Incubation Of Cell Cultures Research Articles

A human ex vivo skin model breaking boundaries

Ex vivo human skin models are valuable tools in skin research due to their physiological relevance. Traditionally, standard cultivation is performed in a cell culture incubator with a defined temperature of 37 °C and a specific atmosphere enriched with CO2 to ensure media stability. Maintaining the model under these specific conditions limits its flexibility in assessing exposures to which the skin is exposed to in daily life, for example changes in atmospheric compositions. In this study we demonstrated that the foreskin-derived skin model can be successfully cultured at room temperature outside a CO2 incubator using a CO2-independent, serum-free media. Over a cultivation period of three days, the integrity of the tissue and the preservation of immune cells is well maintained, indicating the model’s stability and resilience under the given conditions. Exposing our Medical University of Graz – human Organotypic Skin Explant Culture (MUG-hOSEC) model to cytotoxic and inflammatory stimuli results in responses analyzable within the supernatant. Besides the common analysis of released proteins upon treatment, such as cytokines and enzymes, we have included extracellular vesicle to obtain a more comprehensive picture of cell communication.

Long-term Monitoring of Oxygen Consumption Rates in Highly Differentiated and Polarized Retinal Pigment Epithelial Cultures.

Mitochondrial metabolism is critical for the normal function of the retinal pigment epithelium (RPE), a monolayer of cells in the retina important for photoreceptor survival. RPE mitochondrial dysfunction is a hallmark of age-related macular degeneration (AMD), the leading cause of irreversible blindness in the developed world, and proliferative vitreoretinopathy (PVR), a blinding complication of retinal detachments. RPE degenerative conditions have been well-modeled by RPE culture systems that are highly differentiated and polarized to mimic in vivo RPE. However, monitoring oxygen consumption rates (OCR), a proxy for mitochondrial function, has been difficult in such culture systems because the conditions that promote ideal RPE polarization and differentiation do not allow for easy OCR measurements. Here, we introduce a novel system, Resipher, to monitor OCR for weeks at a time in well-differentiated RPE cultures while maintaining the RPE on optimal growth substrates and physiologic culture media in a standard cell culture incubator. This system calculates OCR by measuring the oxygen concentration gradient present in the media above cells. We discuss the advantages of this system over other methods for detecting OCR and how to set up the system for measuring OCR in RPE cultures. We cover key tips and tricks for using the system, caution about interpreting the data, and guidelines for troubleshooting unexpected results. We also provide an online calculator for extrapolating the level of hypoxia, normoxia, or hyperoxia RPE cultures experience based on the oxygen gradient in the media above cells detected by the system. Finally, we review two applications of the system, measuring the metabolic state of RPE cells in a PVR model and understanding how the RPE metabolically adapts to hypoxia. We anticipate that the use of this system on highly polarized and differentiated RPE cultures will enhance our understanding of RPE mitochondrial metabolism both under physiologic and disease states.

Controlling Pericellular Oxygen Tension in Cell Culture Reveals Distinct Breast Cancer Responses to Low Oxygen Tensions.

In oxygen (O2)-controlled cell culture, an indispensable tool in biological research, it is presumed that the incubator setpoint equals the O2 tension experienced by cells (i.e., pericellular O2). However, it is discovered that physioxic (5% O2) and hypoxic (1% O2) setpoints regularly induce anoxic (0% O2) pericellular tensions in both adherent and suspension cell cultures. Electron transport chain inhibition ablates this effect, indicating that cellular O2 consumption is the driving factor. RNA-seq analysis revealed that primary human hepatocytes cultured in physioxia experience ischemia-reperfusion injury due to cellular O2 consumption. A reaction-diffusion model is developed to predict pericellular O2 tension a priori, demonstrating that the effect of cellular O2 consumption has the greatest impact in smaller volume culture vessels. By controlling pericellular O2 tension in cell culture, it is found that hypoxia vs. anoxia induce distinct breast cancer transcriptomic and translational responses, including modulation of the hypoxia-inducible factor (HIF) pathway and metabolic reprogramming. Collectively, these findings indicate that breast cancer cells respond non-monotonically to low O2, suggesting that anoxic cell culture is not suitable for modeling hypoxia. Furthermore, it is shown that controlling atmospheric O2 tension in cell culture incubators is insufficient to regulate O2 in cell culture, thus introducing the concept of pericellular O2-controlled cell culture.

Design and experiment of high-performance small magnetic shielding box

Investigating the state of cells in zero magnetic or near-zero magnetic environments is an important scientific issue. However, standard cell culture incubators can only provide general conditions such as constant temperature, constant humidity, sterility, and carbon dioxide, and cannot provide zero magnetic or near-zero magnetic environments for cell culture. To address this issue, an optimization method was proposed in this paper based on the combination of the particle swarm optimization algorithm (PSO) and the finite element method (FEM), achieving the optimization design of a magnetic shielding box (MSB) with a small volume, high shielding factor, and low residual field. Firstly, the high-permeability layer and high-conductivity layer were optimized respectively using the PSO and FEM. Then, the effectiveness of this method was analyzed through experiments. The experimental results show that the shielding factors in three directions at the center point of the optimized MSB are 786.8 (east-west), 2182.7 (north-south), and 1389.4 (vertical) respectively at 0.01 Hz. Meanwhile, the maximum residual field in the cubic region with a side length of 10 cm inside the MSB is 11.9 nT. Finally, the MSB designed by this method was placed in a standard cell culture incubator to cultivate cells in a zero-magnetic or near-zero magnetic environment. Then the Cell Counting Kit-8 (CCK8) experiments to evaluate the cytotoxicity of chemotherapy drugs on tumor cells in a weak magnetic environment (WMF). The results show that the lethality of paclitaxel (PTX) to Epidermal carcinoma cell (A431) increases by 25.96 times and the lethality of gemcitabine (GE) to Lung carcinoma cell (NCI-H460) increases by 24.23 times in a WMF. At the same time, the maximum proliferation inhibition rate of 5-fluorouracil (5-FU) on NCI-H460 cells in a WMF environment increased by 15.69%. That is, WMF could significantly improve the therapeutic effect of chemotherapy drugs on tumor cells.

Abstract 83: NK cell ADCC assays: Leveraging flow cytometry and reporter cell lines for enhanced biological relevance and throughput

Abstract Amid the growing arsenal of cancer treatments, antibody-dependent cellular cytotoxicity (ADCC) assays have emerged as a significant contributor to the quest for more effective therapies. The ADCC-based antibody therapy often exploits Natural Killer (NK) cells as effectors. This immunotherapeutic approach harnesses the innate cytotoxic potential of NK cells, offering a promising solution for the precision targeting of malignant cells. In this study, we employed two methods that can be used to measure ADCC potency and specificity during preclinical drug discovery. The first was a flow cytometry-based approach that focused on enhancing biological relevance by utilizing primary human peripheral blood mononuclear cells (PBMCs) as a source for NK effector cells. To this end, target cells were fluorescently labeled with CFSE and cultured with freshly isolated PBMCs at 3 effector to target (E:T) ratios, in the presence of ADCC antibody. After 16h incubation, a viability dye was added, and percent cytotoxicity was measured in CFSE+ targets cells by flow cytometry. The second method offered a high-throughput plate reader-based approach (ADCC Reporter Bioassay; Promega), that utilized an ADCC reporter effector cell line harboring a luciferase cassette driven by an NFAT promoter, which is activated when effector and targets cells are bridged by a therapeutic antibody. Cytotoxicity was read out by measuring light output on a Cytation 3 plate imaging reader (Agilent Technologies) after a 6h cell culture incubation using the recommended 6:1 E:T ratio. Preclinical application for both assays was demonstrated using an anti-HER2 therapeutic antibody (trastuzumab) against BT-474, which is a HER2 positive cell line. To demonstrate specificity, an anti-VEGF antibody (bevacizumab) and Hs 578T cells (HER2 low) were used as controls. Our results revealed trastuzumab induced a strong dose-dependent increase in cytotoxicity in the BT-474 cells but not the Hs 578T cell line (range: 40-70%) while bevacizumab failed to induce any cytotoxicity above background levels at any concentration. Furthermore, the flow cytometry and plate reader methodologies produced similar trends. In summary, both applications can be used to produce reliable ADCC drug screening assessments. Additional considerations include investigational needs surrounding high throughput capability versus biological relevance. Unlike traditional models using immortalized cell lines, primary human PBMCs more faithfully recapitulate the physiological interactions between effector cells and target cells. On the other hand, the more streamlined reporter cell line approach not only accelerates assay throughput but also ensures consistent and reliable results in an attractive option for large-scale screening and drug development efforts. Citation Format: David William Draper, Yewei Xing, Scott Wise. NK cell ADCC assays: Leveraging flow cytometry and reporter cell lines for enhanced biological relevance and throughput [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 83.

A Heating and Cooling Stage With Fast Temporal Control for Biological Applications.

The study of biological processes involving live microscopy techniques requires adequate temperature control to respect the physiology of the organism under study. We present here a design strategy for a microscope temperature stage based on thermoelectric elements. The design allows the user to access a range of temperatures below and above room temperature and can accommodate samples of different geometries. In addition, by cooling simultaneously the sample insert and the objective, we minimize the temperature gradients along the sample for large magnification objectives requiring immersion oil. We illustrate how this design can be used to study the physiology of the zebrafish embryo over the temperature tolerance of this species. We envision that this device could benefit the communities using model and non-model organisms with physiological temperatures different from typical mammalian cell culture incubation in biomedical research.

Development of a bioreactor for in-vitro compression cycling of tissue engineered meniscal implants

Injuries to the meniscus are common and can impair physical activities. Bioprinted meniscal tissue offers an attractive alternative to donor tissue for meniscal repair but achieving the strength of native tissue is a challenge. Here we report the development of a tissue engineering bioreactor designed to apply repetitive force which may lead to an increase in the compressive modulus and durability of bioprinted meniscal tissues. The modular bioreactor system is composed of a sterilizable tissue culture vessel together with a dock that applies and measures mechanical force. The culture vessel allows for simultaneous compression cycling of two anatomically sized menisci. Using a hybrid linear actuator with a stepper motor, the dock can apply up to 300 N of force at speeds up to 20 mm/s, corresponding to the upper limits of anatomical force and motion in the knee. An interchangeable 22 N load cell was mated between the culture vessel and the dock to log changes in force. Both the culture vessel and dock are maintained in a standard cell culture incubator to provide heat and CO2, while the dock is powered and controlled externally using a step motor drive and customized software.

A growth chamber for chronic exposure of mammalian cells to H2S

H2S is a redox-active signaling molecule that exerts an array of cellular and physiological effects. While intracellular H2S concentrations are estimated to be in the low nanomolar range, intestinal luminal concentrations can be significantly higher due to microbial metabolism. Studies assessing H2S effects are typically conducted with a bolus treatment with sulfide salts or slow releasing sulfide donors, which are limited by the volatility of H2S, and by potential off-target effects of the donor molecules. To address these limitations, we describe the design and performance of a mammalian cell culture incubator for sustained exposure to 20–500 ppm H2S (corresponding to a dissolved sulfide concentrations of ∼4–120 μM in the cell culture medium). We report that colorectal adenocarcinoma HT29 cells tolerate prolonged exposure to H2S with no effect on cell viability after 24 h although ≥50 ppm H2S (∼10 μM) restricts cell proliferation. Even the lowest concentration of H2S used in this study (i.e. ∼4 μM) significantly enhanced glucose consumption and lactate production, revealing a much lower threshold for impacting cellular energy metabolism and activating aerobic glycolysis than has been previously appreciated from studies with bolus H2S treatment regimens.

A User-Centric 3D-Printed Modular Peristaltic Pump for Microfluidic Perfusion Applications.

Microfluidic organ-on-a-chip (OoC) technology has enabled studies on dynamic physiological conditions as well as being deployed in drug testing applications. A microfluidic pump is an essential component to perform perfusion cell culture in OoC devices. However, it is challenging to have a single pump that can fulfil both the customization function needed to mimic a myriad of physiological flow rates and profiles found in vivo and multiplexing requirements (i.e., low cost, small footprint) for drug testing operations. The advent of 3D printing technology and open-source programmable electronic controllers presents an opportunity to democratize the fabrication of mini-peristaltic pumps suitable for microfluidic applications at a fraction of the cost of commercial microfluidic pumps. However, existing 3D-printed peristaltic pumps have mainly focused on demonstrating the feasibility of using 3D printing to fabricate the structural components of the pump and neglected user experience and customization capability. Here, we present a user-centric programmable 3D-printed mini-peristaltic pump with a compact design and low manufacturing cost (~USD 175) suitable for perfusion OoC culture applications. The pump consists of a user-friendly, wired electronic module that controls the operation of a peristaltic pump module. The peristaltic pump module comprises an air-sealed stepper motor connected to a 3D-printed peristaltic assembly, which can withstand the high-humidity environment of a cell culture incubator. We demonstrated that this pump allows users to either program the electronic module or use different-sized tubing to deliver a wide range of flow rates and flow profiles. The pump also has multiplexing capability as it can accommodate multiple tubing. The performance and user-friendliness of this low-cost, compact pump can be easily deployed for various OoC applications.

Incubator-independent perfusion system integrated with microfluidic device for continuous electrophysiology and microscopy readouts

Advances in primary and stem cell derived neuronal cell culture techniques and abundance of available neuronal cell types have enabled in vitro neuroscience as a substantial approach to model in vivo neuronal networks. Survival of the cultured neurons is inevitably dependent on the cell culture incubators to provide stable temperature and humidity and to supply required CO2 levels for controlling the pH of culture medium. Therefore, imaging and electrophysiology recordings outside of the incubator are often limited to the short-term experimental sessions. This restricts our understanding of physiological events to the short snapshots of recorded data while the major part of temporal data is neglected. Multiple custom-made and commercially available platforms like integrated on-stage incubators have been designed to enable long-term microscopy. Nevertheless, long-term high-spatiotemporal electrophysiology recordings from developing neuronal networks needs to be addressed. In the present work an incubator-independent polydimethylsiloxane-based double-wall perfusion chamber was designed and integrated with multi-electrode arrays (MEAs) electrophysiology and compartmentalized microfluidic device to continuously record from engineered neuronal networks at sub-cellular resolution. Cell culture media underwent iterations of conditioning to the ambient CO2 and adjusting its pH to physiological ranges to retain a stable pH for weeks outside of the incubator. Double-wall perfusion chamber and an integrated air bubble trapper reduced media evaporation and osmolality drifts of the conditioned media for two weeks. Aligned microchannel-microfluidic device on MEA electrodes allowed neurite growth on top of the planar electrodes and amplified their extracellular activity. This enabled continuous sub-cellular resolution imaging and electrophysiology recordings from developing networks and their growing neurites. The on-chip versatile and self-contained system provides long-term, continuous and high spatiotemporal access to the network data and offers a robust in vitro platform with many potentials to be applied on advanced cell culture systems including organ-on-chip and organoid models.

A Novel Reverberation Chamber for In Vitro Bioelectromagnetic Experiments at 3.5 GHz

In this article, a mode-stirred reverberation chamber (RC) was designed and proposed for the first time as a cell culture incubator for in vitro electromagnetic waves exposure of adherent cells in tissue culture plates (TCPs). Typical cell incubators require specific conditions, such as temperature of 37 °C and humidity rate of 95%, which are challenging conditions for an RC. The chamber was characterized as an RC through an innovative experimental methodology based on the measurements of the <italic xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">S</i> <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">11</sub> parameter of the emitting antenna. The proposed RC is adapted for in vitro bioelectromagnetic experiments for simultaneous exposure of up to 10 TCPs under highly homogeneous exposure conditions at 3.5 GHz, i.e., the mid-frequency band of the 5G telecommunication networks. Experimental results showed that the specific absorption rate (SAR) in the exposed samples extracted from temperature measurements was similar (an acceptable maximum variation lower than 30% was observed) in reason of the homogeneity and the uniformity of the field within the chamber. Specifically, measured SAR values were around 1.5 and 1 W/kg per 1 W incident, in 6-well or 96-well plates used for biological exposure, respectively. To validate our system, numerical simulations were performed. Overall, we showed that experimental and numerical SARs are in good agreement with differences <30% considering the standard deviation.

Fabrication and validation of an LED array microscope for multimodal, quantitative imaging

The combination of multiple imaging modalities in a single microscopy system can enable new insights into biological processes. In this work, we describe the construction and rigorous characterization of a custom microscope with multimodal imaging in a single, cost-effective system. Our design utilizes advances in LED technology, robotics, and open-source software, along with existing optical components and precision optomechanical parts to offer a modular and versatile design. This microscope is operated using software written in Arduino and Python and has the ability to run multi-day automated imaging experiments when placed inside of a cell culture incubator. Additionally, we provide and demonstrate methods to validate images taken in brightfield and darkfield, along with validation and optimization for differential phase contrast (DPC) quantitative phase imaging.

Real-time monitoring of oxygen levels within thermoplastic Organ-on-Chip devices

Organ-on-chip (OOC) systems involve culturing of biological tissues within dynamic and controlled microenvironments, in order to mimic as closely as possible, in vivo conditions. PDMS has allowed the majority of the pioneering work in the field of OOC, due to its ease of fabrication. While the high oxygen permeability of PDMS is generally regarded as an advantage, there is growing evidence that this could be a major drawback for OOC systems. The aim of OOC system is to replicate as closely as possible physiological conditions with the human body, such as the oxygen levels, which varies between 0% and 14%. PDMS organ-on-chip systems placed within standard cell culture incubators are exposed to hyperoxic conditions, which do not reflect the conditions within the body. In this research paper, we demonstrate the ease of integration of optical oxygen sensors and efficient control of oxygen levels within thermoplastic organ-on-chip systems, compared to their PDMS counterparts. We also demonstrate that culturing spheroids in a thermoplastic microfluidic chip for 4 days does not decrease their viability, despite a decrease in oxygen level, due to oxygen consumption by the spheroids, with time.

Mechanical and biological properties of atmospheric plasma-sprayed carbon nanotube-reinforced tantalum pentoxide composite coatings on Ti6Al4V alloy

Ta2O5 coatings containing 0, 3, 5 and 7 vol% multi-walled carbon nanotubes (CNTs) were deposited on Ti6Al4V substrates by atmospheric plasma spraying (APS). The addition of CNTs had no obvious effect on the surface morphology and roughness of the coatings, but increased the coating porosity. The coatings consisted primarily of β-Ta2O5 phase, a small amount of α-Ta2O5, and minor C peaks corresponding to the CNTs. As the CNT content increased, the elastic modulus (E) and indentation fracture toughness also increased. However, the higher CNT content increased the coating porosity and reduced the microhardness. Following immersion in simulated body fluid (SBF) for 14 days, the surfaces of all the coatings were completely covered with hemispherical bone-like apatite. Furthermore, after incubation in osteoblast-like osteosarcoma MG-63 cell culture for 7 days, all of the coatings showed excellent cell attachment, growth and spreading. Overall, the present results show that the addition of CNTs to Ta2O5 raw powder improves the indentation fracture toughness of the resulting coatings without degrading their biological properties.

Antidepressant-like Effects of BDNF and NGF Individual Loop Dipeptide Mimetics Depend on the Signal Transmission Patterns Associated with Trk.

Neurotrophins are considered as an attractive target for the development of antidepressants with a novel mechanism of action. Previously, the dimeric dipeptide mimetics of individual loops of nerve growth factor, NGF (GK-6, loop 1; GK-2, loop 4) and brain-derived neurotrophic factor, BDNF (GSB-214, loop 1; GTS-201, loop 2; GSB-106, loop 4) were designed and synthesized. All the mimetics of NGF and BDNF in vitro after a 5–180 min incubation in a HT-22 cell culture were able to phosphorylate the tropomyosin-related kinase A (TrkA) or B (TrkB) receptors, respectively, but had different post-receptor signaling patterns. In the present study, we conduct comparative research of the antidepressant-like activity of these mimetics at acute and subchronic administration in the forced swim test in mice. Only the dipeptide GSB-106 that in vitro activates mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK), phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) and phospholipase C-gamma (PLCγ) post-receptor pathways exhibited antidepressant-like activity (0.1 and 1.0 mg/kg, ip) at acute administration. At the same time, the inhibition of any one of these signaling pathways completely prevented the antidepressant-like effects of GSB-106 in the forced swim test. All the NGF mimetics were inactive after a single injection regardless of post-receptor in vitro signaling patterns. All the investigated dipeptides, except GTS-201, not activating PI3K/AKT in vitro unlike the other compounds, were active at subchronic administration. The data obtained demonstrate that the low-molecular weight BDNF mimetic GSB-106 that activates all three main post-receptor TrkB signaling pathways is the most promising for the development as an antidepressant.

Quantitative, traceable determination of cell viability using absorbance microscopy.

Cell viability, an essential measurement for cell therapy products, lacks traceability. One of the most common cell viability tests is trypan blue dye exclusion where blue-stained cells are counted via brightfield imaging. Typically, live and dead cells are classified based on their pixel intensities which may vary arbitrarily making it difficult to compare results. Herein, a traceable absorbance microscopy method to determine the intracellular uptake of trypan blue is demonstrated. The intensity pixels of the brightfield images are converted to absorbance images which are used to calculate moles of trypan blue per cell. Trypan blue cell viability measurements, where trypan blue content in each cell is quantified, enable traceable live-dead classifications. To implement the absorbance microscopy method, we developed an open-source AbsorbanceQ application that generates quantitative absorbance images. The validation of absorbance microscopy is demonstrated using neutral density filters. Results from four different microscopes demonstrate a mean absolute deviation of 3% from the expected optical density values. When assessing trypan blue-stained Jurkat cells, the difference in intracellular uptake of trypan blue in heat-shock-killed cells using two different microscopes is 3.8%. Cells killed with formaldehyde take up ~50% less trypan blue as compared to the heat-shock-killed cells, suggesting that the killing mechanism affects trypan blue uptake. In a test mixture of approximately 50% live and 50% dead cells, 53% of cells were identified as dead (±6% standard deviation). Finally, to mimic batches of low-viability cells that may be encountered during a cell manufacturing process, viability was assessed for cells that were 1) overgrown in the cell culture incubator for five days or 2) incubated in DPBS at room temperature for five days. Instead of making live-dead classifications using arbitrary intensity values, absorbance imaging yields traceable units of moles that can be compared, which is useful for assuring quality for biomanufacturing processes.

Microscale impeller pump for recirculating flow in organs-on-chip and microreactors.

Fluid flow is an integral part of microfluidic and organ-on-chip technology, ideally providing biomimetic fluid, cell, and nutrient exchange as well as physiological or pathological shear stress. Currently, many of the pumps that actively perfuse fluid at biomimetic flow rates are incompatible with use inside cell culture incubators, require many tubing connections, or are too large to run many devices in a confined space. To address these issues, we developed a user-friendly impeller pump that uses a 3D-printed device and impeller to recirculate fluid and cells on-chip. Impeller rotation was driven by a rotating magnetic field generated by magnets mounted on a computer fan; this pump platform required no tubing connections and could accommodate up to 36 devices at once in a standard cell culture incubator. A computational model was used to predict shear stress, velocity, and changes in pressure throughout the device. The impeller pump generated biomimetic fluid velocities (50-6400 μm s-1) controllable by tuning channel and inlet dimensions and the rotational speed of the impeller, which were comparable to the order of magnitude of the velocities predicted by the computational model. Predicted shear stress was in the physiological range throughout the microchannel and over the majority of the impeller. The impeller pump successfully recirculated primary murine splenocytes for 1 h and Jurkat T cells for 24 h with no impact on cell viability, showing the impeller pump's feasibility for white blood cell recirculation on-chip. In the future, we envision that this pump will be integrated into single- or multi-tissue platforms to study communication between organs.

Enhancing in vitro oocyte maturation competence and embryo development in farm animals: roles of vitamin-based antioxidants – A review

Abstract Oocyte/embryo in vitro culture is one of the most important assisted reproductive technologies used as a tool for maintaining genetic resources biodiversity and the inheritance of valuable genetic resources through generations. The success of such processes affects the final goal of the in vitro culture, getting viable and healthy offspring. In common in vitro oocyte maturation and/or embryo development techniques, the development of oocytes/embryos is carried out at 5% carbon dioxide and roughly 20% atmosphere-borne oxygen ratios in cell culture incubators due to their reduced cost in comparison with low atmospheric oxygen-tension incubators. These conditions are usually accompanied by the emergence of reactive oxygen species (ROS), which can extremely damage cell membrane integrity and other vital cellular organelles, as well as genetic material. The present review mainly focuses on the antioxidant roles of different vitamins on in vitro oocyte maturation competence and embryo development in farm animals. The use of antioxidant agents may prevent the extreme augmentation of ROS generation and enhance in vitro matured oocyte competence and embryo development. Therefore, this review aimed to provide an updated outline of the impact of antioxidant vitamin (Vit) supplementations during in vitro maturation (IVM) and in vitro fertilization (IVF) on oocyte maturation and consequent embryo development, in various domestic animal species. Thus, the enrichment of the culture media with antioxidant agents may prevent and neutralize the extreme augmentation of ROS generation and enhance the in vitro embryo production (IVEP) outcomes.

Low-cost, open-source cell culture chamber for regulating physiologic oxygen levels

The physiological oxygen levels for several mammalian cell types in vivo are considered to be hypoxic (low oxygen tension), but the vast majority of in vitro mammalian cell culture is conducted at atmospheric oxygen levels of around 21%. In order to understand the impact of low oxygen environments on cells, oxygen levels need to be regulated during in vitro culture. Two common methods for simulating a hypoxic environment are through the regulation of gas composition or chemical induction. Chemically mimicking hypoxia can have adverse effects such as reducing cell viability, making oxygen regulation in cell culture chambers crucial for long-term culture. However, oxygen-regulating cell culture incubators and commercial hypoxia chambers may not always be a viable option due to cost and limited customization. Other low-cost chambers have been developed, but they tend to lack control systems or are fairly small scale. Thus, the objective of this project was to design and develop a low-cost, open-source, controllable, and reproducible hypoxia chamber that can fit inside a standard cell culture incubator. This design allows for the control of O2 between 1 and 21%, while maintaining CO2 levels at 5%, as well as monitoring of temperature, pressure, and relative humidity. Testing showed our hypoxia chamber was able to maintain CO2 levels at 5% and hypoxic O2 levels at 1% and 5% for long-term cell culture. This simple and easy-to-manufacture design uses off the shelf components, and the total material cost was $832.47 (USD).

Optogenetic control of cancer cell survival in ChR2‐transfected HeLa cells

Optogenetics is a molecular biological technique involving transfection of cells with photosensitive proteins and the subsequent study of their biological effects. The aim of this study was to evaluate the effect of blue light on the survival of HeLa cells, transfected with channelrhodopsin-2 (ChR2). HeLa wild-type cells were transfected with a plasmid that contained the gene for ChR2. Transfection and channel function were evaluated by real-time polymerase chain reaction (RT-PCR), fluorescence imaging using green fluorescent protein (GFP) and flow cytometry for intracellular calcium changes using a Fura Red probe. We developed a platform for optogenetic stimulation for use within the cell culture incubator. Different stimulation procedures using blue light (467nm) were applied for up to 24h. Cell survival was determined by flow cytometry using propidium iodide and rhodamine probes. Change in cell survival showed a statistically significant (p<0.05) inverse association with the frequency and time of application of the light stimulus. This change seemed to be associated with the ChR2 cis-trans-isomerization cycle. Cell death was associated with high concentrations of calcium in the cytoplasm and stimulation intervals less than the period of isomerization. It is possible to transfect HeLa cells with ChR2 and control their survival under blue light stimulation. We suggest that this practice should be considered in the future development of optogenetic systems in biological or biomedical research.