Experimentally-Induced Inhibition of Growth in Melanoma Cell Cultures Separated by ~2 Kilometers When Both Share Excess Correlation Magnetic Fields: Macroscopic Evidence of Free-Space Quantum Teleportation?

In multiple experiments plates of melanoma cells separated by either 3 m or 1.7 km were placed in the centers of toroids. A specific protocol of changing, angular velocity, pulsed magnetic fields that has been shown to produce excess correlation in photon durations and shift in proton concentrations (pH) in spring water were generated around both plates of cells. Serial injections of 50 μL of standard concentrations of hydrogen peroxide into the “local” plates of cells during the 12 min of field activation produced conspicuous cell death (reduction of viable cells by about 50%) with comparable diminishments of cell numbers in the non-local plates of cells within 24 hr but only if both loci separated by either 3 m or 1.7 km had shared the “excess correlation” magnetic field sequence. The non-local effect did not occur if the magnetic fields had not been present. Higher or lower concentrations of peroxide or concentrations that eliminated all of the cells or very few cells in the local dishes were associated with no significant diminishment of non-local cell growth. The data indicate that there must be a critical number of cells remaining viable following the local chemical reaction for the excess correlation to be manifested in the non-local cells. We suggest that this specific spatial-temporal pattern of fields generated within the paired toroidal geometries promotes transposition of virtual chemical reactions as an information field. Calculations of the energy available per cell and per volume of the quantity of reactants injected into the local space from the intensity of the changing velocity toroidal magnetic field support previous measurements and derivations that the units of information transposition may involve discrete quantities that represent equivalents of photons, electrons and protons.

Identification and characterization of cell cultures with various embryogenic/regenerative potential in cotton based on morphological,cytochemical,and cytogenetical assessment

Somatic embryogenesis(SE) plays a vital role in genetic transformation and massive propagation of important agronomical and economical crops.Here,we conducted a systematic assessment of the morphological,cytochemical,and cytogenetical characteristics of six culture strains with various embryogenic/regenerative potential during SE process in cotton.Results indicated that the six cell culture strains had stable ploidy levels,and did not reveal any relationship between the cytogenetic state and their morphogenetic potential.Moreover,the six culture strains were compared via double staining with Evans blue and Acetocarmine to efficiently distinguish embryogenic and non-embryogenic cells and determine the embryogenic nature of the calli.In addition,the kind of auxins added in medium affected not only growth property,color,size of cell clumps but also ploidy level and regeneration ability.By combining analysis of morphological,cytochemical,and cytogenetical characteristics of the cell cultures,we are able to obtain and maintain homogeneous cell population with high morphogenic and regeneration ability and establish efficient somatic embryogenesis and regeneration system from short-term cell cultures in upland cotton,which highlight the application of biotechnological approaches in crop breeding,and above all,to better understand totipotency of cells in higher plants.

3D Collagen Gels:A Promising Platform for Dendritic Cell Culture in Biomaterials Research

The three-dimensional(3D)cell culture system has garnered significant attention in recent years as a means of studying cell behavior and tissue development,as opposed to traditional two-dimensional cultures.These systems can induce specific cell reactions,promote specific tissue functions,and serve as valuable tools for research in tissue engineering,regenerative medicine,and drug discovery.This paper discusses current developments in the field of three-dimensional cell culture and the potential applications of 3D type 1 collagen gels to enhance the growth and maturation of dendritic cells.

MACS-W:A modified optical clearing agent for imaging 3D cell cultures

Three-dimensional(3D)cell cultures have contributed to a variety of biological research fields by filling the gap between monolayers and animal models.The modern optical sectioning microscopic methods make it possible to probe the complexity of 3D cell cultures but are limited by the inherent opaqueness.While tissue optical clearing methods have emerged as powerful tools for investigating whole-mount tissues in 3D,they often have limitations,such as being too harsh for fragile 3D cell cultures,requiring complex handling protocols,or inducing tissue deformation with shrinkage or expansion.To address this issue,we proposed a modified optical clearing method for 3D cell cultures,called MACS-W,which is simple,highly efficient,and morphology-preserving.In our evaluation of MACS-W,we found that it exhibits excellent clearing capability in just 10 min,with minimal deformation,and helps drug evaluation on tumor spheroids.In summary,MACS-W is a fast,minimally-deformative and fluorescence compatible clearing method that has the potential to be widely used in the studies of 3D cell cultures.

Developments in cell culture systems for human pluripotent stem cells

Human pluripotent stem cells(hPSCs)are important resources for cell-based therapies and pharmaceutical applications.In order to realize the potential of hPSCs,it is critical to develop suitable technologies required for specific applications.Most hPSC technologies depend on cell culture,and are critically influenced by culture medium composition,extracellular matrices,handling methods,and culture platforms.This review summarizes the major technological advances in hPSC culture,and highlights the opportunities and challenges in future therapeutic applications.

Three-dimensional cell culture systems as an in vitro platform for cancer and stem cell modeling

Three-dimensional(3D)culture systems are becoming increasingly popular due to their ability to mimic tissue-like structures more effectively than the monolayer cultures.In cancer and stem cell research,the natural cell characteristics and architectures are closely mimicked by the 3D cell models.Thus,the 3D cell cultures are promising and suitable systems for various proposes,ranging from disease modeling to drug target identification as well as potential therapeutic substances that may transform our lives.This review provides a comprehensive compendium of recent advancements in culturing cells,in particular cancer and stem cells,using 3D culture techniques.The major approaches highlighted here include cell spheroids,hydrogel embedding,bioreactors,scaffolds,and bioprinting.In addition,the progress of employing 3D cell culture systems as a platform for cancer and stem cell research was addressed,and the prominent studies of 3D cell culture systems were discussed.

Overexpression of heme oxygenase-1 protects smooth muscle cells against oxidative injury and inhibits cell proliferation

To investigate whether the expression of exogenous heme oxygenase-1 (HO-1) gene within vascular smooth muscle cells (VSMC) could protect the cells from free radical attack and inhibit cell proliferation, we established an in vitro transfection of human HO-1 gene into rat VSMC mediated by a retroviral vector. The results showed that the profound expression of HO-1 protein as well as HO activity was 1.8- and 2.0-fold increased respectively in the transfected cells compared to the non-transfected ones. The treatment of VSMC with different concentrations of H2O2 led to the remarkable cell damage as indicated by survival rate and LDH leakage. However, the resistance of the HO-1 transfected VSMC against H2O2 was significantly raised. This protective effect was dramatically diminished when the transfected VSMC were pretreated with ZnPP-IX, a specific inhibitor of HO, for 24 h. In addition, we found that the growth potential of the transfected cells was significantly inhibited directly by increased activity of HO-1, and this effect might be related to decreased phosphorylation of MAPK. These results suggest that the overexpression of introduced hHO-1 is potentially able to reduce the risk factors of atherosclerosis, partially due to its cellular protection against oxidative injury and to its inhibitory effect on cellular proliferation.

Investigation of Oxidation-Reduction Properties of Growing Mediums for Cell Culture of Mammals under the Effect of Cosmo-Geophysical Factors

In model experiments were studied the effect of cosmo-geophysical factors of environment (hypomagnetic conditions during 2 days ≈ 1 mkT;electromagnetic irradiation (10 min - 2 MHz with amplitude 5 V/m and power 30 mkVt, background 2 - 4 mkVt), γ-quantum (10 min—from the source 137Cs) and its combined effect on the physic-chemical properties (ORP and pH) of growing medium for cell culture of mammals as nutrition medium 199 (PanEco, Russia). It was used a clear solution of medium (solution 1) and with the adding of 10% embryo bull serum—model of bio-medium (solution 2). Hypomagnetic conditions evoked the decreasing of ORP and pH value in both solutions, electromagnetic irradiation in the solution 1 which evoked the decreasing of ORP and the increasing of pH value, and in the solution 2, on the contrary, the increasing of ORP with the unchanging pH value. γ-radiation sharply decreased ORP value and didn’t change pH in solution 1, i.e. the reduction properties increased. There is insignificant increasing of ORP value and the decreasing of pH is noted in the solution 2, that it is characterized with the increasing of oxidative properties of solution. Under the combined effect of hypomagnetic conditions and electromagnetic irradiation, the values of investigating parameters in the solution 1 decreased and in the solution 2 increased. It was observed acute decreasing of ORP value in both solutions under the combined effect of hypomagnetic conditions and γ-radiation, i.e. the reductive properties of the solutions increased sharply. In this the concentration H+ significantly decreased, (p γ-radiation led to the decreasing of ORP and pH values in both solutions. Thus, the studying factors significantly change the oxidation-reduction properties of growing mediums. The investigation of the processes in biological mediums plays the important role in the assessment of environment effect during the flight in inter-planet space.

Identification of microRNAs and messenger RNAs involved in human umbilical cord mesenchymal stem cell treatment of ischemic cerebral infarction using integrated bioinformatics analysis

In recent years,a large number of differentially expressed genes have been identified in human umbilical cord mesenchymal stem cell(hUMSC)transplants for the treatment of ischemic cerebral infarction.These genes are involved in various biochemical processes,but the role of microRNAs(miRNAs)in this process is still unclear.From the Gene Expression Omnibus(GEO)database,we downloaded two microarray datasets for GSE78731(messenger RNA(mRNA)profile)and GSE97532(miRNA profile).The differentially expressed genes screened were compared between the hUMSC group and the middle cerebral artery occlusion group.Gene ontology enrichment and pathway enrichment analyses were subsequently conducted using the online Database for Annotation,Visualization,and Integrated Discovery.Identified genes were applied to perform weighted gene co-suppression analyses,to establish a weighted co-expression network model.Furthermore,the protein-protein interaction network for differentially expressed genes from turquoise modules was built using Cytoscape(version 3.40)and the most highly correlated subnetwork was extracted from the protein-protein interaction network using the MCODE plugin.The predicted target genes for differentially expressed miRNAs were also identified using the online database starBase v3.0.A total of 3698 differentially expressed genes were identified.Gene ontology analysis demonstrated that differentially expressed genes that are related to hUMSC treatment of ischemic cerebral infarction are involved in endocytosis and inflammatory responses.We identified 12 differentially expressed miRNAs in middle cerebral artery occlusion rats after hUMSC treatment,and these differentially expressed miRNAs were mainly involved in signaling in inflammatory pathways,such as in the regulation of neutrophil migration.In conclusion,we have identified a number of differentially expressed genes and differentially expressed mRNAs,miRNA-mRNAs,and signaling pathways involved in the hUMSC treatment of ischemic cerebral infarction.Bioinformatics and interaction analyses can provide novel clues for further research into hUMSC treatment of ischemic cerebral infarction.

Circulating Tumor Cell Cultures as a Predictive Marker during Salvage Therapy of Refractory Merkel Cell Carcinoma with Chemotherapy and Electron Beam Radiation

Metastatic Merkel Cell carcinoma (MCC) is a highly unusual and aggressive skin cancer that presents as a small, pink to violet skin lesion and metastasizes early in its growth. Metastatic MCC is generally treated with small cell lung cancer chemotherapy regimens, because the tumor consists of neuroendocrine cells, but patients generally do not have durable responses. The pathogenesis of MCC has recently been attributed to the Merkel Cell polyoma virus. This virus activates the cellular retinoblastoma oncoprotein and cell cycle machinery, triggering continual cellular proliferation. A 77-year-old man developed extensive MCC metastases, involving more than one fourth of his scalp and numerous cervical lymph nodes. Following failure of initial chemotherapy and radiation, effective palliation was achieved by using a sequence of electron-beam radiotherapy, low dose gemcitabine, and etoposide, resulting in significant periods of tumor regression and prolonged survival. A novel circulating tumor cell (CTC) culture assay was performed on four separate clinic visits during the treatment period. Tumor colonies were cultured from the patient’s peripheral blood and CTC colony counts were correlated with clinical treatment response. Not only did the patient respond to palliative cell cycle directed chemotherapy and electron beam radiation, but we demonstrated that CTC can be cultured from peripheral blood of MCC patients and serve as a predictive marker to monitor treatment response.

Renal cell carcinoma: Evolving and emerging subtypes

Our knowledge of renal cell carcinoma(RCC) is rapidly expanding. For those who diagnose and treat RCC, it is important to understand the new developments. In recent years, many new renal tumors have been described and defined, and our understanding of the biology and clinical correlates of these tumors is changing. Evolving concepts in Xp11 translocation carcinoma, mucinous tubular and spindle cell carcinoma, multilocular cystic clear cell RCC, and carcinoma associated with neuroblastoma are addressed within this review. Tubulocystic carcinoma, thyroid-like follicular carcinoma of kidney, acquired cystic disease-associated RCC, and clear cell papillary RCC are also described. Finally, candidate entities, including RCC with t(6;11) translocation, hybrid oncocytoma/chromophobe RCC, hereditary leiomyomatosis and RCC syndrome, and renal angiomyoadenomatous tumor are reviewed. Knowledge of these new entities is important for diagnosis, treatment and subsequent prognosis. This review provides a targeted summary of new developments in RCC.

Identification of various cell culture models for the study of Zika virus

AIM To identify cell culture models supportive for Zika virus(ZIKV) replication.METHODS Various human and non-human cell lines were infected with a defined amount of ZIKV Polynesia strain. Cells were analyzed 48 h post infection for the amount of intracellular and extracellular viral genomes and infectious viral particles by quantitative real-time PCR and virus titration assay. The extent of replication was monitored by immunofluorescence and western blot analysis by using Env and NS1 specific antibodies. Innate immunity was assayed by luciferase reporter assay and immunofluorescence analysis.RESULTS All investigated cell lines except CHO cells supported infection, replication and release of ZIKV. While in infected A549 and Vero cells a pronounced cytopathic effect was observed COS7, 293 T and Huh7.5 cells were most resistant. Although the analyzed cell lines released comparable amounts of viral genomes to the supernatant significant differences were found for the number of infectious viral particles. The neuronal cell lines N29.1 and SH-SY5 Y released 100 times less infectious viral particles than Vero-, A549-or 293 T-cells. However there is no strict correlation between the amount of produced viral particles and the induction of an interferon response in the analyzed cell lines.CONCLUSION The investigated cell lines with their different tissue origins and diverging ZIKV susceptibility display a toolbox for ZIKV research.

Which form of collagen is suitable for nerve cell culture?

In this study, we investigated the effects of hydrolyzed and non-hydrolyzed collagen and two-di- mensional and three-dimensional collagen matrices on cell survival, attachment and neurite out- growth of primary cultured nerve cells using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide （MTT） colorimetric assay and inverted microscopy. Hydrolyzed collagen facilitated nerve cell survival and neurite outgrowth, but it had no obvious influences on cell attachment. In contrast, non-hydrolyzed two-dimensional collagen matrix had no obvious effects on neurite outgrowth. These findings suggest that hydrolyzed collagen is an ideal nerve cell culture media.

Cladogynos orientalis Zipp. extracts inhibit cell culture-derived hepatitis C virus genotype 2a replication in Huh-7 cells through NS5B inhibition

Objective: To evaluate the potential anti-hepatitis C virus (HCV) activities of Cladogynos orientalis Zipp. ex Span and to investigate the molecular mode of action. Methods: Ethanolic and water extracts from various parts of Cladogynos orientalis were examined for cytotoxicity by MTT assay. Sub-cytotoxic concentrations of the extracts were used for further determining anti-HCV activity using cell culture-derived HCV genotype 2a propagated in HepaRG cell line. Immunofluorescence assay was performed to observe the effect on viruses at the pre-entry step. Mode of action at the post-entry step was investigated for the viral RNA and protein expressions by real time RT-PCR and Western blotting assays, respectively. Results: Although Cladogynos orientalis water extracts exhibited lower cytotoxicity than ethanolic extracts, all ethanolic extracts from roots, stems, and leaves of Cladogynos orientalis exhibited higher anti-HCV activities than water extracts. The highest anti-HCV activity was observed in infected cells treated with the extracts 5 h after absorption. No extracts showed pre-viral entry effect. At the post-viral entry step, only leaf ethanolic extracts inhibited NS5B expression, while all extracts did not inhibit HCV NS3 expression. Conclusions: Cladogynos orientalis ethanolic extracts could be further studied and the major active compound needs to be identified as a promising source for anti-HCV agents.

Effects of aminoguanidine on nitric oxide production induced by inflammatory cytokines and endotoxin in cultured rat hepatocytes

AIM: To study the effects of aminoguanidine (AG) and two L-arginine analogues N(omega)-nitro-L-arginine methyl ester (L-NAME) and N(omega)-nitro-L-arginine (L-NNA) on nitric oxide (NO) production induced by cytokines (TNF-alpha, IL-1 beta, and IFN-gamma) and bacterial lipopolysaccharide (LPS) mixture (CM) in the cultured rat hepatocytes, and examine their mechanisms action. METHODS: Rat hepatocytes were incubated with AG, L-NAME, L-NNA, Actinomycin D (ActD) and dexamethasone in a medium containing CM (LPS plus TNF-alpha, IL-1 beta, and IFN-gamma) for 24h. NO production in the cultured supernatant was measured with the Griess reaction. Intracellular cGMP level was detected with radioimmunoassy. RESULTS: NO production was markedly blocked by AG and L-NAME in a dose-dependent manner under inflammatory stimuli condition triggered by CM in vitro. The rate of the maximum inhibitory effects of L-NAME (38.9%) was less potent than that obtained with AG(53.7%, P 【 0.05). There was no significant difference between the inhibitory effects of AG and two L-arginine analogues on intracellular cGMP accumulation in rat cultured hepatocytes. Non-specific NOS expression inhibitor dexamethasone (DEX)and iNOS mRNA transcriptional inhibitor ActD also significantly inhibited CM-induced NO production. AG(0.1 mmol x L(-1)) and ActD (0.2 ng x L(-1)) were equipotent in decreasing NO production induced by inflammatory stimuli in vitro, and both effects were more potent than that induced by non-selectivity NOS activity inhibitor L-NAME (0.1 mmol x L(-1)) under similar stimuli conditions (P【0.01). CONCLUSION: AG is a potent selective inhibitor of inducible isoform of NOS,and the mechanism of action may be not only competitive inhibition in the substrate level, but also the gene expression level in rat hepatocytes.

Chemical Constituents of the Suspension Cell Cultures of Maytenus hookeri

Suspension cell cultures of Maytenus hookeri Loos. (Celastraceae) in SH media were established from the calli induced from the leaves and young steins of M. hookeri on MS media with the supplement of 2 mg/L 2,4-D and 0.1 mg/L KIN (kinetin). Ethyl acetate extract of the cultures showed inhibitory activities against Penicillium avellaneum UC-4376 which was sensitive to maytansinoids. Exhaustive isolation of natural products from a large scale of suspension cell cultures did not yield maytansine instead of affording nine compounds including one novel triterpenoid, named 2, 3-diacetoxyl maytenusone (1), and eight known ones including squalene (2), beta-sitosterol (3), 2', 3', 4-triacetyl-sitoindoside I (4), salaspermic acid (5), maytenonic acid (6), 2alpha-hydroxy-maytenonic acid (7), 6, 11,12-trihydroxy-8, 11, 13-abietrien-7-one (8) and 11, 12-dihydroxy-8, 11, 13-abietatrien-7-one (9) elucidated on the basis of 1D and 2D NMR data. The H-1-NMR and C-13-NMR assignments were made for 1, 5, 6 and 7, while the C-13-NMR assignments for 5 and 6 were revised. The chemical results suggested that the suspension cell cultures of M. hookeri did not produce maytansinoids under the reported experiment conditions.

Microfluidic three-dimensional cell culture of stem cells for high-throughput analysis

Although the recent advances in stem cell engineering have gained a great deal of attention due to their high potential in clinical research,the applicability of stem cells for preclinical screening in the drug discovery process is still challenging due to difficulties in controlling the stem cell microenvironment and the limited availability of high-throughput systems.Recently,researchers have been actively developing and evaluating three-dimensional(3D)cell culture-based platforms using microfluidic technologies,such as organ-on-a-chip and organoid-on-a-chip platforms,and they have achieved promising breakthroughs in stem cell engineering.In this review,we start with a comprehensive discussion on the importance of microfluidic 3D cell culture techniques in stem cell research and their technical strategies in the field of drug discovery.In a subsequent section,we discuss microfluidic 3D cell culture techniques for high-throughput analysis for use in stem cell research.In addition,some potential and practical applications of organ-on-a-chip or organoid-on-a-chip platforms using stem cells as drug screening and disease models are highlighted.

Total Phenolic Content and Antioxidant Activity of Standardized Extracts from Leaves and Cell Cultures of Three Callistemon Species

A comparative study was carried out with ethanolic (80%) extracts from leaves and cell cultures of three Callistemon species, namely C. lanceolatus (CL), C. viridiflorous (CV), and C. comboynensis (CC). Cell suspensions of the three species were grown in liquid Murashige and Skoog (MS) medium (100 ml) supplemented with 0.9 mg·g-1 kinetin in combination with 1.1 mg·g-1 NAA. The CL leaf extract was standardized to contain the highest amount of phenolics (104 ± 2.0 mg·g-1), followed by CC (95.8 ± 1.2 mg·g-1) and CV (79.8 ± 4.6 mg·g-1). On the other hand, cell cultures of CV contained more phenolics (14.9 ± 0.6 mg·g-1) than those of the other two species, CL and CC, which contained 12.2 ± 0.16 and 9.12 ± 0.16 mg·g-1, respectively. Nevertheless, CV leaf extract exhibited the highest antioxidant activity (91.4% ± 0.4%) at a concentration of 1000 μg·ml-1, comparable to 100 μg·ml-1 gallic acid (90.8% ± 1.5%).

Assessment of AMBR^TMas a model for high-throughput cell culture process development strategy

The development and delivery of high quality therapeutic products necessitates the need for highthrough-put (HTP) process development tools. Traditionally, these works requires a combination of shake flask and small-scale stirred tank bioreactor (STR) which are labor and resource intensive and time-consuming. Here we demonstrate a strategy for rapid and robust cell culture process development by evaluating and implementing the use of a new HTP disposable micro bioreactor (MBR) called AMBRTM system (Advanced Microscale Bioreactor) that has the capabilities for automated sampling, feed addition, pH, dissolved oxygen (DO), gassing and agitation controls. In these studies the performance of two monoclonal antibody (MAb) producing cell lines (MAb1 and MAb2) was evaluated both in the AMBR system and 3-L STR. We demonstrated that cell culture performance (growth and viability, production titer and product quality) were similar in both vessel systems. Furthermore, process control and feed optimization were demonstrated in an additional cell line (MAb3) in the disposable MBR and its performance confirmed at STR scale. The results indicate that the AMBR system can be used to streamline the process development effort and facilitate a rapid and robust cell culture process development effort for MAb programs in a HTP manner.