indicate that human periapical cysts contain cells with MSC-like properties. Taken together, our findings indicate that human periapical cysts could be a rich source of MSCs.

Frequency response for electromotility of isolated outer hair cells of the guinea pig

NARCIS (Netherlands)

Wit, HP; vanDijk, P; Segenhout, HM

1996-01-01

Frequency and impulse responses were determined for isolated guinea pig outer hair cells by electrically stimulating the cells between two wire electrodes with white noise. Cells were attached to the bottom of a small culture dish at one end while the other end was freely moving. Results have the

Circulating tumor cell isolation and diagnostics: toward routine clinical use

NARCIS (Netherlands)

Stolpe, van de A.; Pantel, K.; Sleijfer, S.; Terstappen, L.W.; Toonder, den J.M.J.

2011-01-01

From February 7–11, 2011, the multidisciplinary Lorentz Workshop Circulating Tumor Cell (CTC) Isolation and Diagnostics: Toward Routine Clinical Use was held in Leiden (The Netherlands) to discuss progress and define challenges and potential solutions for development of clinically useful circulating

Ligand activation of peroxisome proliferator-activated receptor-β/δ suppresses liver tumorigenesis in hepatitis B transgenic mice

International Nuclear Information System (INIS)

Balandaram, Gayathri; Kramer, Lance R.; Kang, Boo-Hyon; Murray, Iain A.; Perdew, Gary H.; Gonzalez, Frank J.; Peters, Jeffrey M.

2016-01-01

Highlights: • The role of PPARβ/δ in HBV-induced liver cancer was examined. • PPARβ/δ inhibits steatosis, inflammation, tumor multiplicity and promotes apoptosis. • Kupffer cell PPARβ/δ mediates these effects independent of DNA binding. - Abstract: Peroxisome proliferator-activated receptor-β/δ (PPARβ/δ) inhibits steatosis and inflammation, known risk factors for liver cancer. In this study, the effect of ligand activation of PPARβ/δ in modulating liver tumorigenesis in transgenic hepatitis B virus (HBV) mice was examined. Activation of PPARβ/δ in HBV mice reduced steatosis, the average number of liver foci, and tumor multiplicity. Reduced expression of hepatic CYCLIN D1 and c-MYC, tumor necrosis factor alpha (Tnfa) mRNA, serum levels of alanine aminotransaminase, and an increase in apoptotic signaling was also observed following ligand activation of PPARβ/δ in HBV mice compared to controls. Inhibition of Tnfa mRNA expression was not observed in wild-type hepatocytes. Ligand activation of PPARβ/δ inhibited lipopolysaccharide (LPS)-induced mRNA expression of Tnfa in wild-type, but not in Pparβ/δ-null Kupffer cells. Interestingly, LPS-induced expression of Tnfa mRNA was also inhibited in Kupffer cells from a transgenic mouse line that expressed a DNA binding mutant form of PPARβ/δ compared to controls. Combined, these results suggest that ligand activation of PPARβ/δ attenuates hepatic tumorigenesis in HBV transgenic mice by inhibiting steatosis and cell proliferation, enhancing hepatocyte apoptosis, and modulating anti-inflammatory activity in Kupffer cells.

Propranolol inhibits the in vitro conversion of thyroxine into triiodothyronine by isolated rat liver parenchymal cells

NARCIS (Netherlands)

van Noorden, C. J.; Wiersinga, W. M.; Touber, J. L.

1979-01-01

A model for the in vitro study of the conversion of thyroxine into triiodothyronine using isolated rat liver parenchymal cells is described. Isolated liver cells (mean protein content 18 mg/ml) convert approximately 0.8% of 1.3 microM exogenously added T4 into T3 during thirty minutes incubation.

Impact of cell lines included in enterovirus isolation protocol on perception of nonpolio enterovirus species C diversity.

Science.gov (United States)

Adeniji, Johnson Adekunle; Faleye, Temitope Oluwasegun Cephas

2014-10-01

There has been under-reporting of nonpolio enterovirus species Cs (NPESCs) in Nigeria despite the fact that most isolates recovered from the Nigerian vaccine derived poliovirus serotype 2 (VDPV2) outbreak were recombinants with nonstructural region of NPESC origin. It has been suggested that cell lines included in enterovirus isolation protocols might account for this phenomenon and this study examined this suggestion. Fifteen environmental samples concentrated previously and analysed using L20B and RD cell lines as part of the poliovirus environmental surveillance (ES) program in Nigeria were randomly selected and inoculated into two cell lines (MCF-7 and LLC-MK2). Isolates were identified as enteroviruses and species C members using different RT-PCR assays, culture in L20B cell line and sequencing of partial VP1. Forty-eight (48) isolates were recovered from the 15 samples, 47 (97.9%) of which were enteroviruses. Of the enteroviruses, 32 (68.1%) belonged to enterovirus species C (EC) of which 19 (40.4%) were polioviruses and 13 (27.7%) were NPESC members. All 13 NPESC isolates were recovered on MCF-7. Results of the study show that NPESCs are circulating in Nigeria and their under-reporting was due to the combination of cell lines used for enterovirus isolation in previous reports. Copyright © 2014 Elsevier B.V. All rights reserved.

Successful Isolation of Viable Adipose-Derived Stem Cells from Human Adipose Tissue Subject to Long-Term Cryopreservation: Positive Implications for Adult Stem Cell-Based Therapeutics in Patients of Advanced Age

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Sean M. Devitt

2015-01-01

Full Text Available We examined cell isolation, viability, and growth in adipose-derived stem cells harvested from whole adipose tissue subject to different cryopreservation lengths (2–1159 days from patients of varying ages (26–62 years. Subcutaneous abdominal adipose tissue was excised during abdominoplasties and was cryopreserved. The viability and number of adipose-derived stem cells isolated were measured after initial isolation and after 9, 18, and 28 days of growth. Data were analyzed with respect to cryopreservation duration and patient age. Significantly more viable cells were initially isolated from tissue cryopreserved 2 years, irrespective of patient age. However, this difference did not persist with continued growth and there were no significant differences in cell viability or growth at subsequent time points with respect to cryopreservation duration or patient age. Mesenchymal stem cell markers were maintained in all cohorts tested throughout the duration of the study. Consequently, longer cryopreservation negatively impacts initial live adipose-derived stem cell isolation; however, this effect is neutralized with continued cell growth. Patient age does not significantly impact stem cell isolation, viability, or growth. Cryopreservation of adipose tissue is an effective long-term banking method for isolation of adipose-derived stem cells in patients of varying ages.

Decreased expression of cell adhesion genes in cancer stem-like cells isolated from primary oral squamous cell carcinomas.

Science.gov (United States)

Mishra, Amrendra; Sriram, Harshini; Chandarana, Pinal; Tanavde, Vivek; Kumar, Rekha V; Gopinath, Ashok; Govindarajan, Raman; Ramaswamy, S; Sadasivam, Subhashini

2018-05-01

The goal of this study was to isolate cancer stem-like cells marked by high expression of CD44, a putative cancer stem cell marker, from primary oral squamous cell carcinomas and identify distinctive gene expression patterns in these cells. From 1 October 2013 to 4 September 2015, 76 stage III-IV primary oral squamous cell carcinoma of the gingivobuccal sulcus were resected. In all, 13 tumours were analysed by immunohistochemistry to visualise CD44-expressing cells. Expression of CD44 within The Cancer Genome Atlas-Head and Neck Squamous Cell Carcinoma RNA-sequencing data was also assessed. Seventy resected tumours were dissociated into single cells and stained with antibodies to CD44 as well as CD45 and CD31 (together referred as Lineage/Lin). From 45 of these, CD44 + Lin - and CD44 - Lin - subpopulations were successfully isolated using fluorescence-activated cell sorting, and good-quality RNA was obtained from 14 such sorted pairs. Libraries from five pairs were sequenced and the results analysed using bioinformatics tools. Reverse transcription quantitative polymerase chain reaction was performed to experimentally validate the differential expression of selected candidate genes identified from the transcriptome sequencing in the same 5 and an additional 9 tumours. CD44 was expressed on the surface of poorly differentiated tumour cells, and within the The Cancer Genome Atlas-Head and Neck Squamous Cell Carcinoma samples, its messenger RNA levels were higher in tumours compared to normal. Transcriptomics revealed that 102 genes were upregulated and 85 genes were downregulated in CD44 + Lin - compared to CD44 - Lin - cells in at least 3 of the 5 tumours sequenced. The upregulated genes included those involved in immune regulation, while the downregulated genes were enriched for genes involved in cell adhesion. Decreased expression of PCDH18, MGP, SPARCL1 and KRTDAP was confirmed by reverse transcription quantitative polymerase chain reaction. Lower expression of

Isolation, Synthesis, and Antisepsis Effects of a C-Methylcoumarinochromone Isolated from Abronia nana Cell Culture.

Science.gov (United States)

Lee, Wonhwa; Lee, Doohyun; Lee, Yuri; Lee, Taeho; Song, Kyung-Sik; Yang, Eun-Ju; Bae, Jong-Sup

2018-05-25

Only a few isoflavones have been isolated from plants of the genus Abronia. The biological properties of compounds isolated from Abronia species have not been well established, and their antisepsis effects have not been reported yet. In the present study, a new C-methylcoumarinochromone, was isolated from Abronia nana suspension cultures. Its structure was deduced as 9,11-dihydroxy-10-methylcoumarinochromone (boeravinone Y, 1) by spectroscopic data analysis and verified by chemical synthesis. The potential inhibitory effects of 1 against high mobility group box 1 (HMGB1)-mediated septic responses were investigated. Results showed that 1 effectively inhibited lipopolysaccharide-induced release of HMGB1 and suppressed HMGB1-mediated septic responses, in terms of reduction of hyperpermeability, leukocyte adhesion and migration, and cell adhesion molecule expression. In addition, 1 increased the phagocytic activity of macrophages and exhibited bacterial clearance effects in the peritoneal fluid and blood of mice with cecal ligation and puncture-induced sepsis. Collectively, these results suggested that 1 might have potential therapeutic activity against various severe vascular inflammatory diseases via inhibition of the HMGB1 signaling pathway.

Cell-swelling-induced taurine release from isolated perfused rat liver

NARCIS (Netherlands)

Brand, H. S.; Meijer, A. J.; Gustafson, L. A.; Jörning, G. G.; Leegwater, A. C.; Maas, M. A.; Chamuleau, R. A.

1994-01-01

Astrocytes and lymphocytes are able to release significant amounts of taurine during periods of hypotonicity to reduce the increase in cell volume. To investigate this mechanism in the liver, we studied the release of free amino acids from isolated perfused rat liver during hypotonicity. The

Liver scanning using indium-113m at the University Teaching Hospital, Lusaka, Zambia

Energy Technology Data Exchange (ETDEWEB)

Mulaisho, C [Nuclear Medicine Unit, Department of Medicine, University of Zambia, Lusaka, Zambia; Mumba, K N [Radio-isotope Research Unit, National Council for Scientific Research, Lusaka, Zambia

1981-11-21

Liver scanning using the radio-isotope indium-113m, can now be routinely perfomed at the University Teaching Hospital, Lusaka, Zambia. The dose used is 1 - 4 mCi. Liver scans have been performed on 48 subjects, including 10 healthy individuals 16 patients with histologically proven hepatocellular carcinoma, 11 with clinical and laboratory evidence of portal hypertension and 11 with miscellaneous illnesses. Seven representative scans are illustrated. The procedure is easy, and gives a fairly accurate functional estimate of Kupffer cell mass. In hepatoma the scan may be either larger than or smaller than normal and reflects more accurately the residual function of the Kupffer cells. In cirrhosis of the liver with portal hypertention, residual Kupffer cell mass is small. Consequently, most of the indium-113m is taken up by the splenic reticulo-endothelial system, resulting in a large spleen scan. This technique, although fraught with major limitations, is a useful additional diagnostic tool in the management of chronic liver disease.

Isolation and characterization of conditionally immortalized mouse glomerular endothelial cell lines.

Science.gov (United States)

Rops, Angelique L; van der Vlag, Johan; Jacobs, Cor W; Dijkman, Henry B; Lensen, Joost F; Wijnhoven, Tessa J; van den Heuvel, Lambert P; van Kuppevelt, Toin H; Berden, Jo H

2004-12-01

The culture and establishment of glomerular cell lines has proven to be an important tool for the understanding of glomerular cell functions in glomerular physiology and pathology. Especially, the recent establishment of a conditionally immortalized visceral epithelial cell line has greatly boosted the research on podocyte biology. Glomeruli were isolated from H-2Kb-tsA58 transgenic mice that contain a gene encoding a temperature-sensitive variant of the SV40 large tumor antigen, facilitating proliferative growth at 33 degrees C and differentiation at 37 degrees C. Glomerular endothelial cells were isolated from glomerular outgrowth by magnetic beads loaded with CD31, CD105, GSL I-B4, and ULEX. Clonal cell lines were characterized by immunofluorescence staining with antibodies/lectins specific for markers of endothelial cells, podocytes, and mesangial cells. Putative glomerular endothelial cell lines were analyzed for (1) cytokine-induced expression of adhesion molecules; (2) tube formation on Matrigel coating; and (3) the presence of fenestrae. As judged by immunostaining for Wilms tumor-1, smooth muscle actin (SMA), podocalyxin, and von Willebrand factor (vWF), we obtained putative endothelial, podocyte and mesangial cell lines. The mouse glomerular endothelial cell clone #1 (mGEnC-1) was positive for vWF, podocalyxin, CD31, CD105, VE-cadherin, GSL I-B4, and ULEX, internalized acetylated-low-density lipoprotein (LDL), and showed increased expression of adhesion molecules after activation with proinflammatory cytokines. Furthermore, mGEnC-1 formed tubes and contained nondiaphragmed fenestrae. The mGEnC-1 represents a conditionally immortalized cell line with various characteristics of differentiated glomerular endothelial cells when cultured at 37 degrees C. Most important, mGEnC-1 contains nondiaphragmed fenestrae, which is a unique feature of glomerular endothelial cells.

Failure of isolated rat tibial periosteal cells to 5 alpha reduce testosterone to 5 alpha-dihydrotestosterone

International Nuclear Information System (INIS)

Turner, R.T.; Bleiberg, B.; Colvard, D.S.; Keeting, P.E.; Evans, G.; Spelsberg, T.C.

1990-01-01

Periosteal cells were isolated from tibiae of adult male rats after collagenase treatment. Northern blot analysis of total cytoplasmic RNA extracted from the isolated periosteal cells was positive for expression of genes encoding the osteoblast marker proteins osteocalcin (BGP) and pre-pro-alpha 2(I) chain of type 1 precollagen. The isolated periosteal cells were incubated with 1 nM [3H]testosterone [( 3 H]T) for up to 240 minutes and the reaction products separated by high-performance liquid chromatography. [ 3 H]5 alpha-dihydrotestosterone [( 3 H]DHT) was not detected in extracts of periosteal cell incubations. In contrast, [ 3 H]DHT was produced in a time-dependent manner by cells from seminal vesicles. These results suggest that testosterone 5 alpha-reductase activity is not expressed by osteoblasts in rat tibial periosteum and that the anabolic effects of androgens in this tissue are not mediated by locally produced DHT

Facilitated Engraftment of Isolated Islets Coated With Expanded Vascular Endothelial Cells for Islet Transplantation.

Science.gov (United States)

Barba-Gutierrez, D Alonso; Daneri-Navarro, A; Villagomez-Mendez, J Jesus Alejandro; Kanamune, J; Robles-Murillo, A Karina; Sanchez-Enriquez, S; Villafan-Bernal, J Rafael; Rivas-Carrillo, J D

2016-03-01

Diabetes is complex disease, which involves primary metabolic changes followed by immunological and vascular pathophysiological adjustments. However, it is mostly characterized by an unbalanced decreased number of the β-cells unable to maintain the metabolic requirements and failure to further regenerate newly functional pancreatic islets. The objective of this study was to analyze the properties of the endothelial cells to facilitate the islet cells engraftment after islet transplantation. We devised a co-cultured engineer system to coat isolated islets with vascular endothelial cells. To assess the cell integration of cell-engineered islets, we stained them for endothelial marker CD31 and nuclei counterstained with DAPI dye. We comparatively performed islet transplantations into streptozotocin-induced diabetic mice and recovered the islet grafts for morphometric analyses on days 3, 7, 10, and 30. Blood glucose levels were measured continuously after islet transplantation to monitor the functional engraftment and capacity to achieve metabolic control. Cell-engineered islets showed a well-defined rounded shape after co-culture when compared with native isolated islets. Furthermore, the number of CD31-positive cells layered on the islet surface showed a direct proportion with engraftment capacities and less TUNEL-positive cells on days 3 and 7 after transplantation. We observed that vascular endothelial cells could be functional integrated into isolated islets. We also found that islets that are coated with vascular endothelial cells increased their capacity to engraft. These findings indicate that islets coated with endothelial cells have a greater capacity of engraftment and thus establish a definitely vascular network to support the metabolic requirements. Copyright © 2016 Elsevier Inc. All rights reserved.

Relationship between plasma cholesterol levels and cholesterol esterification in isolated human mononuclear cells

International Nuclear Information System (INIS)

Dallongeville, J.; Davignon, J.; Lussier-Cacan, S.

1990-01-01

The authors studied the relationship between plasma lipoprotein concentrations and cholesterol esterification in freshly isolated human mononuclear cells from 27 normolipidemic and 32 hyperlipidemic individuals. Cells were either incubated for 5 hours with radiolabeled oleate immediately after isolation or were preincubated for 18 hours in the presence of exogenous cholesterol, and then incubated with [ 14 C]sodium-oleate-albumin complex. In the absence of exogenous cholesterol, control and hypercholesterolemic subjects had similarly low values of intracellular cholesterol esterification. In the presence of exogenous cholesterol, both hypertriglyceridemic and hypercholesterolemic subjects had higher cholesterol esterification than controls. There was a significant correlation between the rate of cholesterol esterification and plasma total cholesterol. These results suggest that plasma cholesterol levels may regulate mononuclear cell intra-cellular cholesterol esterification in humans

Human cultured cells are capable to incorporate isolated plant mitochondria loaded with exogenous DNA

Directory of Open Access Journals (Sweden)

Laktionov P. P.

2012-07-01

Full Text Available Aim. To investigate the possibility of human cultured cells to incorporate isolated mitochondria together with exogenous DNA introduced into organelles. Methods. Two approaches were used for this purpose, fluorescent labelling of mitochondria and/or DNA with subsequent analysis of the cells subjected to incubation by microscopy or by quantitative PCR. Results. We have shown that human cultured cells lines, HeLa and HUVEC, are capable to uptake isolated plant mitochondria and that this process depends on the incubation time and concentration of organelles present in medium. The incorporated mitochondria can serve as vehicles to deliver exogenous DNA into human cells, this DNA is then distributed in different cell compartments. Conclusions. These results are preliminary and need further investigations, including testing the possibility of human cells to incorporate the mitochondria of human or animal origin and creating genetic construction which could provide certain selectivity or stability of the transferred exogenous DNA upon cell uptake of the mitochondria as vectors.

Lack of functional relevance of isolated cell damage in transplants of Parkinson's disease patients

DEFF Research Database (Denmark)

Cooper, Oliver; Astradsson, Arnar; Hallett, Penny

2009-01-01

Postmortem analyses from clinical neural transplantation trials of several subjects with Parkinson's disease revealed surviving grafted dopaminergic neurons after more than a decade. A subset of these subjects displayed isolated dopaminergic neurons within the grafts that contained Lewy body......-like structures. In this review, we discuss why this isolated cell damage is unlikely to affect the overall graft function and how we can use these observations to help us to understand age-related neurodegeneration and refine our future cell replacement therapies....

Differences of isolated dental stem cells dependent on donor age and consequences for autologous tooth replacement.

Science.gov (United States)

Kellner, Manuela; Steindorff, Marina M; Strempel, Jürgen F; Winkel, Andreas; Kühnel, Mark P; Stiesch, Meike

2014-06-01

Autologous therapy via stem cell-based tissue regeneration is an aim to rebuild natural teeth. One option is the use of adult stem cells from the dental pulp (DPSCs), which have been shown to differentiate into several types of tissue in vitro and in vivo, especially into tooth-like structures. DPSCs are mainly isolated from the dental pulp of third molars routinely extracted for orthodontic reasons. Due to the extraction of third molars at various phases of life, DPSCs are isolated at different developmental stages of the tooth. The present study addressed the question whether DPSCs from patients of different ages were similar in their growth characteristics with respect to the stage of tooth development. Therefore DPSCs from third molars of 12-30 year-old patients were extracted, and growth characteristics, e.g. doubling time and maximal cell division potential were analysed. In addition, pulp and hard dental material weight were recorded. Irrespective of the age of patients almost all isolated cells reached 40-60 generations with no correlation between maximal cell division potential and patient age. Cells from patients <22 years showed a significantly faster doubling time than the cells from patients ≥22 years. The age of patients at the time of stem cell isolation is not a crucial factor concerning maximal cell division potential, but does have an impact on the doubling time. However, differences in individuals regarding growth characteristics were more pronounced than age-dependent differences. Copyright © 2014 Elsevier Ltd. All rights reserved.

Failure of isolated rat tibial periosteal cells to 5 alpha reduce testosterone to 5 alpha-dihydrotestosterone

Energy Technology Data Exchange (ETDEWEB)

Turner, R.T.; Bleiberg, B.; Colvard, D.S.; Keeting, P.E.; Evans, G.; Spelsberg, T.C. (Mayo Clinic, Rochester, MN (USA))

1990-07-01

Periosteal cells were isolated from tibiae of adult male rats after collagenase treatment. Northern blot analysis of total cytoplasmic RNA extracted from the isolated periosteal cells was positive for expression of genes encoding the osteoblast marker proteins osteocalcin (BGP) and pre-pro-alpha 2(I) chain of type 1 precollagen. The isolated periosteal cells were incubated with 1 nM (3H)testosterone (({sup 3}H)T) for up to 240 minutes and the reaction products separated by high-performance liquid chromatography. ({sup 3}H)5 alpha-dihydrotestosterone (({sup 3}H)DHT) was not detected in extracts of periosteal cell incubations. In contrast, ({sup 3}H)DHT was produced in a time-dependent manner by cells from seminal vesicles. These results suggest that testosterone 5 alpha-reductase activity is not expressed by osteoblasts in rat tibial periosteum and that the anabolic effects of androgens in this tissue are not mediated by locally produced DHT.

Paired single cell co-culture microenvironments isolated by two-phase flow with continuous nutrient renewal.

Science.gov (United States)

Chen, Yu-Chih; Cheng, Yu-Heng; Kim, Hong Sun; Ingram, Patrick N; Nor, Jacques E; Yoon, Euisik

2014-08-21

Cancer-stromal cell interactions are a critical process in tumorigenesis. Conventional dish-based assays, which simply mix two cell types, have limitations in three aspects: 1) limited control of the cell microenvironment; 2) inability to study cell behavior in a single-cell manner; and 3) have difficulties in characterizing single cell behavior within a highly heterogeneous cell population (e.g. tumor). An innovative use of microfluidic technology is for improving the spatial resolution for single cell assays. However, it is challenging to isolate the paired interacting cells while maintaining nutrient renewal. In this work, two-phase flow was used as a simple isolation method, separating the microenvironment of each individual chamber. As nutrients in an isolated chamber are consumed by cells, media exchange is required. To connect the cell culture chamber to the media exchange layer, we demonstrated a 3D microsystem integration technique using vertical connections fabricated by deep reactive-ion etching (DRIE). Compared to previous approaches, the presented process allows area reduction of vertical connections by an order of magnitude, enabling compact 3D integration. A semi-permeable membrane was sandwiched between the cell culture layer and the media exchange layer. The selectivity of the semi-permeable membrane results in the retention of the signaling proteins within the chamber while allowing free diffusion of nutrients (e.g., glucose and amino acids). Thus, paracrine signals are accumulated inside the chamber without cross-talk between cells in other chambers. Utilizing these innovations, we co-cultured UM-SCC-1 (head and neck squamous cell carcinoma) cells and endothelial cells to simulate tumor proliferation enhancement in the vascular endothelial niche.

Efficient adhesion-based plasma membrane isolation for cell surface N-glycan analysis.

Science.gov (United States)

Mun, Ji-Young; Lee, Kyung Jin; Seo, Hoon; Sung, Min-Sun; Cho, Yee Sook; Lee, Seung-Goo; Kwon, Ohsuk; Oh, Doo-Byoung

2013-08-06

Glycans, which decorate cell surfaces, play crucial roles in various physiological events involving cell surface recognition. Despite the importance of surface glycans, most analyses have been performed using total cells or whole membranes rather than plasma membranes due to difficulties related to isolation. In the present study, we employed an adhesion-based method for plasma membrane isolation to analyze N-glycans on cell surfaces. Cells were attached to polylysine-coated glass plates and then ruptured by hypotonic pressure. After washing to remove intracellular organelles, only a plasma membrane fraction remained attached to the plates, as confirmed by fluorescence imaging using organelle-specific probes. The plate was directly treated with trypsin to digest and detach the glycoproteins from the plasma membrane. From the resulting glycopeptides, N-glycans were released and analyzed using MALDI-TOF mass spectrometry and HPLC. When N-glycan profiles obtained by this method were compared to those by other methods, the amount of high-mannose type glycans mainly contaminated from the endoplasmic reticulum was dramatically reduced, which enabled the efficient detection of complex type glycans present on the cell surface. Moreover, this method was successfully used to analyze the increase of high-mannose glycans on the surface as induced by a mannosidase inhibitor treatment.

Glucose metabolite patterns as markers of functional differentiation in freshly isolated and cultured mouse mammary epithelial cells

International Nuclear Information System (INIS)

Emerman, J.T.; Bartley, J.C.; Bissel, M.J.

1981-01-01

In the mammary gland of non-ruminant animals, glucose is utilized in a characteristic and unique way during lacation. By measuring the incorporation of glucose carbon from [U- 14 C]glucose into intermediary metabolitees and metabolic products in mammary epithelia cells from virgin, pregnant, and lacating mice, we domonstrate that glucose metabolite patterns can be used to recognize stages of differentiated function. For these cells, the rates of synthesis of glycogen and lactose, the ratio of lactate to alanine, and the ratio of citrate to malate are important parameters in identifying the degree of expression of differentiation. We further show that these patterns can be used as markers to determine the differentiated state of cultured mammary epithelial cells. Cells maintained on plastic substrates lose their distinctive glucose metabolite patterns while those on floating collagen gels do not. Cells isolated from pregnant mice and cultured on collagen gels have a pattern similar to that of their freshly isolated counter-parts. When isolated from lacating mice, the metabolite patterns of cells cultured on collagen gels are different from that of the cells of origin, and resembles that of freshly isolated cells from pregnant mice. Our findings suggest that the floating collagen gels under the culture conditions used in these experiments provide an environment for the functional expression of the pregnant state, while additional factors are needed for the expression of the lactating state

Cell cycle disturbances in slowly growing sublines isolated from X-irradiated L5178Y-S cell populations

International Nuclear Information System (INIS)

Beer, J.Z.; Bocian, E.; Budzicka, E.; Szumiel, I.; Ziemba-Zak, B.; Kopec, M.

1974-01-01

Cell cycle was analyzed autoradiographically in a test line of murine leukaemic lymphoblasts L5178Y-S and in two slowly growing sublines isolated from cell cultures irradiated with 300 rad of X-rays. It was found that prolongation of the cell cycle in the slowly growing sublines is connected primarily with delayed progression through G2 phase. This conclusion was further supported by results of determination of DNA content per cell in 13 slowly growing cell sublines and karyotype analysis of 18 sublines. No correlation was found between a sublines' mean doubling time and its chromosome number whereas DNA content per cell was clearly dependent on the growth rate. (author)

Isolate-dependent growth, virulence, and cell wall composition in the human pathogen Aspergillus fumigatus.

Directory of Open Access Journals (Sweden)

Nansalmaa Amarsaikhan

Full Text Available The ubiquitous fungal pathogen Aspergillus fumigatus is a mediator of allergic sensitization and invasive disease in susceptible individuals. The significant genetic and phenotypic variability between and among clinical and environmental isolates are important considerations in host-pathogen studies of A. fumigatus-mediated disease. We observed decreased radial growth, rate of germination, and ability to establish colony growth in a single environmental isolate of A. fumigatus, Af5517, when compared to other clinical and environmental isolates. Af5517 also exhibited increased hyphal diameter and cell wall β-glucan and chitin content, with chitin most significantly increased. Morbidity, mortality, lung fungal burden, and tissue pathology were decreased in neutropenic Af5517-infected mice when compared to the clinical isolate Af293. Our results support previous findings that suggest a correlation between in vitro growth rates and in vivo virulence, and we propose that changes in cell wall composition may contribute to this phenotype.

Isolation and Characterization of Human Lung Lymphatic Endothelial Cells

Science.gov (United States)

Lorusso, Bruno; Falco, Angela; Madeddu, Denise; Frati, Caterina; Cavalli, Stefano; Graiani, Gallia; Gervasi, Andrea; Rinaldi, Laura; Lagrasta, Costanza; Maselli, Davide; Gnetti, Letizia; Silini, Enrico M.; Quaini, Eugenio; Ampollini, Luca; Carbognani, Paolo; Quaini, Federico

2015-01-01

Characterization of lymphatic endothelial cells from the respiratory system may be crucial to investigate the role of the lymphatic system in the normal and diseased lung. We describe a simple and inexpensive method to harvest, isolate, and expand lymphatic endothelial cells from the human lung (HL-LECs). Fifty-five samples of healthy lung selected from patients undergoing lobectomy were studied. A two-step purification tool, based on paramagnetic sorting with monoclonal antibodies to CD31 and Podoplanin, was employed to select a pure population of HL-LECs. The purity of HL-LECs was assessed by morphologic criteria, immunocytochemistry, flow cytometry, and functional assays. Interestingly, these cells retain in vitro several receptor tyrosine kinases (RTKs) implicated in cell survival and proliferation. HL-LECs represent a clinically relevant cellular substrate to study lymphatic biology, lymphoangiogenesis, interaction with microbial agents, wound healing, and anticancer therapy. PMID:26137493

Isolated perifacial lymph node metastasis in oral squamous cell carcinoma with clinically node-negative neck.

Science.gov (United States)

Agarwal, Sangeet Kumar; Arora, Sowrabh Kumar; Kumar, Gopal; Sarin, Deepak

2016-10-01

The incidence of occult perifacial nodal disease in oral cavity squamous cell carcinoma is not well reported. The purpose of this study was to evaluate the incidence of isolated perifacial lymph node metastasis in patients with oral squamous cell carcinoma with a clinically node-negative neck. The study will shed light on current controversies and will provide valuable clinical and pathological information in the practice of routine comprehensive removal of these lymph node pads in selective neck dissection in the node-negative neck. Prospective analysis. This study was started in August 2011 when intraoperatively we routinely separated the lymph node levels from the main specimen for evaluation of the metastatic rate to different lymph node levels in 231 patients of oral squamous cell cancer with a clinically node-negative neck. The current study demonstrated that 19 (8.22%) out of 231 patients showed ipsilateral isolated perifacial lymph node involvement. The incidence of isolated perifacial nodes did not differ significantly between the oral tongue (7.14%) and buccal mucosa (7.75%). Incidence was statistically significant in cases with lower age group (oral squamous cell carcinoma with a clinically node-negative neck. The incidence of isolated perifacial involvement is high in cases of buccal mucosal and tongue cancers. A meticulous dissection of the perifacial nodes seems prudent when treating the neck in oral cavity squamous cell carcinoma. 4 Laryngoscope, 126:2252-2256, 2016. © 2016 The American Laryngological, Rhinological and Otological Society, Inc.

Classification of large circulating tumor cells isolated with ultra-high throughput microfluidic Vortex technology

Science.gov (United States)

Che, James; Yu, Victor; Dhar, Manjima; Renier, Corinne; Matsumoto, Melissa; Heirich, Kyra; Garon, Edward B.; Goldman, Jonathan; Rao, Jianyu; Sledge, George W.; Pegram, Mark D.; Sheth, Shruti; Jeffrey, Stefanie S.; Kulkarni, Rajan P.; Sollier, Elodie; Di Carlo, Dino

2016-01-01

Circulating tumor cells (CTCs) are emerging as rare but clinically significant non-invasive cellular biomarkers for cancer patient prognosis, treatment selection, and treatment monitoring. Current CTC isolation approaches, such as immunoaffinity, filtration, or size-based techniques, are often limited by throughput, purity, large output volumes, or inability to obtain viable cells for downstream analysis. For all technologies, traditional immunofluorescent staining alone has been employed to distinguish and confirm the presence of isolated CTCs among contaminating blood cells, although cells isolated by size may express vastly different phenotypes. Consequently, CTC definitions have been non-trivial, researcher-dependent, and evolving. Here we describe a complete set of objective criteria, leveraging well-established cytomorphological features of malignancy, by which we identify large CTCs. We apply the criteria to CTCs enriched from stage IV lung and breast cancer patient blood samples using the High Throughput Vortex Chip (Vortex HT), an improved microfluidic technology for the label-free, size-based enrichment and concentration of rare cells. We achieve improved capture efficiency (up to 83%), high speed of processing (8 mL/min of 10x diluted blood, or 800 μL/min of whole blood), and high purity (avg. background of 28.8±23.6 white blood cells per mL of whole blood). We show markedly improved performance of CTC capture (84% positive test rate) in comparison to previous Vortex designs and the current FDA-approved gold standard CellSearch assay. The results demonstrate the ability to quickly collect viable and pure populations of abnormal large circulating cells unbiased by molecular characteristics, which helps uncover further heterogeneity in these cells. PMID:26863573

Isolation and Characterization of Current Human Coronavirus Strains in Primary Human Epithelial Cell Cultures Reveal Differences in Target Cell Tropism

Science.gov (United States)

Dijkman, Ronald; Jebbink, Maarten F.; Koekkoek, Sylvie M.; Deijs, Martin; Jónsdóttir, Hulda R.; Molenkamp, Richard; Ieven, Margareta; Goossens, Herman; Thiel, Volker

2013-01-01

The human airway epithelium (HAE) represents the entry port of many human respiratory viruses, including human coronaviruses (HCoVs). Nowadays, four HCoVs, HCoV-229E, HCoV-OC43, HCoV-HKU1, and HCoV-NL63, are known to be circulating worldwide, causing upper and lower respiratory tract infections in nonhospitalized and hospitalized children. Studies of the fundamental aspects of these HCoV infections at the primary entry port, such as cell tropism, are seriously hampered by the lack of a universal culture system or suitable animal models. To expand the knowledge on fundamental virus-host interactions for all four HCoVs at the site of primary infection, we used pseudostratified HAE cell cultures to isolate and characterize representative clinical HCoV strains directly from nasopharyngeal material. Ten contemporary isolates were obtained, representing HCoV-229E (n = 1), HCoV-NL63 (n = 1), HCoV-HKU1 (n = 4), and HCoV-OC43 (n = 4). For each strain, we analyzed the replication kinetics and progeny virus release on HAE cell cultures derived from different donors. Surprisingly, by visualizing HCoV infection by confocal microscopy, we observed that HCoV-229E employs a target cell tropism for nonciliated cells, whereas HCoV-OC43, HCoV-HKU1, and HCoV-NL63 all infect ciliated cells. Collectively, the data demonstrate that HAE cell cultures, which morphologically and functionally resemble human airways in vivo, represent a robust universal culture system for isolating and comparing all contemporary HCoV strains. PMID:23427150

Isolation and characterization of portal branch ligation-stimulated Hmga2-positive bipotent hepatic progenitor cells

International Nuclear Information System (INIS)

Sakai, Hiroshi; Tagawa, Yoh-ichi; Tamai, Miho; Motoyama, Hiroaki; Ogawa, Shinichiro; Soeda, Junpei; Nakata, Takenari; Miyagawa, Shinichi

2010-01-01

Research highlights: → Hepatic progenitor cells were isolated from the portal branch-ligated liver of mice. → Portal branch ligation-stimulated hepatic progenitor cells (PBLHCs) express Hmga2. → PBLHCs have bidirectional differentiation capability in vitro. -- Abstract: Hepatic stem/progenitor cells are one of several cell sources that show promise for restoration of liver mass and function. Although hepatic progenitor cells (HPCs), including oval cells, are induced by administration of certain hepatotoxins in experimental animals, such a strategy would be inappropriate in a clinical setting. Here, we investigated the possibility of isolating HPCs in a portal branch-ligated liver model without administration of any chemical agents. A non-parenchymal cell fraction was prepared from the portal branch-ligated or non-ligated lobe, and seeded onto plates coated with laminin. Most of the cells died, but a small number were able to proliferate. These proliferating cells were cloned as portal branch ligation-stimulated hepatic cells (PBLHCs) by the limiting dilution method. The PBLHCs expressed cytokeratin19, albumin, and Hmga2. The PBLHCs exhibited metabolic functions such as detoxification of ammonium ions and synthesis of urea on Matrigel-coated plates in the presence of oncostatin M. In Matrigel mixed with type I collagen, the PBLHCs became rearranged into cystic and tubular structures. Immunohistochemical staining demonstrated the presence of Hmga2-positive cells around the interlobular bile ducts in the portal branch-ligated liver lobes. In conclusion, successful isolation of bipotent hepatic progenitor cell clones, PBLHCs, from the portal branch-ligated liver lobes of mice provides the possibility of future clinical application of portal vein ligation to induce hepatic progenitor cells.

Isolation of uv-sensitive variants of human FL cells by a viral suicide method

International Nuclear Information System (INIS)

Shiomi, T.; Sato, K.

1979-01-01

A new method (viral suicide method) for the isolation of uv-sensitive mutants is described. Colonies of mutagenized human FL cells were infected with uv-irradiated Herpes simplex viruses and surviving ones which seemed to be deficient in host cell reactivation (HCR) were examined for their uv sensitivity. Nineteen of 238 clones examined were sensitive to uv irradiation at the time of the isolation. After recloning, four of these clones have been studied and two (UVS-1 and UVS-2) of them are stable in their uv sensitivity for 4 months in culture. uv sensitivity of UVS-1, UVS-2, and the parental FL cells are as follows: the extrapolation numbers (n) are 2.2, 2.1, and 1.8 and mean lethal doses (DO) are 2.9, 3.7, and 7.8 J/m 2 for UVS-1, UVS-2, and the parental FL cells, respectively. They are no more sensitive than FL cells to x-irradiation. The ability of HCR in UVS-2 cells is apparently lower than that in FL cells, whereas UVS-1 cells are the same as FL cells in the ability

A comparative study of Mono Mac 6 cells, isolated mononuclear cells and Limulus amoebocyte lysate assay in pyrogen testing

DEFF Research Database (Denmark)

Moesby, Lise; Jensen, S; Hansen, E W

1999-01-01

) and Staphylococcus aureus was comparable to that of MNC. Aspergillus niger and Candida albicans induced IL-6 in isolated MNC, but not in MM6. The detection limit for Salmonella typhimurium in the MM6 assay was comparable to that of the LAL assay. As expected, S. aureus and C. albicans did not show any LAL activity......Pyrogen induced secretion of interleukin 6 (IL-6) in Mono Mac 6 (MM6) cells was measured. The ability of the MM6 cell culture to detect pyrogens was compared to the Limulus amoebocyte lysate (LAL) test and isolated mononuclear cells (MNC). The detection limit of MM6 for lipopolysaccharide (LPS....... A. niger and Influenza virus showed some activity in the LAL test, but could not be detected by MM6 cells. In conclusion, the MM6 assay is a good supplement to the current pyrogen assays for detection of LPS, S. aureus and S. typhimurium, but the MM6 assay could not detect A. niger, C. albicans...

Mast cell density in isolated monkey lungs on exposure to cigarette smoke.

OpenAIRE

Walter, A; Walter, S

1982-01-01

The density and percentage of degranulated cells of the mast cell population were studied in the isolated lungs of 25 monkeys (Macaca radiata radiata) before and after acute exposure to cigarette smoke. In each animal one lung was used as the test lung while the other lung was used as its control. In the control lungs the total mean mast cell count was 9.5/mm2 and the proportion of degranulated cells was 9.7%. In the lungs exposed to smoke the total counts were lower (7.3/mm2) and the percent...

Isolation of stem-like cells from spontaneous feline mammary carcinomas: Phenotypic characterization and tumorigenic potential

Energy Technology Data Exchange (ETDEWEB)

Barbieri, Federica; Wurth, Roberto [Section of Pharmacology, Dept. of Internal Medicine Di.M.I., and Center of Excellence for Biomedical Research - University of Genova, Viale Benedetto XV, 2, 16132 Genova (Italy); Ratto, Alessandra; Campanella, Chiara; Vito, Guendalina [Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle D' Aosta, National Reference Center of Veterinary and Comparative Oncology (CEROVEC), Piazza Borgo Pila, 16129, Genova (Italy); Thellung, Stefano [Section of Pharmacology, Dept. of Internal Medicine Di.M.I., and Center of Excellence for Biomedical Research - University of Genova, Viale Benedetto XV, 2, 16132 Genova (Italy); Daga, Antonio [Laboratory of Translational Oncology, IRCCS Azienda Ospedaliera Universitaria San Martino - IST- Istituto Nazionale Ricerca sul Cancro, L.go R. Benzi, 10, 16132 Genova Italy (Italy); Cilli, Michele [Animal Facility, IRCCS Azienda Ospedaliera Universitaria San Martino - IST- Istituto Nazionale Ricerca sul Cancro, L.go R. Benzi, 10, 16132 Genova Italy (Italy); Ferrari, Angelo [Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle D' Aosta, National Reference Center of Veterinary and Comparative Oncology (CEROVEC), Piazza Borgo Pila, 16129, Genova (Italy); Florio, Tullio, E-mail: tullio.florio@unige.it [Section of Pharmacology, Dept. of Internal Medicine Di.M.I., and Center of Excellence for Biomedical Research - University of Genova, Viale Benedetto XV, 2, 16132 Genova (Italy)

2012-04-15

Current carcinogenesis theory states that only a small subset of tumor cells, the cancer stem cells or tumor initiating cells (TICs), are responsible for tumor formation and progression. Human breast cancer-initiating cells have been identified as CD44-expressing cells, which retain tumorigenic activity and display stem cell-like properties. Spontaneous feline mammary carcinoma (FMC) is an aggressive cancer, which shows biological similarities to the human tumor counterpart. We report the isolation and phenotypic characterization of FMC-derived stem/progenitor cells, showing in vitro self-renewal, long-lasting proliferation and in vivo tumorigenicity. Twenty-one FMC samples were collected, histologically classified and characterized for the expression of Ki67, EGFR, ER-{alpha} and CD44, by immunohistochemistry. By culture in stem cell permissive conditions, we isolated, from 13 FMCs, a CD44-positive subpopulation able to survive and proliferate in vitro as mammospheres of different sizes and morphologies. When injected in NOD/SCID mice, FMC stem-like cells initiate tumors, generating cell heterogeneity and recapitulating the original histotype. In serum-containing medium, spheroid cells showed differentiation properties as shown by morphological changes, the loss of CD44 expression and tumorigenic potential. These data show that stem-defined culture of FMC enriches for TICs and validate the use of these cells as a suitable model for comparative oncology studies of mammary biology and testing therapeutic strategies aimed at eradicating TICs. -- Highlights: Black-Right-Pointing-Pointer Feline mammary carcinoma contain a sub-population of stem-like cells expressing CD44 Black-Right-Pointing-Pointer These grow as spheres in serum-free medium and self-renew Black-Right-Pointing-Pointer Isolated stem-like cancer cells initiate tumor in immunodeficient mice Black-Right-Pointing-Pointer Xenografted tumors are phenotypically similar to the original tumor Black

Isolation of pulmonary artery smooth muscle cells from neonatal mice.

Science.gov (United States)

Lee, Keng Jin; Czech, Lyubov; Waypa, Gregory B; Farrow, Kathryn N

2013-10-19

Pulmonary hypertension is a significant cause of morbidity and mortality in infants. Historically, there has been significant study of the signaling pathways involved in vascular smooth muscle contraction in PASMC from fetal sheep. While sheep make an excellent model of term pulmonary hypertension, they are very expensive and lack the advantage of genetic manipulation found in mice. Conversely, the inability to isolate PASMC from mice was a significant limitation of that system. Here we described the isolation of primary cultures of mouse PASMC from P7, P14, and P21 mice using a variation of the previously described technique of Marshall et al. that was previously used to isolate rat PASMC. These murine PASMC represent a novel tool for the study of signaling pathways in the neonatal period. Briefly, a slurry of 0.5% (w/v) agarose + 0.5% iron particles in M199 media is infused into the pulmonary vascular bed via the right ventricle (RV). The iron particles are 0.2 μM in diameter and cannot pass through the pulmonary capillary bed. Thus, the iron lodges in the small pulmonary arteries (PA). The lungs are inflated with agarose, removed and dissociated. The iron-containing vessels are pulled down with a magnet. After collagenase (80 U/ml) treatment and further dissociation, the vessels are put into a tissue culture dish in M199 media containing 20% fetal bovine serum (FBS), and antibiotics (M199 complete media) to allow cell migration onto the culture dish. This initial plate of cells is a 50-50 mixture of fibroblasts and PASMC. Thus, the pull down procedure is repeated multiple times to achieve a more pure PASMC population and remove any residual iron. Smooth muscle cell identity is confirmed by immunostaining for smooth muscle myosin and desmin.

The notochord breaks bilateral symmetry by controlling cell shapes in the zebrafish laterality organ.

Science.gov (United States)

Compagnon, Julien; Barone, Vanessa; Rajshekar, Srivarsha; Kottmeier, Rita; Pranjic-Ferscha, Kornelija; Behrndt, Martin; Heisenberg, Carl-Philipp

2014-12-22

Kupffer's vesicle (KV) is the zebrafish organ of laterality, patterning the embryo along its left-right (LR) axis. Regional differences in cell shape within the lumen-lining KV epithelium are essential for its LR patterning function. However, the processes by which KV cells acquire their characteristic shapes are largely unknown. Here, we show that the notochord induces regional differences in cell shape within KV by triggering extracellular matrix (ECM) accumulation adjacent to anterior-dorsal (AD) regions of KV. This localized ECM deposition restricts apical expansion of lumen-lining epithelial cells in AD regions of KV during lumen growth. Our study provides mechanistic insight into the processes by which KV translates global embryonic patterning into regional cell shape differences required for its LR symmetry-breaking function. Copyright © 2014 Elsevier Inc. All rights reserved.

Skin cell isolation and expansion for cell transplantation is limited in patients using tobacco, alcohol, or are exhibiting diabetes mellitus.

Science.gov (United States)

Johnen, Christa; Hartmann, Bernd; Steffen, Ingo; Bräutigam, Kirsten; Witascheck, Tom; Toman, Nidal; Küntscher, Markus V; Gerlach, Jörg C

2006-03-01

The aim of this exploratory study was to investigate the isolation and expansion of keratinocytes and fibroblasts from donors with certain medical histories. Biopsies were taken from donors (N=32) falling into one or more of the following categories: a history of heavy smoking and/or alcohol abuse, drug abuse, diabetes mellitus or steroid treatment. Cells from donors who did not fall into any of the above-mentioned categories were used as controls. Proliferation and growth behaviour of cells were analyzed by measurement of passage duration, absorbance (MTT-assay) and light microscopy. Donors with a specific medical history required larger biopsy areas than the control group for isolating a sufficient number of fibroblasts and keratinocytes. Times to confluence were significantly prolonged and absorbances (MTT) were significantly reduced in several donor groups when compared to control cultures. Biopsies from donors with steroid treatment, drug abuse and combined nicotine and alcohol abuse could not be established beyond passage 0 degrees or 1 degree, respectively. We conclude that isolation and expansion of skin cells from donors with certain medical histories may require larger biopsies, prolonged expansion times or may even result in failure. These findings may therefore be of clinical importance in the field of autologous skin cell transplantation.

Isolation of stem-like cells from spontaneous feline mammary carcinomas: Phenotypic characterization and tumorigenic potential

International Nuclear Information System (INIS)

Barbieri, Federica; Wurth, Roberto; Ratto, Alessandra; Campanella, Chiara; Vito, Guendalina; Thellung, Stefano; Daga, Antonio; Cilli, Michele; Ferrari, Angelo; Florio, Tullio

2012-01-01

Current carcinogenesis theory states that only a small subset of tumor cells, the cancer stem cells or tumor initiating cells (TICs), are responsible for tumor formation and progression. Human breast cancer-initiating cells have been identified as CD44-expressing cells, which retain tumorigenic activity and display stem cell–like properties. Spontaneous feline mammary carcinoma (FMC) is an aggressive cancer, which shows biological similarities to the human tumor counterpart. We report the isolation and phenotypic characterization of FMC-derived stem/progenitor cells, showing in vitro self-renewal, long-lasting proliferation and in vivo tumorigenicity. Twenty-one FMC samples were collected, histologically classified and characterized for the expression of Ki67, EGFR, ER-α and CD44, by immunohistochemistry. By culture in stem cell permissive conditions, we isolated, from 13 FMCs, a CD44-positive subpopulation able to survive and proliferate in vitro as mammospheres of different sizes and morphologies. When injected in NOD/SCID mice, FMC stem-like cells initiate tumors, generating cell heterogeneity and recapitulating the original histotype. In serum-containing medium, spheroid cells showed differentiation properties as shown by morphological changes, the loss of CD44 expression and tumorigenic potential. These data show that stem-defined culture of FMC enriches for TICs and validate the use of these cells as a suitable model for comparative oncology studies of mammary biology and testing therapeutic strategies aimed at eradicating TICs. -- Highlights: ► Feline mammary carcinoma contain a sub-population of stem-like cells expressing CD44 ► These grow as spheres in serum-free medium and self-renew ► Isolated stem-like cancer cells initiate tumor in immunodeficient mice ► Xenografted tumors are phenotypically similar to the original tumor ► Upon differentiation, cells grow as monolayers, loosing the tumorigenic potential

Isolation of Human Induced Pluripotent Stem Cell-Derived Dopaminergic Progenitors by Cell Sorting for Successful Transplantation

Directory of Open Access Journals (Sweden)

Daisuke Doi

2014-03-01

Full Text Available Human induced pluripotent stem cells (iPSCs can provide a promising source of midbrain dopaminergic (DA neurons for cell replacement therapy for Parkinson’s disease. However, iPSC-derived donor cells inevitably contain tumorigenic or inappropriate cells. Here, we show that human iPSC-derived DA progenitor cells can be efficiently isolated by cell sorting using a floor plate marker, CORIN. We induced DA neurons using scalable culture conditions on human laminin fragment, and the sorted CORIN+ cells expressed the midbrain DA progenitor markers, FOXA2 and LMX1A. When transplanted into 6-OHDA-lesioned rats, the CORIN+ cells survived and differentiated into midbrain DA neurons in vivo, resulting in significant improvement of the motor behavior, without tumor formation. In particular, the CORIN+ cells in a NURR1+ cell-dominant stage exhibited the best survival and function as DA neurons. Our method is a favorable strategy in terms of scalability, safety, and efficiency and may be advantageous for clinical application.

Isolation, characterization, and differentiation of stem cells for cartilage regeneration.

Science.gov (United States)

Beane, Olivia S; Darling, Eric M

2012-10-01

The goal of tissue engineering is to create a functional replacement for tissues damaged by injury or disease. In many cases, impaired tissues cannot provide viable cells, leading to the investigation of stem cells as a possible alternative. Cartilage, in particular, may benefit from the use of stem cells since the tissue has low cellularity and cannot effectively repair itself. To address this need, researchers are investigating the chondrogenic capabilities of several multipotent stem cell sources, including adult and extra-embryonic mesenchymal stem cells (MSCs), embryonic stem cells (ESCs), and induced pluripotent stem cells (iPSCs). Comparative studies indicate that each cell type has advantages and disadvantages, and while direct comparisons are difficult to make, published data suggest some sources may be more promising for cartilage regeneration than others. In this review, we identify current approaches for isolating and chondrogenically differentiating MSCs from bone marrow, fat, synovium, muscle, and peripheral blood, as well as cells from extra-embryonic tissues, ESCs, and iPSCs. Additionally, we assess chondrogenic induction with growth factors, identifying standard cocktails used for each stem cell type. Cell-only (pellet) and scaffold-based studies are also included, as is a discussion of in vivo results.

Isolation of Precursor Cells from Waste Solid Fat Tissue

Science.gov (United States)

Byerly, Diane; Sognier, Marguerite A.

2009-01-01

A process for isolating tissue-specific progenitor cells exploits solid fat tissue obtained as waste from such elective surgical procedures as abdominoplasties (tummy tucks) and breast reductions. Until now, a painful and risky process of aspiration of bone marrow has been used to obtain a limited number of tissue- specific progenitor cells. The present process yields more tissue-specific progenitor cells and involves much less pain and risk for the patient. This process includes separation of fat from skin, mincing of the fat into small pieces, and forcing a fat saline mixture through a sieve. The mixture is then digested with collagenase type I in an incubator. After centrifugation tissue-specific progenitor cells are recovered and placed in a tissue-culture medium in flasks or Petri dishes. The tissue-specific progenitor cells can be used for such purposes as (1) generating three-dimensional tissue equivalent models for studying bone loss and muscle atrophy (among other deficiencies) and, ultimately, (2) generating replacements for tissues lost by the fat donor because of injury or disease.

Isolation of two chloroethylnitrosourea-sensitive Chinese hamster cell lines

International Nuclear Information System (INIS)

Hata, H.; Numata, M.; Tohda, H.; Yasui, A.; Oikawa, A.

1991-01-01

1-[(4-Amino-2-methylpyrimidin-5-yl)methyl]-3-(2-chloroethyl)-3- nitrosourea hydrochloride (ACNU), a cancer chemotherapeutic bifunctional alkylating agent, causes chloroethylation of DNA and subsequent DNA strand cross-linking through an ethylene bridge. We isolated and characterized two ACNU-sensitive mutants from mutagenized Chinese hamster ovary cells and found them to be new drug-sensitive recessive Chinese hamster mutants. Both mutants were sensitive to various monofunctional alkylating agents in a way similar to that of the parental cell lines CHO9. One mutant (UVS1) was cross-sensitive to UV and complemented the UV sensitivity of all Chinese hamster cell lines of 7 established complementation groups. Since UV-induced unscheduled DNA synthesis was very low, a new locus related to excision repair is thought to be defective in this cell line. Another ACNU-sensitive mutant, CNU1, was slightly more sensitive to UV than the parent cell line. CNU1 was cross-sensitive to 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea and slightly more sensitive to mitomycin C. No increased accumulation of ACNU and a low level of UV-induced unscheduled DNA synthesis in this cell as compared with the parental cell line suggest that there is abnormality in a repair response of this mutant cell to some types of DNA cross-links

Comparison of the usefulness of the CACO-2 cell line with standard substrates for isolation of swine influenza A viruses.

Science.gov (United States)

Chiapponi, Chiara; Zanni, Irene; Garbarino, Chiara; Barigazzi, Giuseppe; Foni, Emanuela

2010-01-01

Influenza A virus isolation is undertaken routinely in embryonated chicken eggs, but to improve virus detection various cell lines can be used. The CACO-2 cell line was compared to the MDCK cell line and embryonated chicken eggs for the isolation of H1N1, H1N2, H3N2 swine influenza A virus subtypes from clinical specimens. From 2006 to 2008, 104 influenza A samples found positive by PCR from 42 respiratory outbreaks in Italian swine farms were examined by virus isolation. Sixty swine influenza A viruses were isolated (16 H1N1, 28 H1N2 and 16 H3N2) and their growth behaviour on the different substrates was examined. 16/16 H1N1, 28/28 H1N2 and 8/16 of H3N2 viruses were isolated from the CACO-2 cell line, while 7/16 H1N1, 3/28 H1N2 and 16/16 H3N2 viruses were isolated using embryonated chicken eggs. Only 9/16 H1N1, 1/28 H1N2 and 6/16 H3N2 viruses replicated in MDCK cells. A link was found between viral hemagglutinin and the isolation rate on the various substrates. The CACO-2 line was statistically more sensitive (Fisher's exact test, pH1N2 subtypes. In contrast influenza A H3N2 virus was isolated more readily in embryonated chicken eggs than in cultured cells (Fisher's exact test, p<0.01).

Successful isolation, in vitro expansion and characterization of stem cells from Human Dental Pulp

OpenAIRE

Preethy SP; Srinivasan T; Tholcopiyan L; Thamaraikannan P; Srinivasan V; Murugan P; Manjunath S; Kannan TA; Shalini R; Sunil PM; Manikandhan R; Muthu MS; Abraham S

2010-01-01

BACKGROUND: Recent studies have shown that mesenchymal stem cells isolated from post natal human dental pulp, (Dental pulp stem cells-DPSCs) which is from permanent teeth and SHED (stem cells from human exfoliated deciduous teeth),the Periodontal ligament stem cells (PDLSC) and Stem cells from root Apical papilla(SCAP)have the potential to differentiate into cells of a variety of tissues including heart, muscle, cartilage, bone, nerve, salivary glands, teeth etc(1,2,3,4).This multipotential a...

Successful isolation, in vitro expansion and characterization of stem cells from Human Dental Pulp

Directory of Open Access Journals (Sweden)

Preethy SP

2010-01-01

Full Text Available BACKGROUND: Recent studies have shown that mesenchymal stem cells isolated from post natal human dental pulp, (Dental pulp stem cells-DPSCs which is from permanent teeth and SHED (stem cells from human exfoliated deciduous teeth,the Periodontal ligament stem cells (PDLSC and Stem cells from root Apical papilla(SCAPhave the potential to differentiate into cells of a variety of tissues including heart, muscle, cartilage, bone, nerve, salivary glands, teeth etc(1,2,3,4.This multipotential ability of DPSCs is being researched for clinical application for treating a variety of diseases like myocardial infarction, muscular dystrophy, neuro-degenerative disorders, cartilage replacement, tooth regeneration and for repair of bone defects to mention a few. Moreover, the isolation of stem cells from teeth is minimally invasive, readily accessible and the non immunogenic characteristic of dental stem cells has paved the way for efforts to store the exfoliated deciduous teeth or milk teeth which is usually discarded, for use in the future. In this study we have isolated and expanded in vitro, the cells obtained from human dental pulp. MATERIALS AND METHODS: After obtaining written informed consent, 24 teeth that were extracted for therapeutic or cosmetic reasons from 16 patients were used in this study. The specimens were transported from the clinic to NCRM lab taking 6 to 48 Hrs. For removal of the pulp tissue, the teeth were split obliquely at the Cementoenamel junction and the pulp tissue was isolated using brooches. The extracted pulp tissues were subjected to digestion using Collagenase type-I and type II at 37˚C for 15- 30 minutes. The digested cells were filtered with 70µm filter and centrifuged at 1800 rpm for 10 minutes. The pellet was then suspended in Dulbecco’s modified Eagle’s medium (DMEM/Ham’s F12 supplemented with 15% fetal bovine serum , 100 U/ml penicillin, 100 µg/ml streptomycin,2 m M L -glutamine, and 2 m M nonessential amino

Prospective Isolation of Murine and Human Bone Marrow Mesenchymal Stem Cells Based on Surface Markers

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Yo Mabuchi

2013-01-01

Full Text Available Mesenchymal stem cells (MSCs are currently defined as multipotent stromal cells that undergo sustained in vitro growth and can give rise to cells of multiple mesenchymal lineages, such as adipocytes, chondrocytes, and osteoblasts. The regenerative and immunosuppressive properties of MSCs have led to numerous clinical trials exploring their utility for the treatment of a variety of diseases (e.g., acute graft-versus-host disease, Crohn’s disease, multiple sclerosis, osteoarthritis, and cardiovascular diseases including heart failure and myocardial infarction. On the other hand, conventionally cultured MSCs reflect heterogeneous populations that often contain contaminating cells due to the significant variability in isolation methods and the lack of specific MSC markers. This review article focuses on recent developments in the MSC research field, with a special emphasis on the identification of novel surface markers for the in vivo localization and prospective isolation of murine and human MSCs. Furthermore, we discuss the physiological importance of MSC subtypes in vivo with specific reference to data supporting their contribution to HSC niche homeostasis. The isolation of MSCs using selective markers (combination of PDGFRα and Sca-1 is crucial to address the many unanswered questions pertaining to these cells and has the potential to enhance their therapeutic potential enormously.

Protoplast isolation from Ulmus americana l. Pollen mother cells, tetrads, and microspores

Energy Technology Data Exchange (ETDEWEB)

Redenbaugh, M K; Westfall, R D; Karnosky, D F

1980-01-01

Meiotic protoplasts of U. amerciana are potentially valuable for producing interspecific elm hybrids through protoplast fusion. Meiotic cells(pollen mother cells, tetrads, and microspores) were incubated in either a cellulase, hemicellylase and pectinase enzyme solution of a beta-1,3-glucanase (lainarinase) solution. Respective protoplast isolation frequencies for the three meiotic cell types were 100, 50, and 10%. Exclusion staining with 0.2% Evans blue and 0.1% methyl blue suggested protoplast viability. Some of the microspore protoplasts were vacuolated, which is an important condition for cell division. Although attempts of regenerating cell walls and inducing cell division were unsuccessful, these two problems may be superceded by protoplast fusion with more regenerative protoplasts.

Methods to isolate a large amount of generative cells, sperm cells and vegetative nuclei from tomato pollen for omics analysis

Directory of Open Access Journals (Sweden)

Yunlong eLu

2015-06-01

Full Text Available The development of sperm cells from microspores involves a set of finely regulated molecular and cellular events and the coordination of these events. The mechanisms underlying these events and their interconnections remain a major challenge. Systems analysis of genome-wide molecular networks and functional modules with high-throughput omics approaches is crucial for understanding the mechanisms; however, this study is hindered because of the difficulty in isolating a large amount of cells of different types, especially generative cells (GCs, from the pollen. Here, we optimized the conditions of tomato pollen germination and pollen tube growth to allow for long-term growth of pollen tubes in vitro with sperm cells (SCs generated in the tube. Using this culture system, we developed methods for isolating GCs, SCs and vegetative-cell nuclei (VN from just-germinated tomato pollen grains and growing pollen tubes and their purification by Percoll density gradient centrifugation. The purity and viability of isolated GCs and SCs were confirmed by microscopy examination and fluorescein diacetate staining, respectively, and the integrity of VN was confirmed by propidium iodide staining. We could obtain about 1.5 million GCs and 2.0 million SCs each from 180 mg initiated pollen grains, and 10 million VN from 270 mg initiated pollen grains germinated in vitro in each experiment. These methods provide the necessary preconditions for systematic biology studies of SC development and differentiation in higher plants.

Method for isolation and molecular characterization of extracellular microvesicles released from brain endothelial cells

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Haqqani Arsalan S

2013-01-01

Full Text Available Abstract Background In addition to possessing intracellular vesicles, eukaryotic cells also produce extracellular microvesicles, ranging from 50 to 1000 nm in diameter that are released or shed into the microenvironment under physiological and pathological conditions. These membranous extracellular organelles include both exosomes (originating from internal vesicles of endosomes and ectosomes (originating from direct budding/shedding of plasma membranes. Extracellular microvesicles contain cell-specific collections of proteins, glycoproteins, lipids, nucleic acids and other molecules. These vesicles play important roles in intercellular communication by acting as carrier for essential cell-specific information to target cells. Endothelial cells in the brain form the blood–brain barrier, a specialized interface between the blood and the brain that tightly controls traffic of nutrients and macromolecules between two compartments and interacts closely with other cells forming the neurovascular unit. Therefore, brain endothelial cell extracellular microvesicles could potentially play important roles in ‘externalizing’ brain-specific biomarkers into the blood stream during pathological conditions, in transcytosis of blood-borne molecules into the brain, and in cell-cell communication within the neurovascular unit. Methods To study cell-specific molecular make-up and functions of brain endothelial cell exosomes, methods for isolation of extracellular microvesicles using mass spectrometry-compatible protocols and the characterization of their signature profiles using mass spectrometry -based proteomics were developed. Results A total of 1179 proteins were identified in the isolated extracellular microvesicles from brain endothelial cells. The microvesicles were validated by identification of almost 60 known markers, including Alix, TSG101 and the tetraspanin proteins CD81 and CD9. The surface proteins on isolated microvesicles could potentially

New Method to Disaggregate and Analyze Single Isolated Helminthes Cells Using Flow Cytometry: Proof of Concept

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Karen Nava-Castro

2011-01-01

Full Text Available In parasitology, particularly in helminthes studies, several methods have been used to look for the expression of specific molecules, such as RT-PCR, western blot, 2D-electrophoresis, and microscopy, among others. However, these methods require homogenization of the whole helminth parasite, preventing evaluation of individual cells or specific cell types in a given parasite tissue or organ. Also, the extremely high interaction between helminthes and host cells (particularly immune cells is an important point to be considered. It is really hard to obtain fresh parasites without host cell contamination. Then, it becomes crucial to determine that the analyzed proteins are exclusively from parasitic origin, and not a consequence of host cell contamination. Flow cytometry is a fluorescence-based technique used to evaluate the expression of extra-and intracellular proteins in different type cells, including protozoan parasites. It also allows the isolation and recovery of single-cell populations. Here, we describe a method to isolate and obtain purified helminthes cells.

Human adipose-derived stem cells: definition, isolation, tissue-engineering applications.

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Nae, S; Bordeianu, I; Stăncioiu, A T; Antohi, N

2013-01-01

Recent researches have demonstrated that the most effective repair system of the body is represented by stem cells - unspecialized cells, capable of self-renewal through successive mitoses, which have also the ability to transform into different cell types through differentiation. The discovery of adult stem cells represented an important step in regenerative medicine because they no longer raises ethical or legal issues and are more accessible. Only in 2002, stem cells isolated from adipose tissue were described as multipotent stem cells. Adipose tissue stem cells benefits in tissue engineering and regenerative medicine are numerous. Development of adipose tissue engineering techniques offers a great potential in surpassing the existing limits faced by the classical approaches used in plastic and reconstructive surgery. Adipose tissue engineering clinical applications are wide and varied, including reconstructive, corrective and cosmetic procedures. Nowadays, adipose tissue engineering is a fast developing field, both in terms of fundamental researches and medical applications, addressing issues related to current clinical pathology or trauma management of soft tissue injuries in different body locations.

Antimicrobial Activity of Cell Free Supernatant of Irradiated Lactic Acid Bacteria Isolates

International Nuclear Information System (INIS)

Abdelaleem, M.A.; AL-Hagar, O.E.Aa.

2015-01-01

Attempts were made to isolate bio preservatives using food wastes with no value and low cost. Whey is the raw material achieved that value. Whey and many other food wastes are used in our study to isolate Lactic acid bacteria (LAB). Cell free supernatants (CFS) of isolates are used to evaluate their antimicrobial activity against indicator pathogenic bacterial strains. CFS-9 isolate from whey has the highest inhibitory activity compared to all other isolates. The inhibitory activity of CFS-9, Nisin (400 IU / ml) and the standard Lactococcus Lactis Subsp. Lactis ATCC 11454 (Lacto) were determined. Furthermore, isolate-9 and Lacto strains were exposed to irradiation at different doses. The inhibition zones of; control isolate-9 (non-irradiated) showed the highest values against all indicator strains, CFS of irradiated Lacto at dose 250 Gy was the highest value against Bacillus cereus and Escherichia coli compared to other irradiation treatments, CFS of irradiated Lacto at dose 100 Gy was the highest value against Staph aureus, while the inhibition zone was in the highest value in CFS of irradiated Lacto at dose 500 Gy against Salmonella typhimurium. Nisin (400 IU / ml) was significantly higher than all CFS of irradiated isolate-9 while, the inhibition zones of all CFS-Lacto (irradiated and nonirradiated) are better and higher than nisin-400

Isolation and Characterization of Human Mesenchymal Stem Cells From Facet Joints and Interspinous Ligaments.

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Kristjánsson, Baldur; Limthongkul, Worawat; Yingsakmongkol, Wicharn; Thantiworasit, Pattarawat; Jirathanathornnukul, Napaphat; Honsawek, Sittisak

2016-01-01

A descriptive in vitro study on isolation and differentiation of human mesenchymal stem cells (MSCs) derived from the facet joints and interspinous ligaments. To isolate cells from the facet joints and interspinous ligaments and investigate their surface marker profile and differentiation potentials. Lumbar spinal canal stenosis and ossification of the posterior longitudinal ligament are progressive conditions characterized by the hypertrophy and ossification of ligaments and joints within the spinal canal. MSCs are believed to play a role in the advancement of these diseases and the existence of MSCs has been demonstrated within the ligamentum flavum and posterior longitudinal ligament. The aim of this study was to investigate whether these cells could also be found within facet joints and interspinous ligaments. Samples were harvested from 10 patients undergoing spinal surgery. The MSCs from facet joints and interspinous ligaments were isolated using direct tissue explant technique. Cell surface antigen profilings were performed via flow cytometry. Their lineage differentiation potentials were analyzed. The facet joints and interspinous ligaments-derived MSCs have the tri-lineage potential to be differentiated into osteogenic, adipogenic, and chondrogenic cells under appropriate inductions. Flow cytometry analysis revealed both cell lines expressed MSCs markers. Both facet joints and interspinous ligaments-derived MSCs expressed marker genes for osteoblasts, adipocytes, and chondrocytes. The facet joints and interspinous ligaments may provide alternative sources of MSCs for tissue engineering applications. The facet joints and interspinous ligaments-derived MSCs are part of the microenvironment of the human ligaments of the spinal column and might play a crucial role in the development and progression of degenerative spine conditions.

Selected Lactobacillus strains isolated from sugary and milk kefir reduce Salmonella infection of epithelial cells in vitro.

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Zavala, L; Golowczyc, M A; van Hoorde, K; Medrano, M; Huys, G; Vandamme, P; Abraham, A G

2016-09-01

The isolation of potentially probiotic strains and the subsequent study of their properties are very important steps to gain insight in the health benefits ascribed to sugary and milk kefir. The aim of the present study was to characterise fifteen Lactobacillus strains isolated from these beverages by determining some surface properties and their ability to antagonise enterocyte cell damage after Salmonella infection in vitro. Lactobacillus surface properties were determined by hydrophobicity, autoaggregation, and coaggregation assays with Salmonella. In addition, lactobacilli adhesion to Caco-2/TC-7 cells and the effect on Salmonella invasion were evaluated. Finally, the disassembly of F-actin cytoskeleton on intestinal epithelial cells was assayed in vitro when Salmonella infection was performed in the presence of selected Lactobacillus strains. Ten out of the 15 strains showed a high adhesion capacity to Caco-2/TC-7 cells. Most of the strains were hydrophilic and non-autoaggregating. Strains isolated from sugary kefir were non-coaggregating with Salmonella, while strains Lactobacillus paracasei CIDCA 83120, 83121, 83123, 83124, 8339, 83102 isolated from milk kefir were able to coaggregate after 1 h. L. paracasei CIDCA 8339 and Lactobacillus kefiri CIDCA 83102 were able to diminish Salmonella invasion to the enterocytes. An antagonistic effect on cytoskeleton disruption elicited by the pathogen was also demonstrated. Our results suggest that both strains isolated from milk kefir could be considered as appropriate probiotic candidates.

Single Nisoldipine-Sensitive Calcium Channels in Smooth Muscle Cells Isolated from Rabbit Mesenteric Artery

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Worley, Jennings F.; Deitmer, Joachim W.; Nelson, Mark T.

1986-08-01

Single smooth muscle cells were enzymatically isolated from the rabbit mesenteric artery. At physiological levels of external Ca, these cells were relaxed and contracted on exposure to norepinephrine, caffeine, or high levels of potassium. The patch-clamp technique was used to measure unitary currents through single channels in the isolated cells. Single channels were selective for divalent cations and exhibited two conductance levels, 8 pS and 15 pS. Both types of channels were voltage-dependent, and channel activity occurred at potentials positive to -40 mV. The activity of both channel types was almost completely inhibited by 50 nM nisoldipine. These channels appear to be the pathways for voltage-dependent Ca influx in vascular smooth muscle and may be the targets of the clinically used dihydropyridines.

Fundamentals and Application of Magnetic Particles in Cell Isolation and Enrichment

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Plouffe, Brian D.; Murthy, Shashi K.; Lewis, Laura H.

2014-01-01

Magnetic sorting using magnetic beads has become a routine methodology for the separation of key cell populations from biological suspensions. Due to the inherent ability of magnets to provide forces at a distance, magnetic cell manipulation is now a standardized process step in numerous processes in tissue engineering, medicine, and in fundamental biological research. Herein we review the current status of magnetic particles to enable isolation and separation of cells, with a strong focus on the fundamental governing physical phenomena, properties and syntheses of magnetic particles and on current applications of magnet-based cell separation in laboratory and clinical settings. We highlight the contribution of cell separation to biomedical research and medicine and detail modern cell separation methods (both magnetic and non-magnetic). In addition to a review of the current state-of-the-art in magnet-based cell sorting, we discuss current challenges and available opportunities for further research, development and commercialization of magnetic particle-based cell separation systems. PMID:25471081

Isolation of cancer cells by "in situ" microfluidic biofunctionalization protocols

KAUST Repository

De Vitis, Stefania; Matarise, Giuseppina; Pardeo, Francesca; Catalano, Rossella; Malara, Natalia Maria; Trunzo, Valentina; Tallerico, Rossana; Gentile, Francesco T.; Candeloro, Patrizio; Coluccio, Maria Laura; Massaro, Alessandro S.; Viglietto, Giuseppe; Carbone, Ennio; Kutter, Jö rg Peter; Perozziello, Gerardo; Di Fabrizio, Enzo M.

2014-01-01

The aim of this work is the development of a microfluidic immunosensor for the immobilization of cancer cells and their separation from healthy cells by using "in situ" microfluidic biofunctionalization protocols. These protocols allow to link antibodies on microfluidic device surfaces and can be used to study the interaction between cell membrane and biomolecules. Moreover they allow to perform analysis with high processing speed, small quantity of reagents and samples, short reaction times and low production costs. In this work the developed protocols were used in microfluidic devices for the isolation of cancer cells in heterogeneous blood samples by exploiting the binding of specific antibody to an adhesion protein (EpCAM), overexpressed on the tumor cell membranes. The presented biofunctionalization protocols can be performed right before running the experiment: this allows to have a flexible platform where biomolecules of interest can be linked on the device surface according to the user's needs. © 2014 Elsevier B.V. All rights reserved.

Isolation of cancer cells by "in situ" microfluidic biofunctionalization protocols

KAUST Repository

De Vitis, Stefania

2014-07-01

The aim of this work is the development of a microfluidic immunosensor for the immobilization of cancer cells and their separation from healthy cells by using "in situ" microfluidic biofunctionalization protocols. These protocols allow to link antibodies on microfluidic device surfaces and can be used to study the interaction between cell membrane and biomolecules. Moreover they allow to perform analysis with high processing speed, small quantity of reagents and samples, short reaction times and low production costs. In this work the developed protocols were used in microfluidic devices for the isolation of cancer cells in heterogeneous blood samples by exploiting the binding of specific antibody to an adhesion protein (EpCAM), overexpressed on the tumor cell membranes. The presented biofunctionalization protocols can be performed right before running the experiment: this allows to have a flexible platform where biomolecules of interest can be linked on the device surface according to the user\\'s needs. © 2014 Elsevier B.V. All rights reserved.

Methods to isolate a large amount of generative cells, sperm cells and vegetative nuclei from tomato pollen for "omics" analysis.

Science.gov (United States)

Lu, Yunlong; Wei, Liqin; Wang, Tai

2015-01-01

The development of sperm cells (SCs) from microspores involves a set of finely regulated molecular and cellular events and the coordination of these events. The mechanisms underlying these events and their interconnections remain a major challenge. Systems analysis of genome-wide molecular networks and functional modules with high-throughput "omics" approaches is crucial for understanding the mechanisms; however, this study is hindered because of the difficulty in isolating a large amount of cells of different types, especially generative cells (GCs), from the pollen. Here, we optimized the conditions of tomato pollen germination and pollen tube growth to allow for long-term growth of pollen tubes in vitro with SCs generated in the tube. Using this culture system, we developed methods for isolating GCs, SCs and vegetative cell nuclei (VN) from just-germinated tomato pollen grains and growing pollen tubes and their purification by Percoll density gradient centrifugation. The purity and viability of isolated GCs and SCs were confirmed by microscopy examination and fluorescein diacetate staining, respectively, and the integrity of VN was confirmed by propidium iodide staining. We could obtain about 1.5 million GCs and 2.0 million SCs each from 180 mg initiated pollen grains, and 10 million VN from 270 mg initiated pollen grains germinated in vitro in each experiment. These methods provide the necessary preconditions for systematic biology studies of SC development and differentiation in higher plants.

Milk fat globule membrane isolate induces apoptosis in HT-29 human colon cancer cells.

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Zanabria, Romina; Tellez, Angela M; Griffiths, Mansel; Corredig, Milena

2013-02-01

A native milk fat globule membrane (MFGM) isolate obtained from raw milk was assessed for its anticarcinogenic capacity using a colon cancer cell line (HT-29). To prevent microbial contamination and eliminate the presence of lipopolysaccharide (LPS) in the milk used for MFGM isolation, the milk was obtained from the mammary glands of cows using a catheter. Cell proliferation assays demonstrated a reduction of exponentially growing cancer cells of up to 53%, expressed as DNA synthesis (BrdU test), after 72 h stimulation with 100 μg of MFGM protein per mL. Using a similar MFGM concentration, the sulforhodamine B assay resulted in 57% reduction of cell density after 48 h incubation. This bioactivity was comparable to that of known anticancer drugs, 0.1 mM melphalan and 20 μM C2-ceramide, which achieved a cell division reduction of 25 and 40%, respectively, under the same experimental conditions. The toxic effect of the MFGM extracts on HT-29 cells was confirmed by the significant reduction in lactate dehydrogenase enzyme (LDH) by the residual viable cells. An increase of caspase-3 activity (up to 26%) led to the conclusion that MFGM has an apoptotic effect on HT-29 cancer cells.

Isolation and characterization of dental epithelial cells derived from amelogenesis imperfecta rat.

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Adiningrat, A; Tanimura, A; Miyoshi, K; Hagita, H; Yanuaryska, R D; Arinawati, D Y; Horiguchi, T; Noma, T

2016-03-01

Disruption of the third zinc finger domain of specificity protein 6 (SP6) presents an enamel-specific defect in a rat model of amelogenesis imperfecta (AMI rats). To understand the molecular basis of amelogenesis imperfecta caused by the Sp6 mutation, we established and characterized AMI-derived rat dental epithelial (ARE) cells. ARE cell clones were isolated from the mandibular incisors of AMI rats, and amelogenesis-related gene expression was analyzed by reverse transcription polymerase chain reaction (RT-PCR). Localization of wild-type SP6 (SP6WT) and mutant-type SP6 (SP6AMI) was analyzed by immunocytochemistry. SP6 transcriptional activity was monitored by rho-associated protein kinase 1 (Rock1) promoter activity with its specific binding to the promoter region in dental (G5 and ARE) and non-dental (COS-7) epithelial cells. Isolated ARE cells were varied in morphology and gene expression. Both SP6WT and SP6AMI were mainly detected in nuclei. The promoter analysis revealed that SP6WT and SP6AMI enhanced Rock1 promoter activity in G5 cells but that enhancement by SP6AMI was weaker, whereas no enhancement was observed in the ARE and COS-7 cells, even though SP6WT and SP6AMI bound to the promoter in all instances. ARE cell clones can provide a useful in vitro model to study the mechanism of SP6-mediated amelogenesis imperfecta. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Intracellular recordings from isolated rabbit retinal Müller (glial) cells.

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Reichenbach, A; Eberhardt, W

1986-09-01

Müller (glial) cells were isolated from rabbit retinae by papaine and mechanical dissociation. The cells were fixed on a gelatine-covered glass slide by means of concanavalin A, and the slide was mounted in a perfusion chamber under a light microscope with modified optics. Besides the recording microelectrode, two other micropipettes could be adjusted with their tips near the cell. These micropipettes were used for application of test solutions into the environment of the cells. On application of high K+ solutions, the cell depolarized strongly but during prolonged application there was a marked repolarization. After the end of high K+ application the cells showed a hyperpolarization which was enhanced in both amplitude and duration with prolongation of the K+ exposure. Both repolarization and afterhyperpolarization disappeared under ouabain. Ouabain application itself caused a small reversible depolarization. Na+ free solution caused hyperpolarization. The results suggest the existence of an active membrane pump mechanism in our cells. This pump seems to be electrogenic under our experimental conditions and seems to be activated even in the absence of sodium. The cell membrane is demonstrated to contain a significant Na+ conductance.

Primary microglia isolation from mixed glial cell cultures of neonatal rat brain tissue.

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Tamashiro, Tami T; Dalgard, Clifton Lee; Byrnes, Kimberly R

2012-08-15

Microglia account for approximately 12% of the total cellular population in the mammalian brain. While neurons and astrocytes are considered the major cell types of the nervous system, microglia play a significant role in normal brain physiology by monitoring tissue for debris and pathogens and maintaining homeostasis in the parenchyma via phagocytic activity. Microglia are activated during a number of injury and disease conditions, including neurodegenerative disease, traumatic brain injury, and nervous system infection. Under these activating conditions, microglia increase their phagocytic activity, undergo morpohological and proliferative change, and actively secrete reactive oxygen and nitrogen species, pro-inflammatory chemokines and cytokines, often activating a paracrine or autocrine loop. As these microglial responses contribute to disease pathogenesis in neurological conditions, research focused on microglia is warranted. Due to the cellular heterogeneity of the brain, it is technically difficult to obtain sufficient microglial sample material with high purity during in vivo experiments. Current research on the neuroprotective and neurotoxic functions of microglia require a routine technical method to consistently generate pure and healthy microglia with sufficient yield for study. We present, in text and video, a protocol to isolate pure primary microglia from mixed glia cultures for a variety of downstream applications. Briefly, this technique utilizes dissociated brain tissue from neonatal rat pups to produce mixed glial cell cultures. After the mixed glial cultures reach confluency, primary microglia are mechanically isolated from the culture by a brief duration of shaking. The microglia are then plated at high purity for experimental study. The principle and protocol of this methodology have been described in the literature. Additionally, alternate methodologies to isolate primary microglia are well described. Homogenized brain tissue may be separated

New technology for ultrasensitive detection and isolation of rare cells for clinical diagnostics and therapeutics

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Leary, James F.; McLaughlin, Scott R.

1995-04-01

A high-speed, 11-parameter, 6-color fluorescence, laser flow cytometer/cell sorter with a number of special and unique features has been built for ultrasensitive detection and isolation of rare cells for clinical diagnostics and therapeutics. The software for real-time data acquisition and sort control, written as C++ programming language modules with a WindowsTM graphical user interface, runs on a 66-MHz 80486 computer joined by an extended bus to 23 sophisticated multi-layered boards of special data acquisition and sorting electronics. Special features include: high-speed (> 100,000 cells/sec) real-time data classification module (U.S. Patent 5,204,884 (1993)); real-time principal component cell sorting; multi-queue signal-processing system with multiple hardware and software event buffers to reduce instrument dead time, LUT charge-pulse definition, high-resolution `flexible' sorting for optimal yield/purity sort strategies (U.S. Patent 5,199,576); pre-focusing optical wavelength correction for a second laser beam; and two trains of three fluorescence detectors-- each adjustable for spatial separation to interrogate only one of two laser beams, syringe- driven or pressure-driven fluidics, and time-windowed parameters. The system has been built to be both expandable and versatile through the use of LUT's and a modular hardware and software design. The instrument is especially useful at detection and isolation of rare cell subpopulations for which our laboratory is well-known. Cell subpopulations at frequencies as small as 10-7 have been successfully studied with this system. Current applications in clinical diagnostics and therapeutics include detection and isolation of (1) fetal cells from material blood for prenatal diagnosis of birth defects, (2) hematopoietic stem and precursor cells for autologous bone marrow transplantation, (3) metastatic breast cancer cells for molecular characterization, and (4) HIV-infected maternal cells in newborn blood to study mother

Impact of isolation method on doubling time and the quality of chondrocyte and osteoblast differentiated from murine dental pulp stem cells

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Rohaya Megat Abdul Wahab

2017-06-01

Full Text Available Background Stem cells are normally isolated from dental pulps using the enzymatic digestion or the outgrowth method. However, the effects of the isolation method on the quality of the isolated stem cells are not studied in detail in murine models. The aim of this study was to compare the matrices secreted by osteoblast and chondrocytes differentiated from dental pulp stem cells isolated through different means. Method DPSC from murine incisors were isolated through either the outgrowth (DPSC-OG or the enzymatic digestion (DPSC-ED method. Cells at passage 4 were used in this study. The cells were characterized through morphology and expression of cell surface markers. The cells’ doubling time when cultured using different seeding densities was calculated and analyzed using one-way ANOVA and Tukey’s multiple comparison post-test. The ability of cells to differentiate to chondrocyte and osteoblast was evaluated through staining and analysis on the matrices secreted. Results Gene expression analysis showed that DPSC-OG and DPSC-ED expressed dental pulp mesenchymal stem cell markers, but not hematopoietic stem cell markers. The least number of cells that could have been used to culture DPSC-OG and DPSC-ED with the shortest doubling time was 5 × 102 cells/cm2 (11.49 ± 2.16 h and 1 × 102 cells/cm2 (10.55 h ± 0.50, respectively. Chondrocytes differentiated from DPSC-ED produced  2 times more proteoglycan and at a faster rate than DPSC-OG. FTIR revealed that DPSC-ED differentiated into osteoblast also secreted matrix, which more resembled a calvaria. Discussion Isolation approaches might have influenced the cell populations obtained. This, in turn, resulted in cells with different proliferation and differentiation capability. While both DPSC-OG and DPSC-ED expressed mesenchymal stem cell markers, the percentage of cells carrying each marker might have differed between the two methods. Regardless, enzymatic digestion clearly yielded cells

Isolation of UV-sensitive mutants of mouse L5178Y cells by a cell suspension spotting method

International Nuclear Information System (INIS)

Shiomi, T.; Hieda-Shiomi, N.; Sato, K.

1982-01-01

We have isolated 56 UV-sensitive mutant clones from a mouse L51 T/t line of L5178Y cells by a cell suspension spotting method. Five mutants have also been isolated from L51 T/t and L5178Y cells by the method reported by Thompson and coworkers. We divided the mutants into two groups, highly sensitive and moderately sensitive mutants, according to their sensitivity to UV irradiation. Fifty-eight mutants were highly sensitive and three were moderately sensitive to UV. The reconstruction experiments indicate that more than 90% of highly sensitive mutants were recovered by the cell suspension spotting method. Frequencies of recovered mutants highly sensitive to UV increased with increasing dose of mutagens. Recovered mutant frequency reached 10(-2) after treatment with 1.5 micrograms/ml of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) (survival 0.2%). Eight UV-sensitive mutants were divided into four complementation groups. These mutants were 2-6 times more sensitive to UV than parental L51 T/t cells in terms of D37 (dose required to reduce survival to 37%). Four representative UV-sensitive mutants which are classified into different complementation groups were examined for their sensitivity to killing by UV, 4-nitroquinoline-1-oxide (4NQO), mitomycin C (MMC), X-rays, and MNNG. All four classes of mutants were found to be cross-sensitive to UV, 4NQO, and MMC, but not sensitive to X-rays and MNNG

[Isolation and identification of brain tumor stem cells from human brain neuroepithelial tumors].

Science.gov (United States)

Fang, Jia-sheng; Deng, Yong-wen; Li, Ming-chu; Chen, Feng-Hua; Wang, Yan-jin; Lu, Ming; Fang, Fang; Wu, Jun; Yang, Zhuan-yi; Zhou, Xang-yang; Wang, Fei; Chen, Cheng

2007-01-30

To establish a simplified culture system for the isolation of brain tumor stem cells (BTSCs) from the tumors of human neuroepithelial tissue, to observe the growth and differentiation pattern of BTSCs, and to investigate their expression of the specific markers. Twenty-six patients with brain neuroepithelial tumors underwent tumor resection. Two pieces of tumor tissues were taken from each tumor to be dissociated, triturated into single cells in sterile DMEM-F12 medium, and then filtered. The tumor cells were seeded at a concentration of 200,000 viable cells per mL into serum-free DMEM-F12 medium simply supplemented with B27, human basic fibroblast growth factor (20 microg/L), human epidermal growth factor (20 microg /L), insulin (4 U/L), L-glutamine, penicillin and streptomycin. After the primary brain tumor spheres (BTSs) were generated, they were triturated again and passed in fresh medium. Limiting dilution assay was performed to observe the monoclone formation. 5-bromodeoxyuridine (BrdU) incorporation test was performed to observe the proliferation of the BTS. The BTSCs were cultured in mitogen-free DMEM-F12 medium supplemented with 10% fetal bovine serum to observe their differentiation. Immunocytochemistry was used to examine the expression of CD133 and nestin, specific markers of BTSC, and the rate of CD133 positive cells. Only a minority of subsets of cells from the tumors of neuroepithelial tissue had the capacity to survive, proliferate, and generate free-floating neurosphere-like BTSs in the simplified serum-free medium. These cells attached to the poly-L-lysine coated coverslips in the serum-supplemented medium and differentiated. The BTSCs were CD133 and nestin positive. The rate of CD133 positive cells in the tumor specimens was (21 +/- 6.2)% - (38 +/- 7.0)%. A new simplified culture system for the isolation of BTSCs is established. The tumors of human neuroepithelial tissue contain CD133 and nestin positive tumor stem cells which can be isolated

Cytotoxic activity of isolated constituents from leaves of Premna serratifolia on MCF-7 and HT-29 cell lines

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Mahesh Biradi

2015-03-01

Full Text Available Premna serratifolia (Syn: Premna integrifolia is an important medicinal herb known as “Agnimantha” in Ayurveda and traditionally used for anticancer activity. The objective of present study was to isolate the cytotoxic phytoconstituents from the n-hexane soluble fraction of P. serratifolia leaf extract. Unsaponifiable portion of n-hexane soluble fraction was subjected to silica based column chromatography. The major constituents present in all the sub-fractions were identified by TLC and phytochemical tests. Two constituents were isolated and they were purified. Sub-fractions with isolates were tested for cytotoxic effect by BSL bioassay. Two isolates were found to be active and which were tested on cancer cell lines MCF-7 and HT-29 for their cytotoxicity. Among two isolates, one compound has shown significant cytotoxicity. From the results we conclude that the plant isolates showed cytotoxicity against selected human cancer cell lines.

Electricity Generation in Microbial Fuel Cell (MFC) by Bacterium Isolated from Rice Paddy Field Soil

Science.gov (United States)

Fakhirruddin, Fakhriah; Amid, Azura; Salim, Wan Wardatul Amani Wan; Suhaida Azmi, Azlin

2018-03-01

Microbial fuel cell (MFC) is an alternative approach in generating renewable energy by utilising bacteria that will oxidize organic or inorganic substrates, producing electrons yielded as electrical energy. Different species of exoelectrogenic bacteria capable of generating significant amount of electricity in MFC has been identified, using various organic compounds for fuel. Soil sample taken from rice paddy field is proven to contain exoelectrogenic bacteria, thus electricity generation using mixed culture originally found in the soil, and pure culture isolated from the soil is studied. This research will isolate the exoelectrogenic bacterial species in the rice paddy field soil responsible for energy generation. Growth of bacteria isolated from the MFC is observed by measuring the optical density (OD), cell density weight (CDW) and viable cell count. Mixed bacterial species found in paddy field soil generates maximum power of 77.62 μW and 0.70 mA of current. In addition, the research also shows that the pure bacterium in rice paddy field soil can produce maximum power and current at 51.32 μW and 0.28 mA respectively.

Differentiation of isolated human umbilical cord mesenchymal stem cells into neural stem cells

Science.gov (United States)

Chen, Song; Zhang, Wei; Wang, Ji-Ming; Duan, Hong-Tao; Kong, Jia-Hui; Wang, Yue-Xin; Dong, Meng; Bi, Xue; Song, Jian

2016-01-01

AIM To investigate whether umbilical cord human mesenchymal stem cell (UC-MSC) was able to differentiate into neural stem cell and neuron in vitro. METHODS The umbilical cords were obtained from pregnant women with their written consent and the approval of the Clinic Ethnics Committee. UC-MSC were isolated by adherent culture in the medium contains 20% fetal bovine serum (FBS), then they were maintained in the medium contain 10% FBS and induced to neural cells in neural differentiation medium. We investigated whether UC-MSC was able to differentiate into neural stem cell and neuron in vitro by using flow cytometry, reverse transcriptase-polymerase chain reaction (RT-PCR) and immunofluorescence (IF) analyzes. RESULTS A substantial number of UC-MSC was harvested using the tissue explants adherent method at about 2wk. Flow cytometric study revealed that these cells expressed common markers of MSCs, such as CD105 (SH2), CD73 (SH3) and CD90. After induction of differentiation of neural stem cells, the cells began to form clusters; RT-PCR and IF showed that the neuron specific enolase (NSE) and neurogenic differentiation 1-positive cells reached 87.3%±14.7% and 72.6%±11.8%, respectively. Cells showed neuronal cell differentiation after induced, including neuron-like protrusions, plump cell body, obviously and stronger refraction. RT-PCR and IF analysis showed that microtubule-associated protein 2 (MAP2) and nuclear factor-M-positive cells reached 43.1%±10.3% and 69.4%±19.5%, respectively. CONCLUSION Human umbilical cord derived MSCs can be cultured and proliferated in vitro and differentiate into neural stem cells, which may be a valuable source for cell therapy of neurodegenerative eye diseases. PMID:26949608

Differentiation of isolated human umbilical cord mesenchymal stem cells into neural stem cells

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Song Chen

2016-01-01

Full Text Available AIM: To investigate whether umbilical cord human mesenchymal stem cell (UC-MSC was able to differentiate into neural stem cell and neuron in vitro. METHODS: The umbilical cords were obtained from pregnant women with their written consent and the approval of the Clinic Ethnics Committee. UC-MSC were isolated by adherent culture in the medium contains 20% fetal bovine serum (FBS, then they were maintained in the medium contain 10% FBS and induced to neural cells in neural differentiation medium. We investigated whether UC-MSC was able to differentiate into neural stem cell and neuron in vitro by using flow cytometry, reverse transcriptase-polymerase chain reaction (RT-PCR and immunofluorescence (IF analyzes. RESULTS: A substantial number of UC-MSC was harvested using the tissue explants adherent method at about 2wk. Flow cytometric study revealed that these cells expressed common markers of MSCs, such as CD105 (SH2, CD73 (SH3 and CD90. After induction of differentiation of neural stem cells, the cells began to form clusters; RT-PCR and IF showed that the neuron specific enolase (NSE and neurogenic differentiation 1-positive cells reached 87.3%±14.7% and 72.6%±11.8%, respectively. Cells showed neuronal cell differentiation after induced, including neuron-like protrusions, plump cell body, obviously and stronger refraction. RT-PCR and IF analysis showed that microtubule-associated protein 2 (MAP2 and nuclear factor-M-positive cells reached 43.1%±10.3% and 69.4%±19.5%, respectively. CONCLUSION: Human umbilical cord derived MSCs can be cultured and proliferated in vitro and differentiate into neural stem cells, which may be a valuable source for cell therapy of neurodegenerative eye diseases.

Isolation and identification of porcine reproductive and respiratory syndrome virus in cell cultures.

Science.gov (United States)

Valícek, L; Psikal, I; Smíd, B; Rodák, L; Kubalíková, R; Kosinová, E

1997-10-01

Three strains of porcine reproductive and respiratory syndrome virus (PRRSV) were isolated in porcine lung macrophage (PLM) cultures from three swine herds. This has been the first successful isolation of PRRSV in the Czech Republic and the strains received the designations CAPM V-501, CAPM V-502 and CAPM V-503, respectively. All the three isolates in PLM were identified by immunofluorescence and immunoperoxidase tests and the strain CAPM V-502 also by electron microscopy using the ultrathin section technique. The strain CAPM V-502 has been adapted to the cell line MARC-145. Viral RNA in PLM cultures infected with any of the isolated PRRSV strains was demonstrated by RT-PCR targeted to the more conserved ORF 7 genomic region encoding the nucleocapsid protein. The assessment of PCR products in agarose gel revealed a uniform size of 394 bp in all the three isolates and the European prototype strain Lelystad used as positive control.

An adhesion-based method for plasma membrane isolation: evaluating cholesterol extraction from cells and their membranes.

Science.gov (United States)

Bezrukov, Ludmila; Blank, Paul S; Polozov, Ivan V; Zimmerberg, Joshua

2009-11-15

A method to isolate large quantities of directly accessible plasma membrane from attached cells is presented. The method is based on the adhesion of cells to an adsorbed layer of polylysine on glass plates, followed by hypotonic lysis with ice-cold distilled water and subsequent washing steps. Optimal conditions for coating glass plates and time for cell attachment were established. No additional chemical or mechanical treatments were used. Contamination of the isolated plasma membrane by cell organelles was less than 5%. The method uses inexpensive, commercially available polylysine and reusable glass plates. Plasma membrane preparations can be made in 15 min. Using this method, we determined that methyl-beta-cyclodextrin differentially extracts cholesterol from fibroblast cells and their plasma membranes and that these differences are temperature dependent. Determination of the cholesterol/phospholipid ratio from intact cells does not reflect methyl-beta-cyclodextrin plasma membrane extraction properties.

Three methods for isolating viable anthozoan endoderm cells with their intracellular symbiotic dinoflagellates

Science.gov (United States)

Gates, R. D.; Muscatine, L.

1992-09-01

Three maceration methods are described for the isolation of single endoderm cells from marine cnidarians. Two are enzymatic treatments suitable for fleshy anthozoans such as sea anemones and zoanthids. The third employs calcium free sea water and is suitable for stony corals. The viability and morphology of the endoderm cells is described using fluorogenic dyes and scanning and transmission electron microscopy.

A FISH-based method for assessment of HER-2 amplification status in breast cancer circulating tumor cells following CellSearch isolation

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Frithiof H

2016-11-01

Full Text Available Henrik Frithiof,1 Kristina Aaltonen,1 Lisa Rydén2,3 1Division of Oncology and Pathology, 2Division of Surgery, Department of Clinical Sciences Lund, Lund University, Lund, 3Department of Surgery, Skåne University Hospital, Malmö, Sweden Introduction: Amplification of the HER-2/neu (HER-2 proto-oncogene occurs in 10%–15% of primary breast cancer, leading to an activated HER-2 receptor, augmenting growth of cancer cells. Tumor classification is determined in primary tumor tissue and metastatic biopsies. However, malignant cells tend to alter their phenotype during disease progression. Circulating tumor cell (CTC analysis may serve as an alternative to repeated biopsies. The Food and Drug Administration-approved CellSearch system allows determination of the HER-2 protein, but not of the HER-2 gene. The aim of this study was to optimize a fluorescence in situ hybridization (FISH-based method to quantitatively determine HER-2 amplification in breast cancer CTCs following CellSearch-based isolation and verify the method in patient samples. Methods: Using healthy donor blood spiked with human epidermal growth factor receptor 2 (HER-2-positive breast cancer cell lines, SKBr-3 and BT-474, and a corresponding negative control (the HER-2-negative MCF-7 cell line, an in vitro CTC model system was designed. Following isolation in the CellSearch system, CTC samples were further enriched and fixed on microscope slides. Immunocytochemical staining with cytokeratin and 4',6-diamidino-2'-phenylindole dihydrochloride identified CTCs under a fluorescence microscope. A FISH-based procedure was optimized by applying the HER2 IQFISH pharmDx assay for assessment of HER-2 amplification status in breast cancer CTCs. Results: A method for defining the presence of HER-2 amplification in single breast cancer CTCs after CellSearch isolation was established using cell lines as positive and negative controls. The method was validated in blood from breast cancer patients

A retinoic acid receptor β2 agonist reduces hepatic stellate cell activation in nonalcoholic fatty liver disease.

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Trasino, Steven E; Tang, Xiao-Han; Jessurun, Jose; Gudas, Lorraine J

2016-10-01

Hepatic stellate cells (HSCs) are an important cellular target for the development of novel pharmacological therapies to prevent and treat nonalcoholic fatty liver diseases (NAFLD). Using a high fat diet (HFD) model of NAFLD, we sought to determine if synthetic selective agonists for retinoic acid receptor β2 (RARβ2) and RARγ can mitigate HSC activation and HSC relevant signaling pathways during early stages of NAFLD, before the onset of liver injury. We demonstrate that the highly selective RARβ2 agonist, AC261066, can reduce the activation of HSCs, marked by decreased HSC expression of α-smooth muscle actin (α-SMA), in mice with HFD-induced NAFLD. Livers of HFD-fed mice treated with AC261066 exhibited reduced steatosis, oxidative stress, and expression of pro-inflammatory mediators, such as tumor necrosis factor-alpha (TNFα), interleukin 1β (IL-1β), and monocyte chemotactic protein-1 (MCP-1). Kupffer cell (macrophage) expression of transforming growth factor-β1 (TGF-β1), which plays a critical role in early HSC activation, was markedly reduced in AC261066-treated, HFD-fed mice. In contrast, HFD-fed mice treated with an RARγ agonist (CD1530) showed no decreases in steatosis, HSC activation, or Kupffer cell TGF-β1 levels. In conclusion, our data demonstrate that RARβ2 is an attractive target for development of NAFLD therapies. • Hepatic stellate cells (HSCs) are an important pharmacological target for the prevention of nonalcoholic fatty liver diseases (NAFLD). • Retinoids and retinoic acid receptors (RARs) possess favorable metabolic modulating properties. • We show that an agonist for retinoic acid receptor-β2 (RARβ2), but not RARγ, mitigates HSC activation and NAFLD.

Isolation of cell nuclei using inert macromolecules to mimic the crowded cytoplasm.

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Ronald Hancock

Full Text Available Cell nuclei are commonly isolated and studied in media which include millimolar concentrations of cations, which conserve the nuclear volume by screening the negative charges on chromatin and maintaining its compaction. However, two factors question if these ionic conditions correctly reproduce the environment of nuclei in vivo: the small-scale motion and conformation of chromatin in vivo are not reproduced in isolated nuclei, and experiments and theory suggest that small ions in the cytoplasm are not free in the soluble phase but are predominantly bound to macromolecules. We studied the possible role in maintaining the structure and functions of nuclei in vivo of a further but frequently overlooked property of the cytoplasm, the crowding or osmotic effects caused by diffusible macromolecules whose concentration, measured in several studies, is in the range of 130 mg/ml. Nuclei which conserved their volume in the cell and their ultrastructure seen by electron microscopy were released from K562 cells in media containing the inert polymer 70 kDa Ficoll (50% w/v or 70 kDa dextran (35% w/v to replace the diffusible cytoplasmic molecules which were dispersed on cell lysis with digitonin, with 100 microM K-Hepes buffer as the only source of ions. Immunofluorescence labelling and experiments using cells expressing GFP-fusion proteins showed that internal compartments (nucleoli, PML and coiled bodies, foci of RNA polymerase II were conserved in these nuclei, and nascent RNA transcripts could be elongated. Our observations are consistent with the hypothesis that crowding by diffusible cytoplasmic macromolecules is a crucial but overlooked factor which supports the nucleus in vivo by equilibrating the opposing osmotic pressure cause by the high concentration of macromolecules in the nucleus, and suggest that crowded media provide more physiological conditions to study nuclear structure and functions. They may also help to resolve the long-standing paradox

Measurement of bacterial capture and phagosome maturation of Kupffer cells by intravital microscopy

NARCIS (Netherlands)

Surewaard, Bas G.J.; Kubes, Paul

2017-01-01

It is central to the field of bacterial pathogenesis to define how bacteria are killed by phagocytic cells. During phagocytosis, the microbe is localized to the phagolysosome where crucial defense mechanisms such as acidification and production of reactive oxygen species (ROS) are initiated. This

Anti-inflammatory liposomes have no impact on liver regeneration in rats

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Betina Norman Jepsen

2015-12-01

Conclusion: Low dose dexamethasone targeted to Kupffer cells does not affect histological liver cell regeneration after 70% hepatectomy in rats, but reduces the inflammatory response judged by circulating markers of inflammation.

A method for the isolation and culture of adult rat retinal pigment epithelial (RPE cells to study retinal diseases

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Janosch Peter Heller

2015-11-01

Full Text Available Diseases such as age-related macular degeneration (AMD affect the retinal pigment epithelium (RPE and lead to the death of the epithelial cells and ultimately blindness. RPE transplantation is currently a major focus of eye research and clinical trials using human stem cell-derived RPE cells are ongoing. However, it remains to be established to which extent the source of RPE cells for transplantation affects their therapeutic efficacy and this needs to be explored in animal models. Autotransplantation of RPE cells has attractions as a therapy, but existing protocols to isolate adult RPE cells from rodents are technically difficult, time-consuming, have a low yield and are not optimized for long-term cell culturing. Here, we report a newly devised protocol which facilitates reliable and simple isolation and culture of RPE cells from adult rats. Incubation of a whole rat eyeball in 20 U/ml papain solution for 50 minutes yielded 4 x 104 viable RPE cells. These cells were hexagonal and pigmented upon culture. Using immunostaining, we demonstrated that the cells expressed RPE cell-specific marker proteins including cytokeratin 18 and RPE65, similar to RPE cells in vivo. Additionally, the cells were able to produce and secrete Bruch’s membrane matrix components similar to in vivo situation. Similarly, the cultured RPE cells adhered to isolated Bruch’s membrane as has previously been reported. Therefore, the protocol described in this article provides an efficient method for the rapid and easy isolation of high quantities of adult rat RPE cells. This provides a reliable platform for studying the therapeutic targets, testing the effects of drugs in a preclinical setup and to perform in vitro and in vivo transplantation experiments to study retinal diseases.

Towards an advanced therapy medicinal product based on mesenchymal stromal cells isolated from the umbilical cord tissue: quality and safety data.

Science.gov (United States)

Martins, José Paulo; Santos, Jorge Miguel; de Almeida, Joana Marto; Filipe, Mariana Alves; de Almeida, Mariana Vargas Teixeira; Almeida, Sílvia Cristina Paiva; Água-Doce, Ana; Varela, Alexandre; Gilljam, Mari; Stellan, Birgitta; Pohl, Susanne; Dittmar, Kurt; Lindenmaier, Werner; Alici, Evren; Graça, Luís; Cruz, Pedro Estilita; Cruz, Helder Joaquim; Bárcia, Rita Nogueira

2014-01-17

Standardization of mesenchymal stromal cells (MSCs) manufacturing is urgently needed to enable translational activities and ultimately facilitate comparison of clinical trial results. In this work we describe the adaptation of a proprietary method for isolation of a specific umbilical cord tissue-derived population of MSCs, herein designated by its registered trademark as UCX®, towards the production of an advanced therapy medicinal product (ATMP). The adaptation focused on different stages of production, from cell isolation steps to cell culturing and cryopreservation. The origin and quality of materials and reagents were considered and steps for avoiding microbiological and endotoxin contamination of the final cell product were implemented. Cell isolation efficiency, MSCs surface markers and genetic profiles, originating from the use of different medium supplements, were compared. The ATMP-compliant UCX® product was also cryopreserved avoiding the use of dimethyl sulfoxide, an added benefit for the use of these cells as an ATMP. Cells were analyzed for expansion capacity and longevity. The final cell product was further characterized by flow cytometry, differentiation potential, and tested for contaminants at various passages. Finally, genetic stability and immune properties were also analyzed. The isolation efficiency of UCX® was not affected by the introduction of clinical grade enzymes. Furthermore, isolation efficiencies and phenotype analyses revealed advantages in the use of human serum in cell culture as opposed to human platelet lysate. Initial decontamination of the tissue followed by the use of mycoplasma- and endotoxin-free materials and reagents in cell isolation and subsequent culture, enabled the removal of antibiotics during cell expansion. UCX®-ATMP maintained a significant expansion potential of 2.5 population doublings per week up to passage 15 (P15). They were also efficiently cryopreserved in a DMSO-free cryoprotectant medium with

Isolated (localized) idiopathic granulomatous (giant cell) vasculitis in an intramuscular lipoma.

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Fernando Val-Bernal, J; Val, Daniel; Calvo, Ignacio; Francisca Garijo, M

2006-01-01

Isolated (localized) idiopathic granulomatous vasculitis (IGV) is an uncommon, heterogeneous, and poorly defined group of disorders characterized by infiltration of the arterial wall caused by compactly grouped mononuclear phagocytes, with or without giant cells, in segmental distribution. We report on a 55-year-old woman with IGV limited to an intramuscular lipoma of the left thigh. The vasculitis was identified incidentally upon microscopic examination of the removed tumor. The IGV was centered on two medium-sized arteries, accompanied by narrowing of the lumens, and not associated with secondary changes such as infart or postinfart fibrosis. The inflammatory infiltrate was rich in T-lymphocytes and macrophages, with the presence of giant cells. The patient was asymptomatic and well in a follow-up period of 2 months, during which she was not treated. To our knowledge, this is the first report of lipoma involvement in localized IGV. It is important to distinguish cases of isolated intratumorous IGV from systemic disease, because the latter implies a poor prognosis and requires an aggressive treatment.

Polyphosphoinositides are present in plasma membranes isolated from fusogenic carrot cells

International Nuclear Information System (INIS)

Wheeler, J.J.; Boss, W.F.

1987-01-01

Fusogenic carrot cells grown in suspension culture were labeled 12 hours with myo-[2- 3 H]inositol. Plasma membranes were isolated from the prelabeled fusogenic carrot cells by both aqueous polymer two-phase partitioning and Renografin density gradients. With both methods, the plasma membrane-enriched fractions, as identified by marker enzymes, were enriched in [ 3 H]inositol-labeled phosphatidylinositol monophosphate (PIP) and phosphatidylinositol bisphosphate (PIP 2 ). An additional [ 3 H]inositol-labeled lipid, lysophosphatidylinositol monophosphate, which migrated between PIP and PIP 2 on thin layer plates, was found primarily in the plasma membrane-rich fraction of the fusogenic cells. This was in contrast to lysophosphatidylinositol which is found primarily in the lower phase, microsomal/mitchrondrial-rich fraction

Live Staining and Isolation of Specific Hormone-Producing Cells from Rat Anterior Pituitary by Cytochemistry with Lectins and Cholera Toxin B Subunit

International Nuclear Information System (INIS)

Kikuchi, Motoshi; Kusumoto, Kenji; Fujiwara, Ken; Takahashi, Kozue; Tando, Yukiko; Yashiro, Takashi

2011-01-01

Anterior pituitary glands contain five types of hormone-producing cells. Distinguishing and isolating specific types of living cells are essential for studying their function. Although many such attempts have been made, the results have been disappointing. In the present study, we labeled specific types of living hormone-producing cells by using potential differences in sugar chains on the cell surfaces. Cytochemical analysis with lectins and cholera toxin B subunit revealed that PNA, S-WGA, and cholera toxin B subunit recognized sugar chains specific to prolactin cells, ACTH cells, and GH cells, respectively, and that UEA-I recognized most of prolactin cells and GH cells. Next, fluorescence-activated cell sorting was used to isolate GH cells labeled by fluoresceinated cholera toxin B. The purity of the GH cell fraction estimated by immunocytochemistry and quantitative real-time PCR for cell type-specific genes was more than 98%, which was higher than that reported in earlier studies, including those using transgenic animals. We conclude that cytochemistry with lectins and cholera toxin B subunit is a straightforward, acceptable method of isolating specific types of anterior pituitary cells and that the cells isolated by this method can serve as useful materials in the study of anterior pituitary cells

High efficiency isolated DC/DC converter inherently optimized for fuel cell applications

DEFF Research Database (Denmark)

Petersen, Lars Press; Jensen, Lasse Crone; Larsen, Martin Norgaard

2013-01-01

The isolated full-bridge boost converter has been suggested as the best choice for fuel cell applications. Comparisons have been carried out in the literature using both stress factors and experimental verified designs to determine the optimal converter. Never the less, this paper suggests...

Muscle-derived stem cells isolated as non-adherent population give rise to cardiac, skeletal muscle and neural lineages

International Nuclear Information System (INIS)

Arsic, Nikola; Mamaeva, Daria; Lamb, Ned J.; Fernandez, Anne

2008-01-01

Stem cells with the ability to differentiate in specialized cell types can be extracted from a wide array of adult tissues including skeletal muscle. Here we have analyzed a population of cells isolated from skeletal muscle on the basis of their poor adherence on uncoated or collagen-coated dishes that show multi-lineage differentiation in vitro. When analysed under proliferative conditions, these cells express stem cell surface markers Sca-1 (65%) and Bcrp-1 (80%) but also MyoD (15%), Neuronal β III-tubulin (25%), GFAP (30%) or Nkx2.5 (1%). Although capable of growing as non-attached spheres for months, when given an appropriate matrix, these cells adhere giving rise to skeletal muscle, neuronal and cardiac muscle cell lineages. A similar cell population could not be isolated from either bone marrow or cardiac tissue suggesting their specificity to skeletal muscle. When injected into damaged muscle, these non-adherent muscle-derived cells are retrieved expressing Pax7, in a sublaminar position characterizing satellite cells and participate in forming new myofibers. These data show that a non-adherent stem cell population can be specifically isolated and expanded from skeletal muscle and upon attachment to a matrix spontaneously differentiate into muscle, cardiac and neuronal lineages in vitro. Although competing with resident satellite cells, these cells are shown to significantly contribute to repair of injured muscle in vivo supporting that a similar muscle-derived non-adherent cell population from human muscle may be useful in treatment of neuromuscular disorders

Muscle-derived stem cells isolated as non-adherent population give rise to cardiac, skeletal muscle and neural lineages.

Science.gov (United States)

Arsic, Nikola; Mamaeva, Daria; Lamb, Ned J; Fernandez, Anne

2008-04-01

Stem cells with the ability to differentiate in specialized cell types can be extracted from a wide array of adult tissues including skeletal muscle. Here we have analyzed a population of cells isolated from skeletal muscle on the basis of their poor adherence on uncoated or collagen-coated dishes that show multi-lineage differentiation in vitro. When analysed under proliferative conditions, these cells express stem cell surface markers Sca-1 (65%) and Bcrp-1 (80%) but also MyoD (15%), Neuronal beta III-tubulin (25%), GFAP (30%) or Nkx2.5 (1%). Although capable of growing as non-attached spheres for months, when given an appropriate matrix, these cells adhere giving rise to skeletal muscle, neuronal and cardiac muscle cell lineages. A similar cell population could not be isolated from either bone marrow or cardiac tissue suggesting their specificity to skeletal muscle. When injected into damaged muscle, these non-adherent muscle-derived cells are retrieved expressing Pax7, in a sublaminar position characterizing satellite cells and participate in forming new myofibers. These data show that a non-adherent stem cell population can be specifically isolated and expanded from skeletal muscle and upon attachment to a matrix spontaneously differentiate into muscle, cardiac and neuronal lineages in vitro. Although competing with resident satellite cells, these cells are shown to significantly contribute to repair of injured muscle in vivo supporting that a similar muscle-derived non-adherent cell population from human muscle may be useful in treatment of neuromuscular disorders.

Relations between fatty acid synthesis, pyruvate concentration and cell concentration of suspensions of isolated rat hepatocytes

NARCIS (Netherlands)

Beynen, A.C.; Geelen, M.J.H.

1984-01-01

1. 1. The cell concentration of suspensions of isolated rat hepatocytes affects both the rate of pyruvate accumulation in the incubation medium and the rate of fatty acid synthesis. 2. 2. At low cell concentrations pyruvate accumulation is directly related to the cell concentration but levels off

Analysis of detergent-free lipid rafts isolated from CD4+ T cell line: interaction with antigen presenting cells promotes coalescing of lipid rafts

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Kennedy Colleen

2011-12-01

Full Text Available Abstract Background Lipid rafts present on the plasma membrane play an important role in spatiotemporal regulation of cell signaling. Physical and chemical characterization of lipid raft size and assessment of their composition before, and after cell stimulation will aid in developing a clear understanding of their regulatory role in cell signaling. We have used visual and biochemical methods and approaches for examining individual and lipid raft sub-populations isolated from a mouse CD4+ T cell line in the absence of detergents. Results Detergent-free rafts were analyzed before and after their interaction with antigen presenting cells. We provide evidence that the average diameter of lipid rafts isolated from un-stimulated T cells, in the absence of detergents, is less than 100 nm. Lipid rafts on CD4+ T cell membranes coalesce to form larger structures, after interacting with antigen presenting cells even in the absence of a foreign antigen. Conclusions Findings presented here indicate that lipid raft coalescence occurs during cellular interactions prior to sensing a foreign antigen.

Adipose mesenchymal stem cells isolated after manual or water jet-assisted liposuction display similar properties

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Claire eBony

2016-01-01

Full Text Available Mesenchymal stem or stromal cells (MSC are under investigation in many clinical trials for their therapeutic potential in a variety of diseases, including autoimmune and inflammatory disorders. One of the main sources of MSCs is the adipose tissue, which is mainly obtained by manual liposuction using a cannula linked to a syringe. However, in the last years, a number of devices for fat liposuction intended for clinical use have been commercialized but few papers have compared these procedures in terms of stromal vascular fraction (SVF or adipose stromal cells (ASC. The objective of the present study was to compare and qualify for clinical use the adipose stromal cells (ASC obtained from fat isolated with the manual or the Bodyjet® waterjet-assisted procedure. Although the initial number of cells after collagenase digestion was higher with the manual procedure, both the percentage of dead cells, the number of CFU-F and the phenotype of cells were identical in the SVF at isolation and in the ASC populations at day 14. We also showed that the osteogenic and adipogenic differentiation potentials of ASCs were identical between preparations while a slight but significant higher in vitro immunosuppressive effect was observed with ASCs isolated from fat removed with a cannula. The difference in the immunomodulatory effect between ASC populations was however not observed in vivo using the delayed-type hypersensitivity model. Our data therefore indicate that the procedure for fat liposuction does not impact the characteristics or the therapeutic function of ASCs.

Compartmentation and equilibration of abscisic acid in isolated Xanthium cells

International Nuclear Information System (INIS)

Bray, E.A.; Zeevaart, J.A.D.

1986-01-01

The compartmentation of endogenous abscisic acid (ABA), applied (+/-)-[ 3 H]ABA, and (+/-)-trans-ABA was measured in isolated mesophyll cells of the Chicago strain of Xanthium strumarium L. The release of ABA to the medium in the presence or absence of DMSO was used to determine the equilibration of ABA in the cells. It was found that a greater percentage of the (+/-)-[ 3 H]ABA and the (+/-)-trans-ABA was released into the medium than of the endogenous ABA, indicating that applied ABA did not equilibrate with the endogenous material. Therefore, in further investigations only the compartmentation of endogenous ABA was studied. Endogenous ABA was released from Xanthium cells according to the pH gradients among the various cellular compartments. Thus, darkness, high external pH, KNO 2 , and drought-stress all increased the efflux of ABA from the cells. Efflux of ABA from the cells in the presence of 0.6 M mannitol occurred within 30 seconds, but only 8% of the endogenous material was released during the 20 minute treatment

Functional study and regional mapping of 44 hormono-regulated genes isolated from a porcine granulosa cell library

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Hatey François

2001-01-01

Full Text Available Abstract cDNA clones from a pig granulosa cell cDNA library were isolated by differential hybridisation for follicle stimulating hormone (FSH regulation in granulosa cells in a previous study. The clones that did not match any known sequence were studied for their expression in granulosa cells (treated or not by FSH and in fresh isolated ovarian follicles mainly by comparative RT-PCR analysis. These results give functional data on genes that may be implicated in follicular growing. These ESTs have been localised on the porcine genome, using a somatic cell hybrid panel, providing new type I markers on the porcine map and information on the comparative map between humans and pigs.

Electrical characterization of single cells using polysilicon wire ion sensor in an isolation window.

Science.gov (United States)

Wu, You-Lin; Hsu, Po-Yen; Hsu, Chung-Ping; Wang, Chih-Cheng; Lee, Li-Wen; Lin, Jing-Jenn

2011-10-01

A polysilicon wire (PSW) sensor can detect the H(+) ion density (pH value) of the medium coated on its surface, and different cells produce different extracellular acidification and hence different H(+) ion densities. Based on this, we used a PSW sensor in combination with a mold-cast polydimethylsiloxane (PDMS) isolation window to detect the adhesion, apoptosis and extracellular acidification of single normal cells and single cancer cells. Single living human normal cells WI38, MRC5, and BEAS-2B as well as non-small-cell lung cancer (NSCLC) cells A549, H1299, and CH27 were cultivated separately inside the isolation window. The current flowing through the PSW channel was measured. From the PSW channel current change as a function of time, we determined the cell adhesion time by observing the time required for the current change to saturate, since a stable extracellular ion density was established after the cells were completely adhered to the PSW surface. The apoptosis of cells can also be determined when the channel current change drops to zero. We found that all the NSCLC cells had a higher channel current change and hence a lower pH value than the normal cells anytime after they were seeded. The corresponding average pH values were 5.86 for A549, 6.00 for H1299, 6.20 for CH27, 6.90 for BEAS-2B, 6.96for MRC5, and 7.02 for WI38, respectively, after the cells were completely adhered to the PSW surface. Our results show that NSCLC cells have a stronger cell-substrate adhesion and a higher extracellular acidification rate than normal cells.

A method for the isolation and characterization of functional murine monoclonal antibodies by single B cell cloning.

Science.gov (United States)

Carbonetti, Sara; Oliver, Brian G; Vigdorovich, Vladimir; Dambrauskas, Nicholas; Sack, Brandon; Bergl, Emilee; Kappe, Stefan H I; Sather, D Noah

2017-09-01

Monoclonal antibody technologies have enabled dramatic advances in immunology, the study of infectious disease, and modern medicine over the past 40years. However, many monoclonal antibody discovery procedures are labor- and time-intensive, low efficiency, and expensive. Here we describe an optimized mAb discovery platform for the rapid and efficient isolation, cloning and characterization of monoclonal antibodies in murine systems. In this platform, antigen-binding splenic B cells from immunized mice are isolated by FACS and cocultured with CD40L positive cells to induce proliferation and mAb production. After 12days of coculture, cell culture supernatants are screened for antigen, and IgG positivity and RNA is isolated for reverse-transcription. Positive-well cDNA is then amplified by PCR and the resulting amplicons can be cloned into ligation-independent expression vectors, which are then used directly to transfect HEK293 cells for recombinant antibody production. After 4days of growth, conditioned medium can be screened using biolayer interferometry for antigen binding and affinity measurements. Using this method, we were able to isolate six unique, functional monoclonal antibodies against an antigen of the human malaria parasite Plasmodium falciparum. Importantly, this method incorporates several important advances that circumvent the need for single-cell PCR, restriction cloning, and large scale protein production, and can be applied to a wide array of protein antigens. Copyright © 2017 Elsevier B.V. All rights reserved.

Flow-through immunomagnetic separation system for waterborne pathogen isolation and detection: Application to Giardia and Cryptosporidium cell isolation

Energy Technology Data Exchange (ETDEWEB)

Ramadan, Qasem, E-mail: qasem.alramadan@epfl.ch [Bioelectronics Program, Institute of Microelectronics, 11 Science Park Road, Singapore 117685 (Singapore); Christophe, Lay; Teo, William; ShuJun, Li; Hua, Feng Han [Bioelectronics Program, Institute of Microelectronics, 11 Science Park Road, Singapore 117685 (Singapore)

2010-07-12

Simultaneous sample washing and concentration of two waterborne pathogen samples were demonstrated using a rotational magnetic system under continuous flow conditions. The rotation of periodically arranged small permanent magnets close to a fluidic channel carrying magnetic particle suspension allows the trapping and release of particles along the fluidic channel in a periodic manner. Each trapping and release event resembles one washing cycle. The performance of the magnetic separation system (MSS) was evaluated in order to test its functionality to isolate magnetic-labelled protozoan cells from filtered, concentrated tap water, secondary effluent water, and purified water. Experimental protocols described in US Environmental Protection Agency method 1623 which rely on the use of a magnetic particle concentrator, were applied to test and compare our continuous flow cell separation system to the standard magnetic bead-based isolation instruments. The recovery efficiencies for Giardia cysts using the magnetic tube holder and our magnetic separation system were 90.5% and 90.1%, respectively, from a tap water matrix and about 31% and 18.5%, respectively, from a spiked secondary effluent matrix. The recovery efficiencies for Cryptosporidium cells using the magnetic tube holder and our magnetic separation system were 90% and 83.3%, respectively, from a tap water matrix and about 38% and 36%, respectively, from a spiked secondary effluent matrix. Recoveries from all matrices with the continuous flow system were typically higher in glass tubing conduits than in molded plastic conduits.

Evaluation of different continuous cell lines in the isolation of mumps virus by the shell vial method from clinical samples

Science.gov (United States)

Reina, J; Ballesteros, F; Mari, M; Munar, M

2001-01-01

Aims—To compare prospectively the efficacy of the Vero, LLC-MK2, MDCK, Hep-2, and MRC-5 cell lines in the isolation of the mumps virus from clinical samples by means of the shell vial method. Methods—During an epidemic outbreak of parotiditis 48 clinical samples (saliva swabs and CSF) were studied. Two vials of the Vero, LLC-MK2, MDCK, MRC-5, and Hep-2 cell lines were inoculated with 0.2 ml of the samples by the shell vial assay. The vials were incubated at 36°C for two and five days. The vials were then fixed with acetone at -20°C for 10 minutes and stained by a monoclonal antibody against mumps virus by means of an indirect immunofluorescence assay. Results—The mumps virus was isolated from 36 samples. The Vero and LLC-MK2 cell lines showed a 100% isolation capacity, MDCK showed 77.7%, MRC-5 showed 44.4%, and Hep-2 showed 22.2%. The Vero and LLC-MK2 lines were significantly different to the other cell lines (p 5 infectious foci) were 94.4% for Vero, 97.2% for LLC-MK2, 5.5% for MDCK, 5.5% for Hep-2, and 0% for MRC-5. Conclusions—The Vero and LLC-MK2 cell lines are equally efficient at two and five days incubation for the isolation of the mumps virus from clinical samples, and the use of the shell vial method considerably shortens the time of aetiological diagnosis with higher specificity. Key Words: mumps virus • Vero cell line • LLC-MK2 cell line • MDCK cell line • Hep-2 cell line • MRC-5 cell line • isolation • shell vial PMID:11729211

Liver inflammation during monocrotaline hepatotoxicity

International Nuclear Information System (INIS)

Copple, Bryan L.; Ganey, Patricia E.; Roth, Robert A.

2003-01-01

Monocrotaline (MCT) is a pyrrolizidine alkaloid (PA) plant toxin that causes hepatotoxicity in humans and animals. Human exposure occurs from consumption of contaminated grains and herbal teas and medicines. Intraperitoneal injection (i.p.) of 300 mg/kg MCT in rats produced time-dependent hepatic parenchymal cell (HPC) injury beginning at 12 h. At this time, an inflammatory infiltrate consisting of neutrophils (PMNs) appeared in areas of hepatocellular injury, and activation of the coagulation system occurred. PMN accumulation was preceded by up-regulation of the PMN chemokines cytokine-induced neutrophil chemoattractant-1 (CINC-1) and macrophage inflammatory protein-2 (MIP-2) in the liver. The monocyte chemokine, monocyte chemoattractant protein-1 (MCP-1), was also upregulated. Inhibition of Kupffer cell function with gadolinium chloride (GdCl 3 ) significantly reduced CINC-1 protein in plasma after MCT treatment but had no effect on hepatic PMN accumulation. Since inflammation can contribute to either pathogenesis or resolution of tissue injury, we explored inflammatory factors as a contributor to MCT hepatotoxicity. To test the hypothesis that PMNs contribute to MCT-induced HPC injury, rats were depleted of PMNs with a rabbit anti-PMN serum prior to MCT treatment. Anti-PMN treatment reduced hepatic PMN accumulation by 80% but had no effect on MCT-induced HPC injury or activation of the coagulation system. To test the hypothesis that Kupffer cells and/or tumor necrosis factor-α (TNF-α) are required for MCT-induced HPC injury, rats were treated with either GdCl 3 to inhibit Kupffer cell function or pentoxifylline (PTX) to prevent synthesis of TNF-α. Neither treatment prevented MCT-induced HPC injury. Results from these studies suggest that PMNs, Kupffer cells and TNF-α are not critical mediators of MCT hepatotoxicity. Accordingly, although inflammation occurs in the liver after MCT treatment, it is not required for HPC injury and possibly occurs secondary to

A novel strategy for enrichment and isolation of osteoprogenitor cells from induced pluripotent stem cells based on surface marker combination.

Directory of Open Access Journals (Sweden)

Hiromi Ochiai-Shino

Full Text Available In this study, we developed a new method to stimulate osteogenic differentiation in tissue-nonspecific alkaline phosphatase (TNAP-positive cells liberated from human induced pluripotent stem cells (hiPSCs-derived embryoid bodies (EBs with 14 days long TGF-β/IGF-1/FGF-2 treatment. TNAP is a marker protein of osteolineage cells. We analyzed and isolated TNAP-positive and E-cadherin-negative nonepithelial cells by fluorescence-activated cell sorting. Treating the cells with a combination of transforming growth factor (TGF-β, insulin-like growth factor (IGF-1, and fibroblast growth factor (FGF-2 for 14 days greatly enhanced TNAP expression and maximized expression frequency up to 77.3%. The isolated cells expressed high levels of osterix, which is an exclusive osteogenic marker. Culturing these TNAP-positive cells in osteoblast differentiation medium (OBM led to the expression of runt-related transcription factor 2, type I collagen, bone sialoprotein, and osteocalcin (OCN. These cells responded to treatment with activated vitamin D3 by upregulating OCN. Furthermore, in OBM they were capable of generating many mineralized nodules with strong expression of receptor activator of NF-kappaB ligand and sclerostin (SOST. Real-time RT-PCR showed a significant increase in the expression of osteocyte marker genes, including SOST, neuropeptide Y, and reelin. Scanning electron microscopy showed dendritic morphology. Examination of semi-thin toluidine blue-stained sections showed many interconnected dendrites. Thus, TNAP-positive cells cultured in OBM may eventually become terminally differentiated osteocyte-like cells. In conclusion, treating hiPSCs-derived cells with a combination of TGF-β, IGF-1, and FGF-2 generated TNAP-positive cells at high frequency. These TNAP-positive cells had a high osteogenic potential and could terminally differentiate into osteocyte-like cells. The method described here may reveal new pathways of osteogenesis and provide a novel

Isolation and characterization of two kinds of stem cells from the same human skin back sample with therapeutic potential in spinal cord injury.

Directory of Open Access Journals (Sweden)

Zhaowen Zong

Full Text Available BACKGROUNDS AND OBJECTIVE: Spinal cord injury remains to be a challenge to clinicians and it is attractive to employ autologous adult stem cell transplantation in its treatment, however, how to harvest cells with therapeutic potential easily and how to get enough number of cells for transplantation are challenging issues. In the present study, we aimed to isolate skin-derived precursors (SKPs and dermal multipotent stem cells (dMSCs simultaneously from single human skin samples from patients with paraplegia. METHODS: Dissociated cells were initially generated from the dermal layer of skin samples from patients with paraplegia and cultured in SKPs proliferation medium. Four hours later, many cells adhered to the base of the flask. The suspended cells were then transferred to another flask for further culture as SKPs, while the adherent cells were cultured in dMSCs proliferation medium. Twenty-four hours later, the adherent cells were harvested and single-cell colonies were generated using serial dilution method. [(3H]thymidine incorporation assay, microchemotaxis Transwell chambers assay, RT-PCR and fluorescent immunocytochemistry were employed to examine the characterizations of the isolated cells. RESULTS: SKPs and dMSCs were isolated simultaneously from a single skin sample. SKPs and dMSCs differed in several respects, including in terms of intermediate protein expression, proliferation capacities, and differentiation tendencies towards mesodermal and neural progenies. However, both SKPs and dMSCs showed high rates of differentiation into neurons and Schwann cells under appropriate inducing conditions. dMSCs isolated by this method showed no overt differences from dMSCs isolated by routine methods. CONCLUSIONS: Two kinds of stem cells, namely SKPs and dMSCs, can be isolated simultaneously from individual human skin sample from paraplegia patients. Both of them show ability to differentiate into neural cells under proper inducing conditions

A rapid, efficient, and economic device and method for the isolation and purification of mouse islet cells.

Directory of Open Access Journals (Sweden)

Yin Zongyi

Full Text Available Rapid, efficient, and economic method for the isolation and purification of islets has been pursued by numerous islet-related researchers. In this study, we compared the advantages and disadvantages of our developed patented method with those of commonly used conventional methods (Ficoll-400, 1077, and handpicking methods. Cell viability was assayed using Trypan blue, cell purity and yield were assayed using diphenylthiocarbazone, and islet function was assayed using acridine orange/ethidium bromide staining and enzyme-linked immunosorbent assay-glucose stimulation testing 4 days after cultivation. The results showed that our islet isolation and purification method required 12 ± 3 min, which was significantly shorter than the time required in Ficoll-400, 1077, and HPU groups (34 ± 3, 41 ± 4, and 30 ± 4 min, respectively; P 1000 islets. In summary, the MCT method is a rapid, efficient, and economic method for isolating and purifying murine islet cell clumps. This method overcomes some of the shortcomings of conventional methods, showing a relatively higher quality and yield of islets within a shorter duration at a lower cost. Therefore, the current method provides researchers with an alternative option for islet isolation and should be widely generalized.

Isolated Post-Transplantation Lymphoproliferative Disease Involving the Breast and Axilla as Peripheral T-cell Lymphoma

OpenAIRE

Hwang, Ji-Young; Cha, Eun Suk; Lee, Jee Eun; Sung, Sun Hee

2013-01-01

Post-transplantation lymphoproliferative disorders (PTLDs) are a heterogeneous group of diseases that represent serious complications following immunosuppressive therapy for solid organ or hematopoietic-cell recipients. In contrast to B-cell PTLD, T-cell PTLD is less frequent and is not usually associated with Epstein Barr Virus infection. Moreover, to our knowledge, isolated T-cell PTLD involving the breast is extremely rare and this condition has never been reported previously in the litera...

Analysis of early mesothelial cell responses to Staphylococcus epidermidis isolated from patients with peritoneal dialysis-associated peritonitis.

Science.gov (United States)

McGuire, Amanda L; Mulroney, Kieran T; Carson, Christine F; Ram, Ramesh; Morahan, Grant; Chakera, Aron

2017-01-01

The major complication of peritoneal dialysis (PD) is the development of peritonitis, an infection within the abdominal cavity, primarily caused by bacteria. PD peritonitis is associated with significant morbidity, mortality and health care costs. Staphylococcus epidermidis is the most frequently isolated cause of PD-associated peritonitis. Mesothelial cells are integral to the host response to peritonitis, and subsequent clinical outcomes, yet the effects of infection on mesothelial cells are not well characterised. We systematically investigated the early mesothelial cell response to clinical and reference isolates of S. epidermidis using primary mesothelial cells and the mesothelial cell line Met-5A. Using an unbiased whole genome microarray, followed by a targeted panel of genes known to be involved in the human antibacterial response, we identified 38 differentially regulated genes (adj. p-value peritonitis. This study provides new insights into early mesothelial cell responses to infection with S. epidermidis, and confirms the importance of validating findings in primary mesothelial cells.

Isolation and characterization of neural stem cells from human fetal striatum

International Nuclear Information System (INIS)

Li Xiaoxia; Xu Jinchong; Bai Yun; Wang Xuan; Dai Xin; Liu Yinan; Zhang Jun; Zou Junhua; Shen Li; Li Lingsong

2005-01-01

This paper described that neural stem cells (hsNSCs) were isolated and expanded rapidly from human fetal striatum in adherent culture. The population was serum- and growth factor-dependent and expressed neural stem cell markers. They were capable of multi-differentiation into neurons, astrocytes, and oligodendrocytes. When plated in the dopaminergic neuron inducing medium, human striatum neural stem cells could differentiate into tyrosine hydroxylase positive neurons. hsNSCs were morphologically homogeneous and possessed high proliferation ability. The population doubled every 44.28 h and until now it has divided for more than 82 generations in vitro. Normal human diploid karyotype was unchanged throughout the in vitro culture period. Together, this study has exploited a method for continuous and rapid expansion of human neural stem cells as pure population, which maintained the capacity to generate almost fifty percent neurons. The availability of such cells may hold great interest for basic and applied neuroscience

Effect of cholera toxin on cAMP levels and Na+ influx in isolated intestinal epithelial cells

International Nuclear Information System (INIS)

Hyun, C.S.; Kimmich, G.A.

1982-01-01

Freshly isolated chicken intestinal cells contain approximately 20 pmol adenosine 3',5'-cyclic monophosphate (cAMP)/mg cellular protein. Incubation with 3 μg/ml cholera toxin (CT) at 37 0 C induces an elevation of cellular cAMP beginning 10-15 min after initial exposure. The response is linear with time for 40-50 min and causes a six- to eightfold increase over control levels at steady state. Dibutyryl cAMP and agents that increase cAMP production inhibit Na + influx into the isolated enterocytes. Chlorpromazine completely abolishes the toxin-induced elevation of cAMP in the isolated cells and also reverses the effect on Na + entry. The data provide evidence for a cAMP-mediated control of intestinal cell Na + uptake, which may represent the mechanistic basis for the antiabsorptive effect of CT on Na + during induction of intestinal secretory activity. Studies on the time-dependent effects of chlorpromazine on both intracellular cAMP concentration and Na + influx suggest that the reactivation of the Na + transport system after cAMP-induced inhibition is slow relative to the disappearance of cAMP

Investigations on the insulin receptor of isolated fat cells

International Nuclear Information System (INIS)

Eichler, W.

1980-01-01

Fat cells, isolated from the epididymal adipose tissue of rats, were incubed with iodine 125 insulin after previous incubation with various antagonists. By varying the antagonist concentration, it was possible to determine the effect these substances have on the insulin receptor, i.e. the insulin similarity. By varying the preincubation time, toxicity of the test substances could be detected, which pretended repression effects; and by finally verying the incubation time the effects on the receptor via the membrane could be distinguished from direct receptor bindings of the antagonist. (orig./MG) [de

Aspirin induces morphological transformation to the secretory state in isolated rabbit parietal cells.

Science.gov (United States)

Murthy, U K; Levine, R A

1991-08-01

The morphological response of rabbit parietal cells to aspirin was evaluated by grading several ultra-structural features including the extent of the tubulovesicular system, intracellular secretory canaliculi, and microvilli. After exposure of isolated parietal cells and gastric glands to aspirin or histamine, there was an approximately twofold increase in the ratio of secretory to nonsecretory parietal cells, and depletion of extracellular Ca2+ abolished the aspirin-induced morphological changes. Morphometry in parietal cells showed that aspirin induced a sixfold increase in secretory canalicular membrane elaboration. Aspirin potentiated histamine-induced parietal cell respiration and aminopyrine uptake ratio but did not increase basal respiration or aminopyrine uptake, suggesting an apparent dissociation from aspirin-induced morphological changes.

Isolation and characterization of a neoplastic epithelial cell line derived from irradiated human submaxillary gland

International Nuclear Information System (INIS)

Shirasuna, Kanemitsu; Sato, Mitsunobu; Yura, Yoshiaki; Yanagawa, Tetuo; Kubo, Kazuko

1979-01-01

Submaxillary tissues taken from a patient whose oral base was irradiated for squamous cell carcinoma were cultured in order to isolate transformed epithelial cells in vitro. The cells showed a fine structure similar to an intermediate duct cell. When they were transplanted in nude mice, salivary tumors developed. It is epidemiologically known that irradiation induces salivary tumors. In this study, the risk of inducement was revealed and a salivary epithelial cell line was used as a model for the analysis of salivary tumors. (Ichikawa, K.)

Transplantation of an alginate-matrigel matrix containing isolated ovarian stromal cells: first step in developing an artificial ovary

OpenAIRE

Vanacker, Julie; Dolmans, Marie-Madeleine; des Rieux, Anne; Jaeger, Jonathan; Luyckx, Valérie; Van Langendonckt, Anne; Donnez, Jacques; Andrade Amorim, Christiani; 3rd International Conference “Strategies in Tissue Engineering”

2012-01-01

Introduction: For women diagnosed with leukemia, transplantation of cryopreserved ovarian tissue after disease remission is not advisable due to the risk of reintroduction of malignant cells. To restore fertility in these patients, a biodegradable artificial ovary that offers an environment allowing isolated follicles and stromal cells (SCs) to survive and grow needs to be developed. The aim of this study is therefore to test the survival and proliferation of isolated SCs encapsulated in an a...

Efficient Isolation and Quantitative Proteomic Analysis of Cancer Cell Plasma Membrane Proteins for Identification of Metastasis-Associated Cell Surface Markers

DEFF Research Database (Denmark)

Lund, Rikke; Leth-Larsen, Rikke; Jensen, Ole N

2009-01-01

Cell surface membrane proteins are involved in central processes such as cell signaling, cell-cell interactions, ion and solute transport, and they seem to play a pivotal role in several steps of the metastatic process of cancer cells. The low abundance and hydrophobic nature of cell surface...... membrane proteins complicate their purification and identification by MS. We used two isogenic cell lines with opposite metastatic capabilities in nude mice to optimize cell surface membrane protein purification and to identify potential novel markers of metastatic cancer. The cell surface membrane...... proteins were isolated by centrifugation/ultracentrifugation steps, followed by membrane separation using a Percoll/sucrose density gradient. The gradient fractions containing the cell surface membrane proteins were identified by enzymatic assays. Stable isotope labeling of the proteome of the metastatic...

Comparison between dissipative snubber and passive regenerative snubber cells as applied to isolated DCM SEPIC converters

NARCIS (Netherlands)

Tibola, G.; Lemmen, E.; Duarte, J.L.

This paper presents the comparison between dissipative RCD and passive regenerative snubber cells for isolated SEPIC converters. The passive cell is intended to improve the converter’s efficiency by transferring the energy stored in the transformer leakage inductance to the converter output. The

All-in-one nanowire-decorated multifunctional membrane for rapid cell lysis and direct DNA isolation.

KAUST Repository

So, Hongyun; Lee, Kunwoo; Murthy, Niren; Pisano, Albert P

2014-01-01

This paper describes a handheld device that uses an all-in-one membrane for continuous mechanical cell lysis and rapid DNA isolation without the assistance of power sources, lysis reagents, and routine centrifugation. This nanowire-decorated multifunctional membrane was fabricated to isolate DNA by selective adsorption to silica surface immediately after disruption of nucleus membranes by ultrasharp tips of nanowires for a rapid cell lysis, and it can be directly assembled with commercial syringe filter holders. The membrane was fabricated by photoelectrochemical etching to create microchannel arrays followed by hydrothermal synthesis of nanowires and deposition of silica. The proposed membrane successfully purifies high-quality DNA within 5 min, whereas a commercial purification kit needs more than an hour.

All-in-one nanowire-decorated multifunctional membrane for rapid cell lysis and direct DNA isolation.

KAUST Repository

So, Hongyun

2014-11-24

This paper describes a handheld device that uses an all-in-one membrane for continuous mechanical cell lysis and rapid DNA isolation without the assistance of power sources, lysis reagents, and routine centrifugation. This nanowire-decorated multifunctional membrane was fabricated to isolate DNA by selective adsorption to silica surface immediately after disruption of nucleus membranes by ultrasharp tips of nanowires for a rapid cell lysis, and it can be directly assembled with commercial syringe filter holders. The membrane was fabricated by photoelectrochemical etching to create microchannel arrays followed by hydrothermal synthesis of nanowires and deposition of silica. The proposed membrane successfully purifies high-quality DNA within 5 min, whereas a commercial purification kit needs more than an hour.

Macrophage and T cell dynamics during the development and disintegration of mycobacterial granulomas.

Science.gov (United States)

Egen, Jackson G; Rothfuchs, Antonio Gigliotti; Feng, Carl G; Winter, Nathalie; Sher, Alan; Germain, Ronald N

2008-02-01

Granulomas play a key role in host protection against mycobacterial pathogens, with their breakdown contributing to exacerbated disease. To better understand the initiation and maintenance of these structures, we employed both high-resolution multiplex static imaging and intravital multiphoton microscopy of Mycobacterium bovis BCG-induced liver granulomas. We found that Kupffer cells directly capture blood-borne bacteria and subsequently nucleate formation of a nascent granuloma by recruiting both uninfected liver-resident macrophages and blood-derived monocytes. Within the mature granuloma, these myeloid cell populations formed a relatively immobile cellular matrix that interacted with a highly dynamic effector T cell population. The efficient recruitment of these T cells was highly dependent on TNF-alpha-derived signals, which also maintained the granuloma structure through preferential effects on uninfected macrophage populations. By characterizing the migration of both innate and adaptive immune cells throughout the process of granuloma development, these studies provide a new perspective on the cellular events involved in mycobacterial containment and escape.

Isolation of a hemidesmosome-rich fraction from a human squamous cell carcinoma cell line

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