In vitro cytocompatibility testing of oxidative degradation products

Implantable medical devices must undergo thorough evaluation to ensure safety and efficacy before use in humans. If a device is designed to degrade, it is critical to understand the rate of degradation and the degradation products that will be released. Oxidative degradation is typically modeled in vitro by immersing materials or devices in hydrogen peroxide, which can limit further analysis of degradation products in many cases. Here we demonstrate a novel approach for testing the cytocompatibility of degradation products for oxidatively-degradable biomaterials where the materials are exposed to hydrogen peroxide, and then catalase enzyme is used to convert the hydrogen peroxide to water and oxygen so that the resulting aqueous solution can be added to cell culture media. To validate our results, expected degradation products are also synthesized then added to cell culture media. We used these methods to evaluate the cytocompatibility of degradation products from an oxidatively-degradable shape memory polyurethane designed in our lab and found that the degradation of these polymers is unlikely to cause a cytotoxic response in vivo based on the guidance provided by ISO 10993-5. These methods may also be applicable to other biocompatibility tests such as tests for mutagenicity or systemic toxicity, and evaluations of cell proliferation, migration, or gene and protein expression.

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How Supraphysiological Oxygen Levels in Standard Cell Culture Affect Oxygen-Consuming Reactions

Oxidative Medicine and Cellular Longevity ◽

10.1155/2018/8238459 ◽

2018 ◽

Vol 2018 ◽

pp. 1-13 ◽

Cited By ~ 8

Author(s):

Jeffrey A. Stuart ◽

Joao Fonseca ◽

Fereshteh Moradi ◽

Cassandra Cunningham ◽

Bishoy Seliman ◽

...

Keyword(s):

Hydrogen Peroxide ◽

Cell Culture ◽

Culture Media ◽

Redox System ◽

Standard Cell ◽

Hypoxia Inducible Factors ◽

Cell Culture Media ◽

Partial Pressures

Most mammalian tissue cells experience oxygen partial pressuresin vivoequivalent to 1–6% O2(i.e., physioxia). In standard cell culture, however, headspace O2levels are usually not actively regulated and under these conditions are ~18%. This drives hyperoxia in cell culture media that can affect a wide variety of cellular activities and may compromise the ability ofin vitromodels to reproducein vivobiology. Here, we review and discuss some specific O2-consuming organelles and enzymes, including mitochondria, NADPH oxidases, the transplasma membrane redox system, nitric oxide synthases, xanthine oxidase, and monoamine oxidase with respect to their sensitivities to O2levels. Many of these produce reactive oxygen and/or nitrogen species (ROS/RNS) as either primary end products or byproducts and are acutely sensitive to O2levels in the range from 1% to 18%. Interestingly, many of them are also transcriptional targets of hypoxia-inducible factors (HIFs) and chronic cell growth at physioxia versus 18% O2may alter their expression. Aquaporins, which facilitate hydrogen peroxide diffusion into and out of cells, are also regulated by HIFs, indicating that O2levels may affect intercellular communication via hydrogen peroxide. The O2sensitivities of these important activities emphasize the importance of maintaining physioxia in culture.

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In Vitro Bioavailability of the Hydrocarbon Fractions of Dimethyl Sulfoxide Extracts of Petroleum Substances

Toxicological Sciences ◽

10.1093/toxsci/kfaa007 ◽

2020 ◽

Vol 174 (2) ◽

pp. 168-177 ◽

Cited By ~ 3

Author(s):

Yu-Syuan Luo ◽

Kyle C Ferguson ◽

Ivan Rusyn ◽

Weihsueh A Chiu

Keyword(s):

Cell Culture ◽

Protein Binding ◽

Aromatic Compounds ◽

Culture Media ◽

In Vitro Testing ◽

Cell Culture Media ◽

Chemical Profiles ◽

In Vivo Extrapolation

Abstract Determining the in vitro bioavailable concentration is a critical, yet unmet need to refine in vitro-to-in vivo extrapolation for unknown or variable composition, complex reaction product or biological material (UVCB) substances. UVCBs such as petroleum substances are commonly subjected to dimethyl sulfoxide (DMSO) extraction in order to retrieve the bioactive polycyclic aromatic compound (PAC) portion for in vitro testing. In addition to DMSO extraction, protein binding in cell culture media and dilution can all influence in vitro bioavailable concentrations of aliphatic and aromatic compounds in petroleum substances. However, these in vitro factors have not been fully characterized. In this study, we aimed to fill in these data gaps by characterizing the effects of these processes using both a defined mixture of analytical standards containing aliphatic and aromatic hydrocarbons, as well as 4 refined petroleum products as prototypical examples of UVCBs. Each substance was extracted with DMSO, and the protein binding in cell culture media was measured by using solid-phase microextraction. Semiquantitative analysis for aliphatic and aromatic compounds was achieved via gas chromatography-mass spectrometry. Our results showed that DMSO selectively extracted PACs from test substances, and that chemical profiles of PACs across molecular classes remained consistent after extraction. With respect to protein binding, chemical profiles were retained at a lower dilution (higher concentration), but a greater dilution factor (ie, lower concentration) resulted in higher protein binding in cell medium, which in turn altered the ultimate chemical profile of bioavailable PACs. Overall, this case study demonstrates that extraction procedures, protein binding in cell culture media, and dilution factors prior to in vitro testing can all contribute to determining the final bioavailable concentrations of bioactive constituents of UVCBs in vitro. Thus, in vitro-to-in vivo extrapolation for UVCBs may require greater attention to the concentration-dependent and compound-specific differences in recovery and bioavailability.

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The Influence of Micronutrients in Cell Culture: A Reflection on Viability and Genomic Stability

BioMed Research International ◽

10.1155/2013/597282 ◽

2013 ◽

Vol 2013 ◽

pp. 1-22 ◽

Cited By ~ 36

Author(s):

Ana Lúcia Vargas Arigony ◽

Iuri Marques de Oliveira ◽

Miriana Machado ◽

Diana Lilian Bordin ◽

Lothar Bergter ◽

...

Keyword(s):

Cell Culture ◽

Cultured Cells ◽

Culture Media ◽

Genomic Stability ◽

Sole Source ◽

Cell Culture Media ◽

The Media ◽

Fetal Bovine

Micronutrients, including minerals and vitamins, are indispensable to DNA metabolic pathways and thus are as important for life as macronutrients. Without the proper nutrients, genomic instability compromises homeostasis, leading to chronic diseases and certain types of cancer. Cell-culture media try to mimic thein vivoenvironment, providingin vitromodels used to infer cells' responses to different stimuli. This review summarizes and discusses studies of cell-culture supplementation with micronutrients that can increase cell viability and genomic stability, with a particular focus on previousin vitroexperiments. In these studies, the cell-culture media include certain vitamins and minerals at concentrations not equal to the physiological levels. In many common culture media, the sole source of micronutrients is fetal bovine serum (FBS), which contributes to only 5–10% of the media composition. Minimal attention has been dedicated to FBS composition, micronutrients in cell cultures as a whole, or the influence of micronutrients on the viability and genetics of cultured cells. Further studies better evaluating micronutrients' roles at a molecular level and influence on the genomic stability of cells are still needed.

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Intestinal and Hepatic Uptake of Dietary Peroxidized Lipids and Their Decomposition Products, and Their Subsequent Effects on Apolipoprotein A1 and Paraoxonase1

Antioxidants ◽

10.3390/antiox10081258 ◽

2021 ◽

Vol 10 (8) ◽

pp. 1258

Author(s):

Xueting Jiang ◽

Pragney Deme ◽

Rajat Gupta ◽

Dmitry Litvinov ◽

Kathryn Burge ◽

...

Keyword(s):

Cell Culture ◽

Culture Media ◽

Degradation Products ◽

Apolipoprotein A1 ◽

Paraoxonase 1 ◽

Pon1 Activity ◽

Atherosclerotic Cardiovascular Disease ◽

Metabolic Fate ◽

Cell Culture Media ◽

Decomposition Products

Both pro- and antiatherosclerotic effects have been ascribed to dietary peroxidized lipids. Confusion on the role of peroxidized lipids in atherosclerotic cardiovascular disease is punctuated by a lack of understanding regarding the metabolic fate and potential physiological effects of dietary peroxidized lipids and their decomposition products. This study sought to determine the metabolic fate and physiological ramifications of 13-hydroperoxyoctadecadienoic acid (13-HPODE) and 13-HODE (13-hydroxyoctadecadienoic acid) supplementation in intestinal and hepatic cell lines, as well as any effects resulting from 13-HPODE or 13-HODE degradation products. In the presence of Caco-2 cells, 13-HPODE was rapidly reduced to 13-HODE. Upon entering the cell, 13-HODE appears to undergo decomposition, followed by esterification. Moreover, 13-HPODE undergoes autodecomposition to produce aldehydes such as 9-oxononanoic acid (9-ONA). Results indicate that 9-ONA was oxidized to azelaic acid (AzA) rapidly in cell culture media, but AzA was poorly absorbed by intestinal cells and remained detectable in cell culture media for up to 18 h. An increased apolipoprotein A1 (ApoA1) secretion was observed in Caco-2 cells in the presence of 13-HPODE, 9-ONA, and AzA, whereas such induction was not observed in HepG2 cells. However, 13-HPODE treatments suppressed paraoxonase 1 (PON1) activity, suggesting the induction of ApoA1 secretion by 13-HPODE may not represent functional high-density lipoprotein (HDL) capable of reducing oxidative stress. Alternatively, AzA induced both ApoA1 secretion and PON1 activity while suppressing ApoB secretion in differentiated Caco-2 cells but not in HepG2. These results suggest oxidation of 9-ONA to AzA might be an important phenomenon, resulting in the accumulation of potentially beneficial dietary peroxidized lipid-derived aldehydes.

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Formation of hydrogen peroxide in cell culture media by apple polyphenols and its effect on antioxidant biomarkers in the colon cell line HT-29

Molecular Nutrition & Food Research ◽

10.1002/mnfr.200800456 ◽

2009 ◽

Vol 53 (10) ◽

pp. 1226-1236 ◽

Cited By ~ 38

Author(s):

Phillip Bellion ◽

Melanie Olk ◽

Frank Will ◽

Helmut Dietrich ◽

Matthias Baum ◽

...

Keyword(s):

Hydrogen Peroxide ◽

Cell Culture ◽

Cell Line ◽

Culture Media ◽

Cell Culture Media ◽

Colon Cell ◽

Colon Cell Line ◽

Ht 29

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Impact of serum in cell culture media on in vitro lactate dehydrogenase (LDH) release determination

Journal of Cellular Biotechnology ◽

10.3233/jcb-179002 ◽

2017 ◽

Vol 3 (1) ◽

pp. 9-13 ◽

Cited By ~ 3

Author(s):

Bernhard Hiebl ◽

Sinem Peters ◽

Ole Gemeinhardt ◽

Stefan M. Niehues ◽

Friedrich Jung

Keyword(s):

Cell Culture ◽

Lactate Dehydrogenase ◽

Culture Media ◽

Cell Culture Media ◽

Ldh Release

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Protein tyrosine phosphatase CD45 as a molecular biosensor of hydrogen peroxide generation in cell culture media

Biochemical and Biophysical Research Communications ◽

10.1016/j.bbrc.2011.10.033 ◽

2011 ◽

Vol 415 (2) ◽

pp. 270-273 ◽

Cited By ~ 3

Author(s):

Alicja Kuban-Jankowska ◽

Narcyz Knap ◽

Magdalena Gorska ◽

Urszula Popowska ◽

Michal Wozniak

Keyword(s):

Hydrogen Peroxide ◽

Cell Culture ◽

Protein Tyrosine Phosphatase ◽

Culture Media ◽

Tyrosine Phosphatase ◽

Cell Culture Media ◽

Protein Tyrosine ◽

Hydrogen Peroxide Generation

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Secretion of glutathione S-transferase isoforms in the seminiferous tubular fluid, tissue distribution and sex steroid binding by rat GSTM1

Biochemical Journal ◽

10.1042/bj3400309 ◽

1999 ◽

Vol 340 (1) ◽

pp. 309-320 ◽

Cited By ~ 11

Author(s):

Sikha Bettina MUKHERJEE ◽

S. ARAVINDA ◽

B. GOPALAKRISHNAN ◽

Sushma NAGPAL ◽

Dinakar M. SALUNKE ◽

...

Keyword(s):

Cell Culture ◽

Germ Cell ◽

Sertoli Cell ◽

Sertoli Cells ◽

Culture Media ◽

Glutathione S Transferase ◽

Steroid Binding ◽

Spent Media

The seminiferous tubular fluid (STF) provides the microenvironment necessary for spermatogenesis in the adluminal compartment of the seminiferous tubule (ST), primarily through secretions of the Sertoli cell. Earlier studies from this laboratory demonstrated the presence of glutathione S-transferase (GST) in STF collected from adult rat testis and in the spent media of ST cultures. This study describes the cellular source, isoform composition and possible function of GSTs in the STF. The major GST isoforms present in STF in vivo share extensive N-terminal similarity with rat GSTM1 (rGSTM1), rGSTM2, rGSTM3 and rGST-Alpha. Molecular masses of rGSTM2, rGSTM3 and rGST-Alpha from liver and testis sources were similar, unlike STF-GSTM1, which was larger by 325 Da than its liver counterpart. Peptide digest analysis profiles on reverse-phase HPLC between liver and STF isoforms were identical, and N-terminal sequences of selected peptides obtained by digestion of the various isoforms were closely similar. The above results confirmed close structural similarity between liver and STF-GST isoforms. Active synthesis and secretion of GSTs by the STs were evident from recovery of radiolabelled GST from the spent media of ST cultures. Analysis of secreted GST isoforms showed that GST-Alpha was not secreted by the STs in vitro, whereas there was an induction of GST-Pi secretion. Detection of immunostainable GST-Mu in Sertoli cells in vitro and during different stages of the seminiferous epithelium in vivo, coupled with the recovery of radiolabelled GST from Sertoli cell-culture media, provided evidence for Sertoli cells as secretors of GST. In addition, STF of ‘Sertoli cell only’ animals showed no change in the profile of GST isoform secretion, thereby confirming Sertoli cells as prime GST secretors. Non-recovery of [35S]methionine-labelled GSTs from germ cell culture supernatants, but their presence in germ cell lysates, confirm the ability of the germ cells to synthesize, but not to release, GSTs. Functionally, STF-GSTM1 appeared to serve as a steroid-binding protein by its ability to bind to testosterone and oestradiol, two important hormones in the ST that are essential for spermatogenesis, with binding constants of < 9.8×10-7 M for testosterone and 9×10-6 M for oestradiol respectively.

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Improving the metabolic fidelity of cancer models with a physiological cell culture medium

Science Advances ◽

10.1126/sciadv.aau7314 ◽

2019 ◽

Vol 5 (1) ◽

pp. eaau7314 ◽

Cited By ~ 64

Author(s):

Johan Vande Voorde ◽

Tobias Ackermann ◽

Nadja Pfetzer ◽

David Sumpton ◽

Gillian Mackay ◽

...

Keyword(s):

Cell Culture ◽

Cancer Cells ◽

Culture Media ◽

Hypoxia Inducible Factor ◽

Commercial Media ◽

Physiological Medium ◽

Cancer Models ◽

Cancer Spheroids

Currently available cell culture media may not reproduce the in vivo metabolic environment of tumors. To demonstrate this, we compared the effects of a new physiological medium, Plasmax, with commercial media. We prove that the disproportionate nutrient composition of commercial media imposes metabolic artifacts on cancer cells. Their supraphysiological concentrations of pyruvate stabilize hypoxia-inducible factor 1α in normoxia, thereby inducing a pseudohypoxic transcriptional program. In addition, their arginine concentrations reverse the urea cycle reaction catalyzed by argininosuccinate lyase, an effect not observed in vivo, and prevented by Plasmax in vitro. The capacity of cancer cells to form colonies in commercial media was impaired by lipid peroxidation and ferroptosis and was rescued by selenium present in Plasmax. Last, an untargeted metabolic comparison revealed that breast cancer spheroids grown in Plasmax approximate the metabolic profile of mammary tumors better. In conclusion, a physiological medium improves the metabolic fidelity and biological relevance of in vitro cancer models.

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