Tissue Culture Incubator Research Articles

Spheroid Drug Sensitivity Screening in Glioma Stem Cell Lines.

In vitro drug sensitivity screens are important tools in the discovery of anti-cancer drug combination therapies. Typically, these in vitro drug screens are performed on cells grown in a monolayer. However, these two-dimensional (2D) models are considered less accurate compared to three-dimensional (3D) spheroid cell models; this is especially true for glioma stem cell lines. Cells grown in spheres activate different signaling pathways and are considered more representative of in vivo models than monolayer cell lines. This protocol describes a method for in vitro drug screening of spheroid lines; mouse and human glioma stem cell lines are used as an example. This protocol describes a 3D spheroid drug sensitivity and synergy assay that can be used to determine if a drug or drug combination induces cell death and if two drugs synergize. Glioma stem cell lines aremodified to express RFP. Cells are plated in low attachment round well bottom 96 plates, and spheres are allowed to form overnight. Drugs are added, and the growth is monitored by measuring the RFP signal over time using the Incucyte live imaging system,a fluorescence microscope embedded in the tissue culture incubator. Half maximal inhibitory concentration (IC50), median lethal dose (LD50), and synergy score are subsequently calculated to evaluate sensitivities to drugs alone or in combination. The three-dimensional nature of this assayprovides a more accurate reflection of tumor growth, behavior, and drug sensitivities in vivo, thus forming the basis for further preclinical investigation.

Successful domestication of Neonothopanus Hygrophanus (Mont.) De Kesel & Degreef and Lentinus Squarrosulus Mont., indigenous saprophytic edible mushrooms from Kibira National Park in Burundi

BackgroundMushroom cultivation in Burundi provides an excellent way to diversify agricultural production although domestication of mushroom species is at an infancy stage. The country is endowed with indigenous forests that harbour a wide diversity of mushrooms with potential for domestication. This study was undertaken to explore opportunities for domestication of saprophytic wild edible mushrooms from the Kibira National Park (KNP) in Burundi.MethodsSamples of Lentinus squarrosulus Mont. and Neonothopanus hygrophanus (Mont.) De Kesel & Degreef were collected from the field, and tissue cultured on Potato Dextrose Agar (PDA) medium. Spawn production and development was performed on sorghum grains and lignocellulosic substrates respectively and the parameters of mycelial growth and mushroom yield were determined.ResultsThe germplasm of L. squarrosulus and N. hygrophanus was successfully isolated with an average tissue culture incubation time of 6.4 ± 0.54 days and 7.6 ± 0.54 days for L. squarrosulus and N. hygrophanus, respectively. Spawn production incubation time on sorghum grains was 12.6 ± 0.89 days and 14.8 ± 0.83 days for L. squarrosulus and N. hygrophanus, respectively. For full colonization of lignocellulosic substrates, spawn production time ranged between 19 ± 1 and 21 ± 1 days for L. squarrosulus and between 17 ± 1.22 and 18 ± 1.22 days for N. hygrophanus. Both species successfully produced fruiting bodies and mushrooms yielded at a rate of 18.24 ± 9.76 to 22.85 ± 9.16% for L. squarrosulus and 12.66 ± 8.95 to 15.3 ± 8.94% for N. hygrophanus which is here reported for the first time to be successfully domesticated. For both species, the cottonseed hulls substrate comparatively showed the best yield followed by the combination of maize cobs/soybean straws (MC + SBS) (50:50), the combination of rice straws/soybean straws (RS + SBS) (50:50) and the maize cobs substrate respectively, while the rice straw showed the least. The combinations of MC + SBS (50:50) and RS + SBS (50:50) showed a yield close to that of cottonseed hulls.ConclusionsFor the first-time, this study presents successful domestication of N. hygrophanus and L. squarrosulus from KNP. It is concluded that the substrates combinations used in the study give good yields, and therefore recommended for use as cost-effective and efficient alternative substrates.

Real‐time and Multichannel Measurement of Contractility of hiPSC‐Derived 3D Skeletal Muscle using Fiber Optics‐Based Sensing

AbstractAs the field of cardiac and skeletal muscle tissue engineering expands, so does the need for accurate and reliable systems to generate in vitro 3D tissues and analyze their functional properties. In this study, the Cuore is introduced, a system that integrates sensors based on optical fibers and uses the principle of light interferometry to detect the contraction of 3D Tissue Engineered Skeletal Muscles (3D‐TESMs). The technology employed in the Cuore allows for reproducible and multichannel force measurements down to a nano‐Newtons resolution while maintaining sterility and permitting continuous non‐invasive recording within and outside standard tissue culture incubators. Thanks to the integrated electrodes for electrical pulse stimulation (EPS), 3D‐TESMs generated from three independent hiPSC‐derived myogenic progenitors (MPs) lines are stimulated and the contractility is recorded over the course of a week. Through the modulation of different EPS parameters, the optimal combination to induce the 3D‐TESMs in producing fully fused tetani without causing damage is determined. Furthermore, 3D‐TESMs from different lines exhibit characteristic signatures of spontaneous contractility and response to caffeine, verapamil, and the β‐agonist clenbuterol. The ease of use, high sensitivity, and the integrated electrodes and sensors make the Cuore an ideal technology to investigate the biology of contractile tissues and their response to drugs.

Studies on intraocular transplantation of pancreatic cells in a large‐eyed preclinical animal model

AbstractPurpose: Pancreatic islet transplantation (PIT) to the anterior chamber of the eye (ACE) has been shown to correct hyperglycemia and restore/improve glycemic control in rodents and non‐human primates. These studies also suggested that the eye may be in several ways a superior host site compared to the conventional hepatic portal system. In order to bring this technology to the clinic, it is essential to establish a large‐eyed non‐primate transplantation model. This proposal aims at establishing a rabbit ACE model of allogeneic PIT. We expect to gain insight into isolated islets function as well as ACE grafting efficiency and histological morphology in the large‐eyed rabbit model.Methods: Pancreatic islets (PI) were isolated from Dutch Belted rabbits by injection of collagenase A into the pancreatic interstitium, digestion and hand picking under a stereo microscope. PI were cultured in RPMI medium overnight in a standard tissue culture incubator prior to assessment of purity. In vitro functional capacity of rabbit PI was assessed by glucose‐stimulated insulin release and intracellular Ca2+ imaging assays. PI were injected into the ACE an allogenic Dutch Belted rabbit using surgical procedures routinely applied in ophthalmology. PI engraftment and cellular content was analysed by anterior segment optical coherence tomography (ASOCT) and immunohistofluorescence (IHF).Results: Isolated PI were of high purity with an approximate yield of 600–800 islets per pancreas. Preliminary results of functional capacity demonstrate that islets are responsive to high glucose stimulus, despite some variability. Allogeneic transplantation of isolated PI into the ACE of a Dutch Belted rabbit was followed by ASOCT. Engraftment was confirmed by IHF for vimentin. IHF suggests ACE engrafted islets are viable and maintain alpha, beta and delta cells.Conclusions: The presented isolation method renders high purity and functional rabbit PI. Allogeneic transplantation of PI into the ACE was successful using clinically relevant procedures and resulted in islets engraftment and long‐term survival. These results may have implications for the clinical treatment of diabetes mellitus, particularly regarding patients with indications for PIT.

P12.06: Effect of Supplementing Taurine to the Developed Lowtemperature Islet Preservation Solution

Introduction: Pancreatic islet transplantation has recently emerged as one of the most promising therapeutic approaches for improving glycemic control in type 1 diabetes patients. However, one of the problems with islet transplantation is that it is impossible to culture the isolated islet for extended periods to while recipients are selected, tested and prepared for surgery. To address this problem, we developed the islet preservation solution, and to improve the function of the preservation solution, we added taurine and conducted a preservation experiment. Taurine is antioxidant activity and it is not a classical free radical scavenger. Therefore, its mechanism remains unclear but it controls osmotic pressure. Our hypothesis is that taurine supplementation can improve islet recovery and enhance islet function after low temperature preservation. Methods: CMRL 1066 medium (Corning) was used as a control group, and a comparison group was prepared some compounds OPTI(Optipharm presevation) solution (with taurine) and OPTI-T solution (without taurine). To confirm the preservation effect of these solutions, we isolated islet from adult Yucatan pig’s pancreas using standard enzymatic digestion (Nordmark) followed by Ficoll purification, these islets were preserved in a humidified tissue culture incubator with preservation solutions and preserved in 4℃ for up to 4 weeks. We compared islet survival in groups of islets. To islet viability and islet functionality, we utilized Glucose Stimulated Insulin Secretion (GSIS) for measuring insulin release, calculated stimulation index (SI) and AO/PI viability staining for during the preservation period. Results: Highest islet recovery was preserved in the group of pig islets cultured with Taurine as compared to the group of islets hypothermically preserved in standard tissue culture media over the 4 week preservation period (p<0.001 using ANOVA). The porcine islet viability/cell number ratio in the OPTI solution maintained a high survival rate for up to 2 weeks (94%). In contrast, porcine islet in OPTI-T solution and CMRL 1066 supplemented media significantly reduced viability after 1 weeks. OPTI solution (with taurine) was significantly higher than OPTI-T solution (without taurine) and CMRL 1066 during 4 weeks after preservation. Glucose stimulation insulin secretion test was performed for functional analysis. As a result, a level of porcine insulin was higher during 2 weeks in the OPTI solution than OPTI-T solution and CMRL 1066. But after 3 weeks preservation, insulin level in high glucose, significantly decreased. Therefore, the viability and functionality of islets were improved by adding taurine to the developed preservation solution. Conclusion: Taurine supplementation has been shown to highest islet recovery, viability and insulin secretion level for 2 weeks. Based on this data, we have performed 2 weeks cold storage of isolated islets for current clinical islet transplantation. Ministry of trade industry and energy, KEIT 20011276, NTIS 1415172664.

Abstract 5327: Characterization of a green, temperature activated nano formulation that drives the mechanism of doxorubicin toxicity from apoptosis to ferroptosis

Abstract Introduction: Nano formulations continue to be a very attractive venue for the development of “smart” drug delivery systems. Emerging green (organic) formulations do not require harsh chemical solvents and are easy to prepare. Solid Core Lipid Formulations: The lipid phase (l.p.) contained a solid fatty acid (lauric acid) and liquid lipid (oleic acid) with or without Doxorubicin. 55° surfactant containing an emulsifying agent (Brij-58) mixed with nonionic surfactant (Span 80) was dropwise added. The emulsion was cooled to form temperature-activated nanoparticles (TAN). Before usage, the emulsion was activated by heating to 41°C. Experiments: B16F10 melanoma, U87-MR glioblastoma, MDA-MB-231 and MCF-7 breast cancer cell lines were analyzed on a variety of quantitative Imaging platforms. Results iCyte (CompuCyte): Laser based instrument for obtaining quantitative fluorescence and light scatter measurements. We obtained single endpoint dosage response curves to compare the TAN with free Dox, and found the TANs were effective at 4-6 times lower equivalent Dox concentrations. Holomonitor (Phase Holographic Imaging): The HM4 resides in tissue culture incubators and allows long-term medium resolution holographic imaging. It offersa complete image processing library including routines for proliferation and cell tracking. We devised methods for 4-dimensional imaging (X pos. vs. Y pos. vs Time, vs. Density). Thus, we are able to monitor the temporal effects of the compounds. Cell Explorer (Nanolive): The Cell Explorer has a a rotating mirror that directs the laser in an orbit around the sample to obtain super-resolution 3D tomographs. It enables long-term time-lapse imaging via stage-top environmental chamber. It is well suited for quantifying sub cellular components such as mitochondria and lipids. HT-2 (Tomocube): The HT-2 is a label-free ultra-high resolution holotomography system complemented by fluorescence capabilities. Combined RI and fluorescence images give the highest information content of any of the platforms. Discussion: In 2012, Dixon et al described ferroptosis as a distinct new form of cell death through the iron dependent accumulation of oxidatively damaged phospholipids. They state that a diagnostic feature is mitochondrial shrinkage and collapse. We confirmed this in our tomographic imaging images. Other features that were detected and consistent with ferroptosis are loss of nuclear contents, a thickening of the nuclear membrane, and peri-nuclear mitochondrial accumulation. In 48-hour HM4 plots control cells show numerous mitotic events. Dox treated cells show cellular enlargement but no mitotic events. In the TAN population at about 20 hours, there is sudden death occurring in the entire population. Conclusion: Our experiments suggest a high potency of the newly developed TANs. Ferroptosis is the most likely mechanism of action. Citation Format: Farzana Parveen, Nina Filipczak, Asadullah Madni, Vladimir P. Torchilin, Ed Luther. Characterization of a green, temperature activated nano formulation that drives the mechanism of doxorubicin toxicity from apoptosis to ferroptosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5327.

LuminoCell: a versatile and affordable platform for real-time monitoring of luciferase-based reporters.

Luciferase reporter assays represent a simple and sensitive experimental system in cell and molecular biology to study multiple biological processes. However, the application of these assays is often limited by the costs of conventional luminometer instruments and the versatility of their use in different experimental conditions. Therefore, we aimed to develop a small, affordable luminometer allowing continuous measurement of luciferase activity, designed for inclusion into various kinds of tissue culture incubators. Here, we introduce LuminoCell-an open-source platform for the construction of an affordable, sensitive, and portable luminometer capable of real-time monitoring in-cell luciferase activity. The LuminoCell costs $40, requires less than 1 h to assemble, and it is capable of performing real-time sensitive detection of both magnitude and duration of the activity of major signalling pathways in cell cultures, including receptor tyrosine kinases (EGF and FGF), WNT/β-catenin, and NF-κB. In addition, we show that the LuminoCell is suitable to be used in cytotoxicity assays as well as for monitoring periodic circadian gene expression.

Multifunctional profiling of triple-negative breast cancer patient-derived tumoroids for disease modeling.

3D cell models derived from patient tumors are highly translational tools that can recapitulate the complex genetic and molecular compositions of solid cancers and accelerate identification of drug targets and drug testing. However, the complexity of performing assays with such models remains a hurdle for their wider adoption. In the present study, we describe methods for processing and multi-functional profiling of tumoroid samples to test compound effects using a novel flowchip system in combination with high content imaging and metabolite analysis. Tumoroids were formed from primary cells isolated from a patient-derived tumor explant, TU-BcX-4IC, that represents metaplastic breast cancer with a triple-negative breast cancer subtype. Assays were performed in a microfluidics-based device (Pu⋅MA System) that allows automated exchange of media and treatments of tumoroids in a tissue culture incubator environment. Multi-functional assay profiling was performed on tumoroids treated with anti-cancer drugs. High-content imaging was used to evaluate drug effects on cell viability and expression of E-cadherin and CD44. Lactate secretion was used to measure tumoroid metabolism as a function of time and drug concentration. Observed responses included loss of cell viability, decrease in E-cadherin expression, and increase of lactate production. Importantly, the tumoroids were sensitive to romidepsin and trametinib, while showed significantly reduced sensitivity to paclitaxel and cytarabine, consistent with the primary tumor response. These methods for multi-parametric profiling of drug effects in patient-derived tumoroids provide an in depth understanding of drug sensitivity of individual tumor types, with important implications for the future development of personalized medicine.

Efficient Method to Differentiate Mouse Embryonic Stem Cells into Macrophages in vitro.

Macrophages are key cells in the innate immune system and play a role in a variety of diseases. However, macrophages are terminally differentiated and difficult to manipulate genetically via transfection or through CRISPR-Cas9 gene editing. To overcome this limitation, we provide a simplified protocol for the generation of mouse embryonic stem cells-derived macrophages (ESDM). Thus, genetic manipulation can be performed using embryonic stem cells, selecting for the desired changes, and finally producing macrophages to study the effects of the previous genetic manipulation. These studies can contribute to many areas of research, including atherosclerosis and inflammation. Production of ESDM has been previously achieved using embryoid body (EB) intermediates. Here, we optimized the EB method using a simplified medium, reducing the number of recombinant proteins and medium recipes required. Our EB-based differentiation protocol consists of three stages: 1) floating EB formation; 2) adherence of EBs and release of floating macrophage progenitors; and, 3) terminal differentiation of harvested macrophage progenitors. The advantages of this protocol include achieving independent floating EBs in stage 1 by using a rocker within the tissue culture incubator, as well as the exclusion of small EBs and cell clusters when harvesting macrophage progenitors via cell filtration.

Simulating microgravity using a random positioning machine for inducing cellular responses to mechanotransduction in human osteoblasts.

The mechanotransduction pathways that mediate cellular responses to contact forces are better understood than those that mediate response to distance forces, especially the force of gravity. Removing or reducing gravity for significant periods of time involves either sending samples to space, inducing diamagnetic levitation with high magnetic fields, or continually reorienting samples for a period, all in a manner that supports cell culturing. Undesired secondary effects due to high magnetic fields or shear forces associated with fluid flow while reorienting must be considered in the designof ground-based devices. We have developed a lab-friendly and compact random positioning machine (RPM) that fits in a standard tissue culture incubator. Using a two-axis gimbal, it continually reorients samples in a manner that produces an equal likelihood that all possible orientations are visited. We contribute a new control algorithm by which the distribution of probabilities over all possible orientations is completely uniform. Rather than randomly varying gimbal axis speed and/or direction as in previous algorithms (which produces non-uniform probability distributions of orientation), we use inverse kinematics to follow a trajectory with a probability distribution of orientations that is uniform by construction. Over a time period of 6 h of operation using our RPM, the average gravity is within 0.001 23% of the gravity of Earth. Shear forces are minimized by limiting the angular speed of both gimbal motors to under 42 °/s. We demonstrate the utility of our RPM by investigating the effects of simulated microgravity on adherent human osteoblasts immediately after retrieving samples from our RPM. Cytoskeletal disruption and cell shape changes were observed relative to samples cultured in a 1 g environment. We also found that subjecting human osteoblasts in suspension to simulated microgravity resulted in less filamentous actin and lower cell stiffness.

Replacing the SpCas9 HNH domain by deaminases generates compact base editors with an alternative targeting scope

Base editors are RNA-guided deaminases that enable site-specific nucleotide transitions. The targeting scope of these Cas-deaminase fusion proteins critically depends on the availability of a protospacer adjacent motif (PAM) at the target locus and is limited to a window within the CRISPR-Cas R-loop, where single-stranded DNA (ssDNA) is accessible to the deaminase. Here, we reason that the Cas9-HNH nuclease domain sterically constrains ssDNA accessibility and demonstrate that omission of this domain expands the editing window. By exchanging the HNH nuclease domain with a monomeric or heterodimeric adenosine deaminase, we furthermore engineer adenine base editor variants (HNHx-ABEs) with PAM-proximally shifted editing windows. This work expands the targeting scope of base editors and provides base editor variants that are substantially smaller. It moreover informs of potential future directions in Cas9 protein engineering, where the HNH domain could be replaced by other enzymes that act on ssDNA.

The incubot: A 3D printer-based microscope for long-term live cell imaging within a tissue culture incubator

Commercial live cell imaging systems represent a large financial burden to research groups, while current open source incubator microscopy systems lack adaptability and are sometimes inadequate for complex imaging experimentation. We present here a low-cost microscope designed for inclusion within a conventional tissue culture incubator. The build is constructed using an entry level 3D printer as the basis for the motion control system, with Raspberry Pi imaging and software integration, allowing for reflected, oblique, and fluorescence imaging of live cell monolayers. The open source nature of the design is aimed to facilitate adaptation by both the community at large and by individual researchers/groups. The development of an adaptable and easy-to-use graphic user interface (GUI) allows for the scientist to be at the core of experimental design through simple modifications of the base GUI code, or generation of an entirely purpose-built script. This adaptability will allow scientists to adapt this equipment for their experimental needs, as opposed to designing experiments to fit their current equipment. The build can be constructed for a cost of roughly €1000 and thus serves as a low-cost and adaptable addition to the open source microscopy community.

Extremely Low Frequency Magnetic Fields Increase the Expression of HIF-1 in Prostate Cancer Cells

Background: Increased levels of hypoxia-inducible factor 1α (HIF-1α) are related to poor prognosis and treatment failure in prostate cancer. The effect of extremely low frequency magnetic fields (ELF-MF), generally emitted from electronic devices on HIF-1α in cancer cells and, specifically, in prostate cancer is not well described. This study aimed to investigate the effects of ELF-MF on HIF- 1α protein expression and HIF-1 transcriptional activity in prostate cancer cells. Methods: Prostate cancer cells were irradiated with ELF-MF by means of a frequency generator at 875 MHz with an intensity of 0.07 mW/cm2 at various time periods and with an exposure system housed in a tissue culture incubator. The expression of HIF-1α protein and HIF-1 transcriptional activity at the different conditions were evaluated. Results: HIF-1α expression in different prostate cancer cells was higher after one hour of exposure to ELF-MF. All exposed cells also demonstrated a significant 50% increase in HIF-1 transcriptional activity as measured by an HRE-dependent reporter gene assay after ELF-MF irradiation. Cells exposed to ELF-MF exhibited 3-fold longer HIF-1α half-life compared to non-exposed control cells, while its mRNA levels were not affected. Immunofluorescence staining confirmed nuclear HIF-1α accumulation following exposure to ELF-MF. Cell proliferation was increased following ELF-MF exposure and significantly reduced when HIF-1α was silenced even after exposure to ELF-MF. Conclusions: Exposure of prostate cancer cells to ELF-MF increases HIF-1α protein stability and upregulates HIF-1 transcriptional activity. These findings provide new insights into the effects of ELF-MF on the hypoxic pathway which are important for guiding future implications.

A novel specialized tissue culture incubator designed and engineered for radiobiology experiments in a sub-natural background radiation research environment

Extensive research has been conducted investigating the effects of ionizing radiation on biological systems, including specific focus at low doses. However, at the surface of the planet, there is the ubiquitous presence of ionizing natural background radiation (NBR) from sources both terrestrial and cosmic. We are currently conducting radiobiological experiments examining the impacts of sub-NBR exposure within SNOLAB. SNOLAB is a deep underground research laboratory in Sudbury, Ontario, Canada located 2 km beneath the surface of the planet. At this depth, significant shielding of NBR components is provided by the rock overburden. Here, we describe a Specialized Tissue Culture Incubator (STCI) that was engineered to significantly reduce background ionizing radiation levels. The STCI was installed 2 km deep underground within SNOLAB. It was designed to allow precise control of experimental variables such as temperature, atmospheric gas composition and humidity. More importantly, the STCI was designed to reduce radiological contaminants present within the underground laboratory. Quantitative measurements validated the STCI is capable of maintaining an appropriate experimental environment for sub-NBR experiments. This included reduction of sub-surface radiological contaminants, most notably radon gas. The STCI presents a truly novel piece of infrastructure enabling future research into the effects of sub-NBR exposure in a highly unique laboratory setting.

High-resolution Imaging of Nuclear Dynamics in Live Cells under Uniaxial Tensile Strain

Extracellular mechanical strain is known to elicit cell phenotypic responses and has physiological relevance in several tissue systems. To capture the effect of applied extracellular tensile strain on cell populations in vitro via biochemical assays, a device has previously been designed which can be fabricated simply and is small enough to fit inside tissue culture incubators, as well as on top of microscope stages. However, the previous design of the polydimethylsiloxane substratum did not allow high-resolution subcellular imaging via oil-immersion objectives. This work describes a redesigned geometry of the polydimethylsiloxane substratum and a customized imaging setup that together can facilitate high-resolution subcellular imaging of live cells while under applied strain. This substratum can be used with the same, earlier designed device and, hence, has the same advantages as listed above, in addition to allowing high-resolution optical imaging. The design of the polydimethylsiloxane substratum can be improved by incorporating a grid which will facilitate tracking the same cell before and after the application of strain. Representative results demonstrate high-resolution time-lapse imaging of fluorescently labeled nuclei within strained cells captured using the method described here. These nuclear dynamics data give insights into the mechanism by which applied tensile strain promotes differentiation of oligodendrocyte progenitor cells.

190 Effects of endocrine-disrupting chemical on calcium signalling in cardiomyocyte differentiation from mouse embryonic stem cells

Endocrine-disrupting chemicals (EDC) are substances similar to steroid hormones that can disturb normal physiological functions of male and female reproductive organs. Endocrine-disrupting chemicals tend to bind to steroid hormone receptors. Sex steroid hormones modulate calcium signalling in the cardiac muscle in early embryo development. Among the steroid hormones, progesterone (P4) has been reported to affect blood pressure and other aspects of the cardiovascular system. The EDC that have similar structure to P4, such as octyl-phenol (OP) and bisphenol A (BPA), are potentially harmful to development of the heart. To confirm the effect of OP and BPA on early differentiation of mouse embryonic stem cells (mES) into cardiomyocytes, the hanging-drop method with mESC cell line (ES-E14TG2a) was used for forming embryoid bodies. Pluripotent mESC were cultured in basal medium with leukemia inhibitory factor and grown on mitomycin C-treated mouse embryonic fibroblasts in a 60-mm plate at 37°C in a 5% CO2 humidified tissue culture incubator with basal medium consisting of DMEM/F-12 supplemented with nonessential amino acids, 10% heat-inactivated and certified fetal bovine serum (FBS), 2-mercaptoethanol, penicillin, and streptomycin. The mouse embryoid bodies (mEB) were suspended onto 6-well plates and cultured with differentiation medium containing steroid-free FBS without leukemia inhibitory factor. Progesterone, OP, and BPA were added on Day 2 from mEB attachment. In addition, mifepristone (RU486), an antagonist for progesterone receptor (PR), was used to confirm the effect of P4 through PR. To determine whether RU486 is capable of attenuating the inhibition effect of P4, RU486 was added for 1 day starting on Day 11. Assessment of cardiomyocyte differentiation was determined by checking the number of beating cell populations divided by the total number of attached mEB. Total RNA was extracted using Trizol reagent and synthesised to cDNA using Moloney murine leukemia virus (MMLV) reverse transcriptase. Messenger RNA level was assessed using quantitative real-time PCR. To investigate calcium signalling, the mRNA levels of calcium channel gene Trpv2 and contraction-related genes Ryr2, Cam2, and Mylk3 were analysed. Beating ratio was decreased in P4, OP, and BPA treatments. Data were analysed by one-way ANOVA followed by Tukey’s multiple comparison tests, and P&amp;lt;0.05 was considered statistically significant in least 3 different replicates. The mRNA level of Pgr was significantly increased in P4, OP, and BPA-treated group. However, the mRNA level of calcium channel gene Trpv2 was significantly decreased in the P4, OP, and BPA-treated group. Expression of contraction-related genes such as Ryr2, Cam2, and Mlck3 were significantly decreased in the P4, OP, and BPA-treated group. In addition, treatment with RU486 rescues altered calcium channel gene and contraction-related genes. Taken together, these results suggest that OP and BPA may affect the differentiation of mESC into cardiomyocyte and disrupts differentiation of cardiomyocytes.

Proton Radioprotection of Fanconi Anemia (Fanca-/-) Mouse Marrow Stromal Cell Lines by Mitochondrial Targeted GS-Nitroxide JP-4-039

The increasing incidence of oral cavity squamous cell carcinomas in radiosensitive Fanconi Anemia (FA) patients suggested that protons might minimize normal tissue toxicity. We tested the radiation protector/mitigator, JP4-039, with Proton irradiated mesenchymal stem cell lines derived from Fanca-/- mouse marrow. Fanca-/- and control Fanca+/+ bone marrow stromal cell lines were irradiated to doses of 0 – 10 Gy via pencil beam scanning proton therapy system. Subgroups of cultures were treated with 100uM JP4-039 for 12 h prior to irradiation and after plating for clonogenic survival curves. Parallel cultures were irradiated using a JL Shepherd Model 68A cesium irradiator at 340 cGy per minute, subgroups treated with JP4-039 in a similar protocol. Cells were plated in 4 well limbro plates and incubated for 10 days in a CO2 tissue culture incubator. Colonies of greater than 50 cells were stained with crystal violet and counted. Data from triplicate experiments was analyzed using Linear Quadratic and Single Hit, Multiple Target model. Cultures irradiated to 5 or 10 Gy using proton or gamma irradiation were plated in T25 flasks and stained 10 days later for β-Galactosidase. Proton irradiation revealed radiosensitivity of Fanca-/- bone marrow stromal cell lines compared to Fanca+/+ cell lines (D0 = 2.11 ± 0.17, vs D0 = 3.28 ± 0.10 p = 0.0027, ñ = 1.1 ± 0.1, vs ñ = 1.0 ± 0.1, respectively). Fanca-/- cells were also radiosensitive to gamma irradiation, (D0 = 2.08 ± 0.17, vs D0 = 3.36 ± 0.05 p = 0.0017, ñ = 2.0 ± 0.5, ñ = 1.3 ± 0.3, respectively). JP4-039 provided significant radiation protection for both Fanca-/- and Fanca+/+ cell lines against both proton irradiation (D0 = 3.01 ± 0.19 p = 0.0125, ñ = 1.0 ± 0.1, vs D0 = 3.84 ± 0.11 p = 0.0196, ñ = 1.0 ± 0.1), and gamma irradiation ((D0 = 3.81 ± 0.72 p = 0.0423, ñ = 1.2 ± 0.2; D0 = 3.70 ± 0.06 p = 0.0133 p = 0.0133, ñ = 1.7 ± 0.7). Following proton irradiation, Fanca+/+ cells showed greater senescence compared to Fanca-/- cells: 28% vs 10% β-galactosidase positive (p < 0.05). In contrast, gamma irradiation of Fanca+/+ compared to Fanca-/- cells showed decreased senescence (7% compared to 17% (p < 0.05). Fanca-/- bone marrow stromal cell lines are radiosensitive to proton and gamma irradiation, but significantly protected by JP4-039. The data support intra-oral JP4-039 for Fanconi Anemia patients receiving proton radiotherapy for oral squamous cell carcinoma.

Novel aspects of live intestinal epithelial cell function revealed using a custom time-lapse video microscopy apparatus.

Many aspects of cell physiology, including migration, membrane function, and cell division, are best understood by observing live cell dynamics over time using video microscopy. To probe these phenomena in colon epithelial cells using simple components with a limited budget, we have constructed an inexpensive (<$410) self-contained apparatus, consisting of a closed-loop, feedback-controlled system regulated by a PID (proportional-integrative-derivative) controller contained within a 0.077 m3 insulated acrylic box. Temperature, humidity, pH, and proliferative capacity of colon epithelial cells in this system mimic those in a standard tissue culture incubator for over four days. Our system offers significant advantages over existing cost-prohibitive commercially available and custom-made devices because of its very low cost, use of PID temperature control, lack of reliance on constant infusion of external humidified, heated air or carbon dioxide, ability to directly measure cell culture medium temperature, and combination of exquisite cellular detail with minimal focus drift under physiological conditions for extended periods of time. Using this apparatus, coupled with an inverted microscope equipped with phase contrast optics and a programmable digital camera, we have observed many events in colon epithelial cells not visible by static imaging, including kinetics of normal and abnormal mitoses, dynamic membrane structures, intracellular vesicle movements, and cell migration. © 2018 International Society for Advancement of Cytometry.

Activation of Signaling Cascades by Weak Extremely Low Frequency Electromagnetic Fields

Background/Aims: Results from recent studies suggest that extremely low frequency magnetic fields (ELF-MF) interfere with intracellular signaling pathways related to proliferative control. The mitogen-activated protein kinases (MAPKs), central signaling components that regulate essentially all stimulated cellular processes, include the extracellular signal-regulated kinases 1/2 (ERK1/2) that are extremely sensitive to extracellular cues. Anti-phospho-ERK antibodies serve as a readout for ERK1/2 activation and are able to detect minute changes in ERK stimulation. The objective of this study was to explore whether activation of ERK1/2 and other signaling cascades can be used as a readout for responses of a variety of cell types, both transformed and non-transformed, to ELF-MF. Methods: We applied ELF-MF at various field strengths and time periods to eight different cell types with an exposure system housed in a tissue culture incubator and followed the phosphorylation of MAPKs and Akt by western blotting. Results: We found that the phosphorylation of ERK1/2 is increased in response to ELF-MF. However, the phosphorylation of ERK1/2 is likely too low to induce ELF-MF-dependent proliferation or oncogenic transformation. The p38 MAPK was very slightly phosphorylated, but JNK or Akt were not. The effect on ERK1/2 was detected for exposures to ELF-MF strengths as low as 0.15 µT and was maximal at ∼10 µT. We also show that ERK1/2 phosphorylation is blocked by the flavoprotein inhibitor diphenyleneiodonium, indicating that the response to ELF-MF may be exerted via NADP oxidase similar to the phosphorylation of ERK1/2 in response to microwave radiation. Conclusions: Our results further indicate that cells are responsive to ELF-MF at field strengths much lower than previously suspected and that the effect may be mediated by NADP oxidase. However, the small increase in ERK1/2 phosphorylation is probably insufficient to affect proliferation and oncogenic transformation. Therefore, the results cannot be regarded as proof of the involvement of ELF-MF in cancer in general or childhood leukemia in particular.

Agitation increases expansion of cord blood hematopoietic cells and promotes their differentiation into myeloid lineage.

Mechanical stress caused by agitation is one of the factors that can affect hematopoietic stem cell expansion in suspension bioreactors. Therefore, we have investigated the effects of agitation on umbilical cord blood hematopoietic stem cell (UCB-HSC) growth and differentiation. A comparison was made between various agitation rates (20, 40 and 60rpm) in spinner-flask and cells cultured in glass petri dish as a static culture. Moreover, the fluid dynamic at various agitation rates of spinner-flask was analyzed to determine shear stress. The spinner-flask contained a rotational moving mixer with glass ball and was kept in tissue culture incubator. To reduce consumption of cytokines, UCB-serum was used which widely decreased the costs. Our results determined that, agitation rate at 40rpm promoted UCB-HSCs expansion and their colony forming potential. Myeloid progenitors were the main type of cells at 40rpm agitation rate. The results of glucose consumption and lactic acid production were in complete agreement with colony assay and expansion data and indicated the superiority of culture in spinner-flask when agitated at 40rpm over to other agitation speeds and also static culture. Cell viability and colony count was affected by changing the agitation speed. We assume that changes in cell growth resulted from the effect of shear stress directly on cell viability, and indirectly on signaling pathways that influence the cells to differentiate.