scholarly journals Environmental cystine drives glutamine anaplerosis and sensitizes cells to glutaminase inhibition

2017 ◽  
Author(s):  
Alexander Muir ◽  
Laura V. Danai ◽  
Dan Y. Gui ◽  
Chiara Y. Waingarten ◽  
Matthew G. Vander Heiden
Keyword(s):  
Cell Culture ◽  
Tissue Culture ◽  
Bovine Serum ◽  
Tca Cycle ◽  
Culture Conditions ◽  
Adult Bovine Serum ◽  
Necessary And Sufficient ◽  
Cells Cultured

AbstractMany cancer cell lines depend on extracellular glutamine as a major tri-carboxylic acid (TCA) cycle anaplerotic substrate to support proliferationin vitro. However, recent studies have suggested that some cells that depend on glutamine anaplerosis in culture rely much less on glutamine catabolism to proliferatein vivo, with environmental differences between tumors and cell culture influencing the extent of glutamine catabolism. Here we sought to better understand the environmental differences that cause differential dependence on glutamine for TCA cycle anaplerosis. We find that cells cultured in adult bovine serum, a condition that more closely reflects the nutrients available to cellsin vivo, leads to decreased glutamine catabolism and reliance on glutamine anaplerosis compared to standard tissue culture conditions. By analyzing the nutrient differences between bovine serum and media, we find that levels of a single nutrient, cystine, can account for the differential dependence on glutamine in these different environmental contexts. Further, we show that cystine levels dictate glutamine dependence via the cystine/glutamate antiporter xCT/SLC7A11, and that environmental cystine levels in conjunction withxCT/SLC7A11expression is necessary and sufficient to drive increased glutamine anaplerosis, defining important determinants of glutamine anaplerosis and glutaminase dependence in cancer cells.

scholarly journals Environmental cystine drives glutamine anaplerosis and sensitizes cancer cells to glutaminase inhibition

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Alexander Muir ◽  
Laura V Danai ◽  
Dan Y Gui ◽  
Chiara Y Waingarten ◽  
Caroline A Lewis ◽  
...  
Keyword(s):  
Tissue Culture ◽  
Carboxylic Acid ◽  
Cancer Cells ◽  
Cell Lines ◽  
Tca Cycle ◽  
Culture Conditions ◽  
Adult Bovine Serum ◽  
Necessary And Sufficient ◽  
Cells Cultured

Many mammalian cancer cell lines depend on glutamine as a major tri-carboxylic acid (TCA) cycle anaplerotic substrate to support proliferation. However, some cell lines that depend on glutamine anaplerosis in culture rely less on glutamine catabolism to proliferate in vivo. We sought to understand the environmental differences that cause differential dependence on glutamine for anaplerosis. We find that cells cultured in adult bovine serum, which better reflects nutrients available to cells in vivo, exhibit decreased glutamine catabolism and reduced reliance on glutamine anaplerosis compared to cells cultured in standard tissue culture conditions. We find that levels of a single nutrient, cystine, accounts for the differential dependence on glutamine in these different environmental contexts. Further, we show that cystine levels dictate glutamine dependence via the cystine/glutamate antiporter xCT/SLC7A11. Thus, xCT/SLC7A11 expression, in conjunction with environmental cystine, is necessary and sufficient to increase glutamine catabolism, defining important determinants of glutamine anaplerosis and glutaminase dependence in cancer.

scholarly journals Enrichment of Melanoma Stem-Like Cells via Sphere Assays

2021 ◽  
pp. 185-199
Author(s):  
Nabanita Mukherjee ◽  
Karoline A. Lambert ◽  
David A. Norris ◽  
Yiqun G. Shellman
Keyword(s):  
Cell Culture ◽  
Tissue Culture ◽  
Stem Cell ◽  
Three Dimensional ◽  
Culture Conditions ◽  
Low Density ◽  
In Vitro Techniques ◽  
Sphere Culture

AbstractSphere assays are widely used in vitro techniques to enrich and evaluate the stem-like cell behavior of both normal and cancer cells. Utilizing three-dimensional in vitro sphere culture conditions provide a better representation of tumor growth in vivo than the more common monolayer cultures. We describe how to perform primary and secondary sphere assays, used for the enrichment and self-renewability studies of melanoma/melanocyte stem-like cells. Spheres are generated by growing melanoma cells at low density in nonadherent conditions with stem cell media. We provide protocols for preparing inexpensive and versatile polyHEMA-coated plates, setting up primary and secondary sphere assays in almost any tissue culture format and quantification methods using standard inverted microscopy. Our protocol is easily adaptable to laboratories with basic cell culture capabilities, without the need for expensive fluidic instruments.

scholarly journals Self-organization and culture of Mesenchymal Stem Cell spheroids in acoustic levitation

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Nathan Jeger-Madiot ◽  
Lousineh Arakelian ◽  
Niclas Setterblad ◽  
Patrick Bruneval ◽  
Mauricio Hoyos ◽  
...  
Keyword(s):  
Cell Culture ◽  
3D Cell Culture ◽  
Culture Conditions ◽  
Acoustic Levitation ◽  
Cell Sheets ◽  
Cell Therapies ◽  
Cellular Models ◽  
Cell Spheroids

AbstractIn recent years, 3D cell culture models such as spheroid or organoid technologies have known important developments. Many studies have shown that 3D cultures exhibit better biomimetic properties compared to 2D cultures. These properties are important for in-vitro modeling systems, as well as for in-vivo cell therapies and tissue engineering approaches. A reliable use of 3D cellular models still requires standardized protocols with well-controlled and reproducible parameters. To address this challenge, a robust and scaffold-free approach is proposed, which relies on multi-trap acoustic levitation. This technology is successfully applied to Mesenchymal Stem Cells (MSCs) maintained in acoustic levitation over a 24-h period. During the culture, MSCs spontaneously self-organized from cell sheets to cell spheroids with a characteristic time of about 10 h. Each acoustofluidic chip could contain up to 30 spheroids in acoustic levitation and four chips could be ran in parallel, leading to the production of 120 spheroids per experiment. Various biological characterizations showed that the cells inside the spheroids were viable, maintained the expression of their cell surface markers and had a higher differentiation capacity compared to standard 2D culture conditions. These results open the path to long-time cell culture in acoustic levitation of cell sheets or spheroids for any type of cells.

scholarly journals Albumin Enhances Caspofungin Activity against Aspergillus Species by Facilitating Drug Delivery to Germinating Hyphae

2015 ◽  
Vol 60 (3) ◽  
pp. 1226-1233 ◽  
Author(s):  
Petros Ioannou ◽  
Aggeliki Andrianaki ◽  
Tonia Akoumianaki ◽  
Irene Kyrmizi ◽  
Nathaniel Albert ◽  
...  
Keyword(s):  
Drug Delivery ◽  
Cell Culture ◽  
Antifungal Agents ◽  
Fold Increase ◽  
Culture Conditions ◽  
The Other ◽  
Related Factors ◽  
Content Type

The modestin vitroactivity of echinocandins againstAspergillusimplies that host-related factors augment the action of these antifungal agentsin vivo. We found that, in contrast to the other antifungal agents (voriconazole, amphotericin B) tested, caspofungin exhibited a profound increase in activity against variousAspergillusspecies under conditions of cell culture growth, as evidenced by a ≥4-fold decrease in minimum effective concentrations (MECs) (P= 0. 0005). Importantly, the enhanced activity of caspofungin againstAspergillusspp. under cell culture conditions was strictly dependent on serum albumin and was not observed with the other two echinocandins, micafungin and anidulafungin. Of interest, fluorescently labeled albumin bound preferentially on the surface of germinatingAspergillushyphae, and this interaction was further enhanced upon treatment with caspofungin. In addition, supplementation of cell culture medium with albumin resulted in a significant, 5-fold increase in association of fluorescently labeled caspofungin withAspergillushyphae (P< 0.0001). Collectively, we found a novel synergistic interaction between albumin and caspofungin, with albumin acting as a potential carrier molecule to facilitate antifungal drug delivery toAspergillushyphae.

The regenerative capacity of the notochordal cell: tissue constructs generated in vitro under hypoxic conditions

2009 ◽  
Vol 10 (6) ◽  
pp. 513-521 ◽  
Author(s):  
W. Mark Erwin ◽  
Facundo Las Heras ◽  
Diana Islam ◽  
Michael G. Fehlings ◽  
Robert D. Inman
Keyword(s):  
Extracellular Matrix ◽  
Tissue Culture ◽  
Sodium Alginate ◽  
Cultured Cells ◽  
3D Culture ◽  
Culture Conditions ◽  
Notochordal Cells ◽  
Collagen Ii

Object The intervertebral disc (IVD) is a highly avascular structure that is occupied by highly specialized cells (nucleus pulposus [NP] cells) that have adapted to survive within an O2 concentration of 2–5%. The object of this study was to investigate the effects of long-term hypoxic and normoxic tissue cultures of nonchondrodystrophic canine notochordal cells—cells that appear to protect the disc NP from degenerative change. Methods The authors obtained notochordal cells from nonchondrodystrophic canines according to their established methods and placed them into monolayer and 3D culture using sodium alginate globules under either hypoxic (3.5% O2) or normoxic (21% O2) conditions. Histological, immunohistochemical, scanning electron microscopy, and histomorphometric methods were used to evaluate the cells within the globules after 5 months in culture. Results Notochordal cells under in vitro hypoxic tissue culture conditions produced a highly complex, organized, 3D cellular construct that was strikingly similar to that observed in vivo. In contrast, traditional normoxic tissue culture conditions resulted in notochordal cells that failed to produce an organized matrix. Hypoxia resulted in a matrix rich in aggrecan and collagen II, whereas normoxic cultured cells did not produce any observable aggrecan or collagen II after 5 months of culture. Conclusions Hypoxia induces notochordal cells to organize a complex 3D cellular/extracellular matrix without an external scaffold other than suspension within sodium alginate. These cells produce an extracellular matrix and large construct that shares exactly the same characteristics as the in vivo condition—robust aggrecan, and type II collagen production. Normoxic tissue culture conditions, however, lead to a failure of these cells to thrive and a lack of extracellular matrix production and significantly smaller cells. The authors suggest that future studies of NP cells and, in particular, notochordal cells should utilize hypoxic tissue culture conditions to derive meaningful, biologically relevant conclusions concerning possible biological/molecular interventions.

scholarly journals Changes in the number of chick ciliary ganglion neuron processes with time in cell culture.

1987 ◽  
Vol 104 (2) ◽  
pp. 363-370 ◽  
Author(s):  
L W Role ◽  
G D Fischbach
Keyword(s):  
Cell Culture ◽  
Time Course ◽  
Culture Conditions ◽  
Ciliary Ganglion ◽  
Intact Cells ◽  
Dissociated Cell Culture ◽  
Ciliary Ganglion Neurons ◽  
Ganglion Neurons

The purpose of this study was to describe the shape of chick ciliary ganglion neurons dissociated from embryonic day 8 or 9 ganglia and maintained in vitro. Most of the neurons were multipolar during the first three days after plating, with an average of 6.0 processes extending directly from the cell body. The neurons became unipolar with time. The remaining primary process accounted for greater than 90% of the total neuritic arbor. This striking change in morphology was not due to the selective loss of multipolar cells, or to an obvious decline in the health of apparently intact cells. The retraction of processes was neither prevented nor promoted by the presence of embryonic muscle cells. Process pruning occurred to the same extent and over the same time course whether the cells were plated on a monolayer of embryonic myotubes or on a layer of lysed fibroblasts. Process retraction is not an inevitable consequence of our culture conditions. Motoneurons dissociated from embryonic spinal cords remained multipolar over the same period of time. We conclude that ciliary ganglion neurons breed true in dissociated cell culture in that the multipolar-unipolar transition reflects their normal, in vivo, developmental program.

A 3 Dimensional in Vitro Model to Test HL-60 Cell Line Sensitivity to Chemotherapy

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4882-4882
Author(s):  
Omar S. Aljitawi ◽  
Dandan Li ◽  
Da Zhang ◽  
Jonathan Mahnken ◽  
Suman Kambhampati ◽  
...  
Keyword(s):  
Cell Culture ◽  
Bone Marrow ◽  
3D Culture ◽  
Culture Conditions ◽  
Cytotoxic Chemotherapy ◽  
3 Dimensional ◽  
3D Microenvironment ◽  
3D Culture System

Abstract Abstract 4882 Introduction: Current in vitro drug testing models are based on 2-dimensional (2D) cell culture systems and therefore do not always predict in vivo responses. This lack of predictability of the 2D assays is believed to be related to the 3-dimensional (3D) microenvironment present in tissues or tumors. This 3D microenvironment, were cell-cell and cell-extracellular matrix (ECM) interactions occur, is fundamental for cell biologic activities. This is especially true for acute myeloid leukemia, were current 2-D cell culture models do not always predict clinical responses. This discrepancy in leukemia cell responses to chemotherapy in vivo, in comparison to in vitro, is at least partly related to leukemia cells interaction with the bone marrow microenvironment and their ability to establish niches. These niches offer partial protection from the effects of cytotoxic chemotherapy, otherwise termed cell adhesion-mediated drug resistance. In these experiments, we investigate the apoptotic effects of cytotoxic chemotherapy on HL-60 cell line cultured in a designed 3D AML cell culture model. In this 3D microenvironment, HL-60 cells were co-cultured with ex vivo expanded bone marrow mesenchyaml stem cells in a 3D synthetic scaffold. Aim: To examine the apoptotic effect of cytotoxic chemotherapy on HL-60 co-cultured with human bone marrow mesenchymal stem cells (huBM-MSCs) in 3D conditions. Methods: After several passages, expanded huBM-MSCs were seeded into PGA/PLLA 90/10 copolymer discs, 5-mm in diameter and 2-mm in thickness and allowed to attach to scaffold fibers and to expand over 2 weeks. Then, HL-60 were added and allowed to grow in the 3D culture system for another 10 days. HL-60 cells in 3D culture system were then exposed to doxorubicin given in two concentrations (25 and 50 μM) and incubated for 24 hours. HL-60 were then retrieved applying a combination of mechanical forces and using cell dissociation solution. FITC Annexin V Apoptosis Detection Kit was used to determine apoptosis. Apoptosis was confirmed by TUNEL assay. Proliferation of HL-60 cells in the 3D scaffold was assessed using Ki-67 stain of scaffold's cryosections. All tests were done in triplicates, and untreated HL-60 served as controls for treatment. Comparison was made with HL-60 cells alone and with HL-60 cells growing on a hu-BM-MSC monolayer. SAS version 9.2 (SAS Institute, Inc., 2002–2008) was used for statistical analysis Results: Virtually, all HL-60 cells treated with 25 or 50 μM underwent late apoptosis. Around.03% of HL-60 cells survived 25 μM concentration, none, however, survived 50 μM concentration. In 2D, most of HL-60 cells underwent necrosis, and to lesser extent late apoptosis. In sharp contrast, 17.8% of HL-60 cells survived 25μM concentration, nevertheless, only.27% of HL-60 cells treated with 50 μM concentration survived. The differences in apoptosis patterns between the three groups was statistically significant (P<.0001). Conclusion: compared to traditional cell culture conditions, the designed 3D culture conditions protected a higher percentage of HL-60 cells from undergoing apoptosis and necrosis. Disclosures: No relevant conflicts of interest to declare.

scholarly journals MICROPROPOGATION AND ANALYSIS OF PHYTOCHEMICAL PROFILE OF TISSUE CULTURE GROWN PLANTAGO OVATA FORSK.

2017 ◽  
Vol 10 (4) ◽  
pp. 202 ◽  
Author(s):  
Mamta Sharma ◽  
Amita Kumari ◽  
Eshita Mahant
Keyword(s):  
Tissue Culture ◽  
Ecological Factors ◽  
Culture Method ◽  
Culture Conditions ◽  
Culture Technique ◽  
Plantago Ovata ◽  
Tissue Culture Technique ◽  
Grown Plant

Objectives : Plantago ovata is an important medicinal plant of Himalayan region greatly used in herbal dugs manufacturing. The plant is multipurpose and strictly present in the Himalaya. Plantago has many medicinal properties such as antioxidant, anti-inflammatory and hematopoiesis effects and protects the liver and is used for the treatment of cancer. The plant being medicinal possesses complex phytochemicals. The investigation of various Plantago organ (leaves, stem etc) revealed their high potential to produce a wide array of bioactive secondary metabolites. In present study the a new method of micropropagation through tissue culture  was developed for Plantago so as to meet the future demand of plant. Futher a morphological and physiochemical comparison of tissue culture grown plant was done with in vivo grown plants.Methods:  Plantago ovata was grown in -vitro through tissue culture technique using MS media and in-vivo in the nursery area of Shoolini University. In vitro culture of  Plantago ovata forsk. were managed to restrict the ecological factors and to control the culture conditions. Experimental culture parameter including germination and phytochemical constituents of Plantago ovata in vivo and in vitro conditions were observed.Results: The result revealed changes in the concentration of phytochemical constituent’s in tissue culture grown Plantago. Phytochemicals constituents (carbohydrate, tannin, chlorophyll, saponin) was reduced in tissue culture grown plant where as some phytochemicals (phenol, alkaloid, flavanoid, protein, phytosterol) increased in tissue culture grown plant than in vivo plant.  A reduction in morphological trait was found in tissue cultured plant.Conclusion: The developed tissue culture method for the micropropagation of  Plantago ovata can be used as milestone to meet the industrial need in near future.Keywords: Plantago, Tissue Culture Technique, germination, phytochemicals.

scholarly journals Multiscale modelling of drug transport and metabolism in liver spheroids

2020 ◽  
Vol 10 (2) ◽  
pp. 20190041 ◽  
Author(s):  
Joseph A. Leedale ◽  
Jonathan A. Kyffin ◽  
Amy L. Harding ◽  
Helen E. Colley ◽  
Craig Murdoch ◽  
...  
Keyword(s):  
Drug Transport ◽  
Temporal Dynamics ◽  
Three Dimensional ◽  
Culture Conditions ◽  
Isolated Cells ◽  
Modelling Framework ◽  
The Impact ◽  
Cells Cultured

In early preclinical drug development, potential candidates are tested in the laboratory using isolated cells. These in vitro experiments traditionally involve cells cultured in a two-dimensional monolayer environment. However, cells cultured in three-dimensional spheroid systems have been shown to more closely resemble the functionality and morphology of cells in vivo . While the increasing usage of hepatic spheroid cultures allows for more relevant experimentation in a more realistic biological environment, the underlying physical processes of drug transport, uptake and metabolism contributing to the spatial distribution of drugs in these spheroids remain poorly understood. The development of a multiscale mathematical modelling framework describing the spatio-temporal dynamics of drugs in multicellular environments enables mechanistic insight into the behaviour of these systems. Here, our analysis of cell membrane permeation and porosity throughout the spheroid reveals the impact of these properties on drug penetration, with maximal disparity between zonal metabolism rates occurring for drugs of intermediate lipophilicity. Our research shows how mathematical models can be used to simulate the activity and transport of drugs in hepatic spheroids and in principle any organoid, with the ultimate aim of better informing experimentalists on how to regulate dosing and culture conditions to more effectively optimize drug delivery.

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