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

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.

Download Full-text

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.

Download Full-text

Application of Three-dimensional (3D) Tumor Cell Culture Systems and Mechanism of Drug Resistance

Current Pharmaceutical Design ◽

10.2174/1381612825666191014163923 ◽

2019 ◽

Vol 25 (34) ◽

pp. 3599-3607 ◽

Cited By ~ 3

Author(s):

Adeeb Shehzad ◽

Vijaya Ravinayagam ◽

Hamad AlRumaih ◽

Meneerah Aljafary ◽

Dana Almohazey ◽

...

Keyword(s):

Cell Culture ◽

Cancer Cells ◽

Anticancer Drugs ◽

Three Dimensional ◽

3D Culture ◽

3D Cell Culture ◽

Cancer Therapeutics ◽

In Vivo Model

: The in-vitro experimental model for the development of cancer therapeutics has always been challenging. Recently, the scientific revolution has improved cell culturing techniques by applying three dimensional (3D) culture system, which provides a similar physiologically relevant in-vivo model for studying various diseases including cancer. In particular, cancer cells exhibiting in-vivo behavior in a model of 3D cell culture is a more accurate cell culture model to test the effectiveness of anticancer drugs or characterization of cancer cells in comparison with two dimensional (2D) monolayer. This study underpins various factors that cause resistance to anticancer drugs in forms of spheroids in 3D in-vitro cell culture and also outlines key challenges and possible solutions for the future development of these systems.

Download Full-text

Morphological Changes in Astrocytes by Self-Oxidation of Dopamine to Polydopamine and Quantification of Dopamine through Multivariate Regression Analysis of Polydopamine Images

Polymers ◽

10.3390/polym12112483 ◽

2020 ◽

Vol 12 (11) ◽

pp. 2483

Author(s):

Anik Karan ◽

Elnaz Khezerlou ◽

Farnaz Rezaei ◽

Leon Iasemidis ◽

Mark A. DeCoster

Keyword(s):

Cell Culture ◽

Regression Analysis ◽

Multivariate Regression ◽

Culture Media ◽

Morphological Changes ◽

Multivariate Regression Analysis ◽

Cell Number ◽

Polymerization Process

Astrocytes, also known as astroglia, are important cells for the structural support of neurons as well as for biochemical balance in the central nervous system (CNS). In this study, the polymerization of dopamine (DA) to polydopamine (PDA) and its effect on astrocytes was investigated. The polymerization of DA, being directly proportional to the DA concentration, raises the prospect of detecting DA concentration from PDA optically using image-processing techniques. It was found here that DA, a naturally occurring neurotransmitter, significantly altered astrocyte cell number, morphology, and metabolism, compared to astrocytes in the absence of DA. Along with these effects on astrocytes, the polymerization of DA to PDA was tracked optically in the same cell culture wells. This polymerization process led to a unique methodology based on multivariate regression analysis that quantified the concentration of DA from optical images of astrocyte cell culture media. Therefore, this developed methodology, combined with conventional imaging equipment, could be used in place of high-end and expensive analytical chemistry instruments, such as spectrophotometry, mass spectrometry, and fluorescence techniques, for quantification of the concentration of DA after polymerization to PDA under in vitro and potentially in vivo conditions.

Download Full-text

In vitro and in vivo inhibitory effects of a Pleurotus eryngii protein on colon cancer cells

Food & Function ◽

10.1039/c7fo00895c ◽

2017 ◽

Vol 8 (10) ◽

pp. 3553-3562 ◽

Cited By ~ 8

Author(s):

Biao Yuan ◽

Ning Ma ◽

Liyan Zhao ◽

Ermin Zhao ◽

Zili Gao ◽

...

Keyword(s):

Cell Culture ◽

Colon Cancer ◽

Cancer Cells ◽

Pleurotus Eryngii ◽

Colon Cancer Cells ◽

Inhibitory Effects ◽

Tumor Models

The inhibitory effects of a protein isolated from Pleurotus eryngii were demonstrated in both cell culture and mouse allograft tumor models.

Download Full-text

Three-Dimensional Culture System of Cancer Cells Combined with Biomaterials for Drug Screening

Cancers ◽

10.3390/cancers12102754 ◽

2020 ◽

Vol 12 (10) ◽

pp. 2754 ◽

Cited By ~ 2

Author(s):

Teruki Nii ◽

Kimiko Makino ◽

Yasuhiko Tabata

Keyword(s):

Cell Culture ◽

Cancer Cells ◽

Drug Screening ◽

Culture System ◽

Three Dimensional ◽

3D Culture ◽

New Drugs ◽

Biological Research

Anticancer drug screening is one of the most important research and development processes to develop new drugs for cancer treatment. However, there is a problem resulting in gaps between the in vitro drug screening and preclinical or clinical study. This is mainly because the condition of cancer cell culture is quite different from that in vivo. As a trial to mimic the in vivo cancer environment, there has been some research on a three-dimensional (3D) culture system by making use of biomaterials. The 3D culture technologies enable us to give cancer cells an in vitro environment close to the in vivo condition. Cancer cells modified to replicate the in vivo cancer environment will promote the biological research or drug discovery of cancers. This review introduces the in vitro research of 3D cell culture systems with biomaterials in addition to a brief summary of the cancer environment.

Download Full-text

Mint3 depletion restricts tumor malignancy of pancreatic cancer cells by decreasing SKP2 expression via HIF-1

Oncogene ◽

10.1038/s41388-020-01423-8 ◽

2020 ◽

Vol 39 (39) ◽

pp. 6218-6230 ◽

Cited By ~ 2

Author(s):

Akane Kanamori ◽

Daisuke Matsubara ◽

Yurika Saitoh ◽

Yuya Fukui ◽

Noriko Gotoh ◽

...

Keyword(s):

Pancreatic Cancer ◽

Cancer Cells ◽

Epithelial Mesenchymal Transition ◽

Hypoxia Inducible Factor ◽

Human Pancreatic Cancer ◽

Mesenchymal Transition ◽

Skp2 Expression ◽

Pancreatic Cancer Cells

Abstract Pancreatic cancer is one of the most fatal cancers without druggable molecular targets. Hypoxia inducible factor-1 (HIF-1) is a heterodimeric transcriptional factor that promotes malignancy in various cancers including pancreatic cancer. Herein, we found that HIF-1 is accumulated in normoxic or moderate hypoxic areas of pancreatic cancer xenografts in vivo and is active even during normoxia in pancreatic cancer cells in vitro. This prompted us to analyze whether the HIF-1 activator Mint3 contributes to malignant features of pancreatic cancer. Mint3 depletion by shRNAs attenuated HIF-1 activity during normoxia and cell proliferation concomitantly with accumulated p21 and p27 protein in pancreatic cancer cells. Further analyses revealed that Mint3 increased transcription of the oncogenic ubiquitin ligase SKP2 in pancreatic cancer cells via HIF-1. This Mint3-HIF-1-SKP2 axis also promoted partial epithelial-mesenchymal transition, stemness features, and chemoresistance in pancreatic cancer cells. Even in vivo, Mint3 depletion attenuated tumor growth of orthotopically inoculated human pancreatic cancer AsPC-1 cells. Database and tissue microarray analyses showed that Mint3 expression is correlated with SKP2 expression in human pancreatic cancer specimens and high Mint3 expression is correlated with poor prognosis of pancreatic cancer patients. Thus, targeting Mint3 may be useful for attenuating the malignant features of pancreatic cancer.

Download Full-text

The Antitumor Effects of Plasma-Activated Saline on Muscle-Invasive Bladder Cancer Cells In Vitro and In Vivo Demonstrate Its Feasibility as a Potential Therapeutic Approach

Cancers ◽

10.3390/cancers13051042 ◽

2021 ◽

Vol 13 (5) ◽

pp. 1042

Author(s):

Hao Zhang ◽

Jishen Zhang ◽

Bo Guo ◽

Hailan Chen ◽

Dehui Xu ◽

...

Keyword(s):

Bladder Cancer ◽

Cancer Cells ◽

Culture Media ◽

Invasive Bladder Cancer ◽

Antitumor Effects ◽

Muscle Invasive Bladder Cancer ◽

Plasma Irradiation ◽

Muscle Invasive

Muscle-invasive bladder cancer (MIBC) is a fast-growing and aggressive malignant tumor in urinary system. Since chemotherapy and immunotherapy are only useable with a few MIBC patients, the clinical treatment of MIBC still faces challenges. Here, we examined the feasibility of plasma-activated saline (PAS) as a fledgling therapeutic strategy for MIBC treatment. Our data showed that plasma irradiation could generate a variety of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in saline. In vivo tests revealed that pericarcinomatous tissue injection with PAS was effective at preventing subcutaneous bladder tumor growth, with no side effects to the visceral organs after long-term administration, as well as having no obvious influence on the various biochemistry indices of the blood in mice. The in vitro studies indicated that adding 30% PAS in cell culture media causes oxidative damage to the bladder transitional cells T24 and J82 through enhancing the intracellular ROS level, and eventually induces cancer cells’ apoptosis by activating the ROS-mediated Fas/CD95 pathway. Therefore, for an intracavity tumor, these initial observations suggest that the soaking of the tumor tissue with PAS by intravesical perfusion may be a novel treatment option for bladder cancer.

Download Full-text

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.

Download Full-text

In vitro cytocompatibility testing of oxidative degradation products

Journal of Bioactive and Compatible Polymers ◽

10.1177/08839115211003115 ◽

2021 ◽

pp. 088391152110031

Author(s):

Scott M Herting ◽

Mary Beth B Monroe ◽

Andrew C Weems ◽

Sam T Briggs ◽

Grace K Fletcher ◽

...

Keyword(s):

Hydrogen Peroxide ◽

Cell Culture ◽

Culture Media ◽

Degradation Products ◽

Oxidative Degradation ◽

Cell Culture Media ◽

Novel Approach ◽

Gene And Protein Expression

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.

Download Full-text

Inclusion of Antibodies to Cell Culture Media Preserves the Integrity of Genes Encoding RL13 and the Pentameric Complex Components during Fibroblast Passage of Human Cytomegalovirus

10.20944/preprints201807.0503.v1 ◽

2018 ◽

Author(s):

Amine Ourahmane ◽

Xiaohong Cui ◽

Li He ◽

Dirk Dittmer ◽

Mark Schleiss ◽

...

Keyword(s):

Cell Culture ◽

Human Cytomegalovirus ◽

Cultured Cells ◽

Culture Media ◽

Adaptive Mutations ◽

Culture Passage ◽

Genes Encoding ◽

Cell Culture Passage

Propagation of human cytomegalovirus (CMV) in cultured cells results in genetic adaptations that confer improved growth in vitro and significant attenuation in vivo. Mutations in RL13 arise quickly during cell culture passage, while mutations in the UL128-131A locus emerge later during fibroblast passage and disrupt expression of a glycoprotein complex that is important for entry into epithelial and endothelial cells. As in vivo CMV replicates in the context of host antibodies, we reasoned that antibodies might mitigate the accumulation of adaptive mutations during cell culture passage. To test this, CMV in infant urine was used to infect replicate fibroblast cultures. One lineage was passaged in the absence of CMV-hyperimmuneglobulin (HIG) while the other was passaged with HIG in the culture medium. The former lost epithelial tropism and aquired mutations disrupting RL13 and UL131A expression, whereas the latter retained epithelial tropism and both gene loci remained intact after 22 passages. An epitheliotropic RL13+/ UL131A+ virus was isolated by limiting-dilution in the presence of HIG and expanded to produce a working stock sufficient to conduct cell tropism experiments. Thus, culture in the presence of antibodies may facilitate in vitro experiments using viruses that are genetically more authentic than has been previously possible.

Download Full-text