scholarly journals Disease-related Metabolites in Culture Medium of Fibroblasts from Patients with d-2-Hydroxyglutaric Aciduria, l-2-Hydroxyglutaric Aciduria, and Combined d/l-2-Hydroxyglutaric Aciduria

2003 ◽  
Vol 49 (7) ◽  
pp. 1133-1138 ◽  
Author(s):  
Eduard A Struys ◽  
Nanda M Verhoeven ◽  
Birthe Roos ◽  
Cornelis Jakobs
Keyword(s):  
Cell Culture ◽  
Glutamic Acid ◽  
Cell Lines ◽  
Culture Medium ◽  
Culture Media ◽  
Control Cell ◽  
Fibroblast Cell ◽  
Gas Chromatography Mass Spectrometry ◽  
Organic Acidurias ◽  
Stable Isotope Dilution

Abstract Background: d-2-Hydroxyglutaric aciduria (D-2-HGA), l-2-hydroxyglutaric aciduria (L-2-HGA), and the combined d/l-2-hydroxyglutaric aciduria (D/L-2-HGA) are poorly understood organic acidurias. To investigate the usefulness of cultured human skin fibroblasts for both diagnostic and research purposes, we measured disease-related metabolites in the cell culture medium. Methods: We measured d-2-hydroxyglutarate (D-2-HG), l-2-hydroxyglutarate (L-2-HG), succinate, 2-ketoglutarate, and citrate in fibroblast cell medium by stable-isotope-dilution gas chromatography–mass spectrometry and glutamine, glutamic acid, and lysine with an amino acid analyzer. We used six cell lines from patients with D-2-HGA, two from patients with L-2-HGA, three from patients with D/L-2-HGA, and seven control cell lines. Culture medium was analyzed after a 96-h incubation period. Results: Culture media from cell lines from D-2-HGA patients contained D-2-HG at concentrations 5- to 30-fold higher than media from controls, whereas the concentration of L-2-HG in media was not increased. Media from L-2-HGA cell lines showed a fivefold increase in L-2-HG compared with controls. Media containing fibroblasts from D/L-2-HGA patients contained moderately increased amounts of both D-2-HG and L-2-HG. For all cell lines, succinate concentrations in the blank medium were higher than after 96 h of incubation with the exception of two of three D/L-2-HGA cell lines. Media of D-2-HGA cell lines had 2-ketoglutarate concentrations that were 40% of that for controls. Glutamic acid concentrations in media of these cell lines were 60% lower than in controls. Conclusions: Cell culture media from fibroblasts from patients with D-2-HGA, L-2-HGA, or D/L-2-HGA contain increased amounts the corresponding 2-HGs, demonstrating the suitability of fibroblasts for both diagnosis of and research concerning 2-HGAs.

scholarly journals Accumulation of Free 3-Hydroxy Fatty Acids in the Culture Media of Fibroblasts from Patients Deficient in Long-Chain l-3-Hydroxyacyl-CoA Dehydrogenase: A Useful Diagnostic Aid

2001 ◽  
Vol 47 (7) ◽  
pp. 1190-1194 ◽  
Author(s):  
Patricia M Jones ◽  
Monica Moffitt ◽  
Delanie Joseph ◽  
Pamela A Harthcock ◽  
Richard L Boriack ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Cell Lines ◽  
Culture Media ◽  
Fibroblast Cell ◽  
Hydroxy Fatty Acids ◽  
Gas Chromatography Mass Spectrometry ◽  
Selected Ion Monitoring ◽  
Cultured Fibroblasts ◽  
Stable Isotope Dilution ◽  
Long Chain

Abstract Background: The diagnosis of long-chain l-3-hydroxy-acyl-coenzyme A dehydrogenase (LCHAD) deficiency frequently requires the study of cultured fibroblasts. We developed such a test that does not require disruption and loss of the cells. Methods: We measured free 3-hydroxy fatty acids (3-OHFAs) in media of skin fibroblasts cultures from 11 patients with a genetic deficiency of LCHAD and the associated disorder of mitochondrial trifunctional protein (MTFP). Fibroblasts were cultured for 24 h with 100 μmol/L nonisotopic palmitate added. 3-OHFAs were measured by selected-ion monitoring, stable-isotope dilution gas chromatography-mass spectrometry with [13C]-labeled internal standards. Results: 3-OH-hexadecanoic and 3-OH-tetradecanoic FAs were increased 14- and 11-fold, respectively, in all patients with LCHAD or MTFP deficiency when compared with control fibroblast cell lines after overnight incubation with palmitate. 3-OH-dodecanoic FA demonstrated a modest, fivefold increase in LCHAD-deficient cells. The concentrations of all 3-OHFAs were similar whether or not the medium samples were hydrolyzed to release conjugated species such as acylcarnitines, suggesting that 3-OHFAs accumulate in the media as free FAs. Conclusions: Measurement of 3-OHFA excretion from LCHAD- or MTFP-deficient cell lines can be used as a diagnostic tool. Free FAs are the predominant form of these abnormal metabolic intermediates in culture media.

Using 3D perfusion bioreactor system to studying cell biology of ovarian cancer.

2021 ◽  
Vol 39 (15_suppl) ◽  
pp. e17558-e17558
Author(s):  
Alba Martínez ◽  
Molly Buckley ◽  
Joel Berry ◽  
Rebecca Christian Arend
Keyword(s):  
Ovarian Cancer ◽  
Cell Culture ◽  
Cell Growth ◽  
Cell Lines ◽  
Culture Media ◽  
Tumor Biology ◽  
Perfusion Bioreactor ◽  
Suitable Model ◽  
Cell Culture Media ◽  
Bioreactor System

e17558 Background: Epithelial Ovarian Cancer (EOC) is the most common cause of death among gynecological malignancies. This is a result of the high rate of recurrence and chemo-resistance in EOC patients. Therefore, the development of new therapeutics is crucial. A major factor contributing to this is the lack of therapeutic candidates is lack of translational accuracy in preclinical models. Recently, 3-dimensional (3-D) models have aided in accurately recreating tumor biology. We have developed an EOC 3-D perfused bioreactor system that recapitulates EOC tumor biology and incorporates tumor biomechanical regulation. This model allows for us to more accurately predict the clinical response of new drug candidates, which aids in elimination of ineffective candidates prior to clinical trials. Methods: EOC cell lines (luciferase-taggedSKOV-3 and OVCAR-8) were embedded in a relevant extracellular matrix (ECM) and injected into a perfused, polydimethylsiloxane (PDMS) bioreactor. Microchannels were embedded in matrigel so that the cell culture media with or without chemotherapy could flow through the perfused PDMS to provide nutrient delivery and gas exchange enhancing viability and function of surrounding cells. The bioreactors were connected to a peristaltic pump that allowed for the cell culture media to perfuse over a 7-day period. We monitored cell viability using bioluminescence imaging (BLI), immunohistochemistry (IHC), and lactate dehydrogenase (LDH) release in media. Results: BLI showed a linear increase in SKOV-3 and OVCAR-8 cell growth over 7 days. These results were confirmed by IHC measuring the number of nucleated cells per micron2. Graphical representation of the region of interest (ROI) showed a high correlation between IHC staining of nucleated cells and BLI score. IHC analysis of PAX8 staining was positive and proved that the perfusion bioreactor system maintains EOC biology over time. In addition, our results suggest that the bioreactor is a suitable model for drug preclinical testing in both cell lines as well as in patients’ samples. Conclusions: Our preliminary results using the 3D EOC perfused, PDMS bioreactor model showed increased EOC cell growth overtime, while maintaining original EOC histology. Moreover, our results suggest that this model could provide a novel platform to study therapeutic interventions in EOC. Our ultimate goal is to implement ovarian cancer microenvironment components (e.g. immune cells) into bioreactor system to study different drug treatments to better determine drug candidate’s translational efficacy.

scholarly journals Non-Mulberry and Mulberry Silk Protein Sericins as Potential Media Supplement for Animal Cell Culture

2016 ◽  
Vol 2016 ◽  
pp. 1-13 ◽  
Author(s):  
Neety Sahu ◽  
Shilpa Pal ◽  
Sunaina Sapru ◽  
Joydip Kundu ◽  
Sarmistha Talukdar ◽  
...  
Keyword(s):  
Cell Culture ◽  
Culture Medium ◽  
Culture Media ◽  
Animal Cell ◽  
Animal Cell Culture ◽  
Silk Protein ◽  
Antheraea Mylitta ◽  
Serum Free ◽  
Growth Supplement ◽  
Fibrosarcoma Cells

Silk protein sericins, in the recent years, find application in cosmetics and pharmaceuticals and as biomaterials. We investigate the potential of sericin, extracted from both mulberryBombyx moriand different non-mulberry sources, namely, tropical tasar,Antheraea mylitta; muga,Antheraea assama; and eri,Samia ricini,as growth supplement in serum-free culture medium. Sericin supplemented media containing different concentrations of sericins from the different species are examined for attachment, growth, proliferation, and morphology of fibrosarcoma cells. The optimum sericin supplementation seems to vary with the source of sericins. The results indicate that all the sericins promote the growth of L929 cells in serum-free culture media; however,S. ricinisericin seems to promote better growth of cells amongst other non-mulberry sericins.

Trichothecene and Moniliformin Production by Fusarium Species from Western Canadian Wheat†

2001 ◽  
Vol 64 (8) ◽  
pp. 1220-1225 ◽  
Author(s):  
D. ABRAMSON ◽  
R. M. CLEAR ◽  
D. GABA ◽  
D. M. SMITH ◽  
S. K. PATRICK ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Gas Chromatography ◽  
Liquid Chromatography ◽  
Culture Medium ◽  
Culture Media ◽  
Fusarium Species ◽  
Fusarium Culmorum ◽  
Fusarium Avenaceum ◽  
Gas Chromatography Mass Spectrometry ◽  
Western Canada

Fusarium graminearum, Fusarium culmorum, and Fusarium avenaceum, isolated from Fusarium-damaged wheat harvested in western Canada, were cultured and evaluated for mycotoxin production. Extracts of the culture media were assayed for trichothecenes by gas chromatography-mass spectrometry and for moniliformin by liquid chromatography. Deoxynivalenol (DON) was found in 28 of 42 isolates of F. graminearum and 42 of 42 isolates of F. culmorum at levels ranging from 0.5 to 25.0 μg/g. 15-AcetylDON was found in 28 of 42 isolates of F. graminearum at levels ranging from 1.0 to 7.1 μg/g. 3-AcetylDON was found in 41 of 42 isolates of F. culmorum at levels ranging from 0.8 to 13.0 μg/g. Several other trichothecenes were assayed but not detected in the culture medium. Moniliformin was present in 40 of 42 isolates of F. avenaceum at levels ranging from 1.3 to 138.1 μg/g, but was not present in any of the isolates of F. graminearum or F. culmorum.

scholarly journals The landscape of metabolic pathway dependencies in cancer cell lines

2021 ◽  
Vol 17 (4) ◽  
pp. e1008942
Author(s):  
James H. Joly ◽  
Brandon T. L. Chew ◽  
Nicholas A. Graham
Keyword(s):  
Cell Culture ◽  
Cancer Cells ◽  
Cell Lines ◽  
Cancer Cell ◽  
Metabolic Pathway ◽  
Culture Medium ◽  
Cancer Cell Lines ◽  
Metabolic Reprogramming ◽  
Cell Culture Medium ◽  
Loss Of Function

The metabolic reprogramming of cancer cells creates metabolic vulnerabilities that can be therapeutically targeted. However, our understanding of metabolic dependencies and the pathway crosstalk that creates these vulnerabilities in cancer cells remains incomplete. Here, by integrating gene expression data with genetic loss-of-function and pharmacological screening data from hundreds of cancer cell lines, we identified metabolic vulnerabilities at the level of pathways rather than individual genes. This approach revealed that metabolic pathway dependencies are highly context-specific such that cancer cells are vulnerable to inhibition of one metabolic pathway only when activity of another metabolic pathway is altered. Notably, we also found that the no single metabolic pathway was universally essential, suggesting that cancer cells are not invariably dependent on any metabolic pathway. In addition, we confirmed that cell culture medium is a major confounding factor for the analysis of metabolic pathway vulnerabilities. Nevertheless, we found robust associations between metabolic pathway activity and sensitivity to clinically approved drugs that were independent of cell culture medium. Lastly, we used parallel integration of pharmacological and genetic dependency data to confidently identify metabolic pathway vulnerabilities. Taken together, this study serves as a comprehensive characterization of the landscape of metabolic pathway vulnerabilities in cancer cell lines.

scholarly journals Mammalian Cell Culture Clarification: A Case Study Using Chimeric Anti-CEA Monoclonal Antibodies

2011 ◽  
Vol 12 (4) ◽  
Author(s):  
Mohamed Ali Abol Hassan ◽  
Abdul Wahab Mohammad ◽  
And Badarulhisam Abdul Rahman
Keyword(s):  
Cell Culture ◽  
Monoclonal Antibodies ◽  
Host Cell ◽  
Cell Lines ◽  
Mammalian Cell ◽  
Mammalian Cells ◽  
Culture Media ◽  
Mammalian Cell Culture ◽  
Host Cell Proteins ◽  
Depth Filtration

The extracellular expression of monoclonal antibodies (mAbs) in mammalian cell culture provides both opportunities and restrictions for the design of robust harvest and clarification operations. With advances in cell culture media and cell lines, it is now possible to achieve high titers of over 5 g/l for mAbs. However, Mammalian cells are sensitive to breakage due to shear stress that can result in release of proteases and other host cell proteins (HCPs) which eventually affects product stability and purity. There is larger number of mAbs undergoing clinical development and it has placed significant importance on platform technologies of process development. Generally, Centrifugation and microfiltration are the primary harvest techniques used in the industry and depth filtration is also used as a step operation on clarification. This study compares the unit operations; centrifugation, microfiltration and depth filtration for maximum recovery of monoclonal antibodies. The results have shown that the depth filtration as more suitable operation for mammalian cell culture clarification since it gives 96% recovery of mAbs in comparison to centrifugation and microfiltration. ABSTRAK: Pengungkapan luar sel dari antibodi monoklon (monoclonal antibodies ((mAbs) dalam kultur sel mamalia memberi ruang dan batasan terhadap reka bentuk penuaian yang cekap dan penerangan operasi. Dengan kemajuan dalam media sel kultur dan cell lines (produk yang berupa sel kekal yang digunakan untuk tujuan kajian biologi), kini adalah berkemungkinan untuk memperolehi titer tinggi melebihi 5g/l untuk mAbs [2]. Walaupun begitu, sel mamalia sensitif terhadap retakan disebabkan tegasan ricih yang menyebabkan pengeluaran protease dan hos sel protein yang lain, (host cell proteins (HCPs)) akhirnya mempengaruhi kestabilan dan keaslian produk. Terdapat mAbs dalam jumlah besar yang masih menjalani pembangunan klinikal dan sesungguhnya ini penting sebagai satu landasan teknologi dalam proses pembangunan. Umumnya pengemparan dan mikropenurasan merupakan teknik asas tuaian dalam industri dan penurasan dalam juga digunakan sebagai satu pengendalian langkah dalam penjelasannya. Kajian ini membandingkan operasi unit: pengemparan, mikropenurasan dan penurasan dalam untuk perolehan antibodi monoklon yang maksima. Keputusan menunjukkan penurasan dalam adalah operasi yang lebih sesuai untuk penjelasan kultur sel mamalia kerana ia memberikan perolehan 96 % mAbs berbandingkan dengan cara pengemparan dan mikropenurasan.

scholarly journals Selectivity of direct plasma treatment and plasma-conditioned media in bone cancer cell lines

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Inès Hamouda ◽  
Cédric Labay ◽  
Uroš Cvelbar ◽  
Maria-Pau Ginebra ◽  
Cristina Canal
Keyword(s):  
Cell Culture ◽  
Plasma Treatment ◽  
Cancer Cell ◽  
Culture Medium ◽  
Culture Media ◽  
Bone Cancer ◽  
Reactive Species ◽  
Conditioned Media ◽  
Cell Culture Medium ◽  
Cell Culture Media

AbstractAtmospheric pressure plasma jets have been shown to impact several cancer cell lines, both in vitro and in vivo. These effects are based on the biochemistry of the reactive oxygen and nitrogen species generated by plasmas in physiological liquids, referred to as plasma-conditioned liquids. Plasma-conditioned media are efficient in the generation of reactive species, inducing selective cancer cell death. However, the concentration of reactive species generated by plasma in the cell culture media of different cell types can be highly variable, complicating the ability to draw precise conclusions due to the differential sensitivity of different cells to reactive species. Here, we compared the effects of direct and indirect plasma treatment on non-malignant bone cells (hOBs and hMSCs) and bone cancer cells (SaOs-2s and MG63s) by treating the cells directly or exposing them to previously treated cell culture medium. Biological effects were correlated with the concentrations of reactive species generated in the liquid. A linear increase in reactive species in the cell culture medium was observed with increased plasma treatment time independent of the volume treated. Values up to 700 µM for H2O2 and 140 µM of NO2− were attained in 2 mL after 15 min of plasma treatment in AdvDMEM cell culture media. Selectivity towards bone cancer cells was observed after both direct and indirect plasma treatments, leading to a decrease in bone cancer cell viability at 72 h to 30% for the longest plasma treatment times while maintaining the survival of non-malignant cells. Therefore, plasma-conditioned media may represent the basis for a potentially novel non-invasive technique for bone cancer therapy.

Cells that overproduce protein kinase C are more susceptible to transformation by an activated H-ras oncogene

1989 ◽  
Vol 9 (6) ◽  
pp. 2641-2647
Author(s):  
W L Hsiao ◽  
G M Housey ◽  
M D Johnson ◽  
I B Weinstein
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Cell Lines ◽  
Cell Transformation ◽  
Synergistic Effects ◽  
Control Cell ◽  
Fold Increase ◽  
Fibroblast Cell ◽  
Ras Oncogene ◽  
Kinase C

We recently developed rat fibroblast cell lines that stably overproduce high levels of the beta 1 form of protein kinase C (PKC). These cells display several disorders in growth control and form small microscopic colonies in agar. In the present study we demonstrate that one of these cell lines, R6-PKC3, is extremely susceptible to transformation by an activated human bladder cancer c-H-ras oncogene (T24). Compared with control cell line R6-C1, T24-transfected R6-PKC3 cells yielded a 10-fold increase in the formation of large colonies in agar. Cell lines established from these colonies displayed a highly transformed morphology, expressed the T24-encoded p21 ras protein, continued to express high levels of PKC, and were highly tumorigenic in nude mice. These results provide genetic evidence that PKC mediates some of the effects of the c-H-ras oncogene on cell transformation. Data are also presented suggesting that optimum synergistic effects between c-H-ras and PKC require critical levels of their respective activities. These findings may be relevant to the process of multistage carcinogenesis in tissues containing cells with an activated c-H-ras oncogene.

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