Cell Incubator Research Articles

A modular and flexible open source cell incubator system for mobile and stationary use

Culturing living cells in vitro requires the maintenance of physiological conditions for extended periods of time. Here, we introduce a versatile and affordable incubation system, addressing the limitations of traditional incubation systems. Conventionally, stationary cell incubators maintain constant temperature and gas levels for in vitro cell culturing. Combining such incubators with additional lab equipment proves challenging. The presented platform offers modularity and adaptability, enabling customization to diverse experimental needs. The system includes a main unit with a user-friendly interface as well as an interchangeable incubation chamber. We present two incubation chambers targeting two completely different use cases. The first chamber, named “inkugo” facilitates the transportation of cells for up to two hours without external power and for more than a day without an external CO2 source. The second chamber termed “inkubox” was designed to enable continuous electrophysiological recordings. Recordings from up to four neural cultures growing on high-density microelectrode arrays can be performed in parallel. The system’s unique feature lies in its separability of control and incubation components, allowing one control unit to manage various custom chambers. The design’s simplicity and the use of widely accessible components make the here proposed incubation system replicable for any laboratory. This platform fosters collaboration and experimentation in both decentralized and traditional laboratory settings, making it an invaluable addition to any cell culturing pipeline.

Oxygenator assisted dynamic microphysiological culture elucidates the impact of hypoxia on valvular interstitial cell calcification

IntroductionMicrophysiological systems (MPS) offer simulation of (patho)physiological parameters. Investigation includes items which lead to fibrosis and calcification in development and progress of calcific aortic valve disease, based e.g. on culturing of isolated valvular interstitial cells (VICs). Hypoxia regulated by hypoxia inducible factors impacts pathological differentiation in aortic valve (AV) disease. This is mimicked via an MPS implemented oxygenator in combination with calcification inducing medium supplementation.MethodsHuman valvular interstitial cells were isolated and dynamically cultured in MPS at hypoxic, normoxic, arterial blood oxygen concentration and cell incubator condition. Expression profile of fibrosis and calcification markers was monitored and calcification was quantified in induction and control media with and without hypoxia and in comparison to statically cultured counterparts.ResultsHypoxic 24-hour culture of human VICs leads to HIF1α nuclear localization and induction of EGLN1, EGLN3 and LDHA mRNA expression but does not directly impact expression of fibrosis and calcification markers. Dependent on medium formulation, induction medium induces monolayer calcification and elevates RUNX2, ACTA2 and FN1 but reduces SOX9 mRNA expression in dynamic and static MPS culture. But combining hypoxic oxygen concentration leads to higher calcification potential of human VICs in calcification and standard medium formulation dynamically cultured for 96 h.ConclusionIn hypoxic oxygen concentration an increased human VIC calcification in 2D VIC culture in an oxygenator assisted MPS was detected. Oxygen regulation therefore can be combined with calcification induction media to monitor additional effects of pathological marker expression. Validation of oxygenator dependent VIC behavior envisions future advancement and transfer to long term aortic valve tissue culture MPS.

Novel Gas Supply System for Multi-Chamber Tri-Gas Cell Culture: Low Gas Consumption and Wide Concentration Range

Gas plays a crucial role in cell culture as cells require a specific gas environment to maintain their growth, reproduction, and function. Here, we propose a gas supply system for tri-gas multi-channel cell incubators to meet the specific needs of various cells. The system utilizes a circulating gas supply method powered by air pumps for each chamber. Gas inflow from the cylinder is independently controlled by Mass Flow Controllers (MFCs), and a quantitative step-by-step adjustment control strategy is employed to calculate the volume of different gases being introduced. Through mixing simulations and experiments, we identified the SV static mixer with an L/D ratio of 2.5 as the optimal choice. To evaluate the concentration accuracy and gas consumption of the gas system, we conduct gas mixing and distribution experiments under different conditions. The results show that the system could achieve a concentration range of 0–100% for O2 with an accuracy of ±0.5%, and a concentration range of 0–10% for CO2 with an accuracy of ±0.1%. The daily gas consumption during cultivation is 3570 mL of N2, 330 mL of CO2, and 115 mL of O2, significantly lower than conventional incubators. Overall, our system can effectively manage dynamic gas concentration changes, particularly in high O2 concentration environments. It offers advantages such as low gas consumption, a wide concentration range, and high accuracy compared to existing incubators.

Automated xeno-free chondrogenic differentiation from human embryonic stem cells: Enhancing efficiency and ensuring high-quality mass production

IntroductionRepairing damaged cartilage poses significant challenges, particularly in cases of congenital cartilage defects such as microtia or congenital tracheal stenosis, or as a consequence of traumatic injury, as the regenerative potential of cartilage is inherently limited. Stem cell therapy and tissue engineering offer promising approaches to overcome these limitations in cartilage healing. However, the challenge lies in the size of cartilage-containing organs, which necessitates a large quantity of cells to fill the damaged areas. Therefore, pluripotent stem cells that can proliferate indefinitely are highly desirable as a cell source. This study aims to delineate the differentiation conditions for cartilage derived from human embryonic stem cells (ESCs) and to develop an automated cell culture system to facilitate mass production for therapeutic applications. MethodsCartilage cell sheets were derived from human ESCs (SEES2, clinical trial-compatible line) by forming embryoid bodies (EBs) with either conventional manual culture or a benchtop multi-pipetter and an automated medium exchange integrated cell incubator, using xeno-free media. Cell sheets were implanted into the subcutaneous tissue of immunodeficient NOG mice to obtain cartilage tissue. The properties of cartilage tissues were examined by histological staining and quantitative PCR analysis. ResultsWe have optimized an efficient xeno-free system for cartilage production with the conventional culture method and successfully transitioned to an automated system. Differentiated cartilage was histologically uniform with cartilage-specific elasticity and strength. The cartilage tissues were stained by Alcian blue, safranin O, and toluidine blue, and quantitative PCR showed an increase in differentiation markers such as ACAN, COL2A1, and Vimentin. Automation significantly enhanced the efficiency of human ESC-derived chondrocyte differentiation. The number of constituent cells within EBs and the seeding density of EBs were identified as key factors influencing chondrogenic differentiation efficiency. By automating the process of chondrogenic differentiation, we achieved scalable production of chondrocytes. ConclusionsBy integrating the differentiation protocol with an automated cell culture system, there is potential to produce cartilage of sufficient size for clinical applications in humans. The resulting cartilage tissue holds promise for clinical use in repairing organs such as the trachea, joints, ears, and nose.

Development of a kidney microphysiological system hardware platform for microgravity studies

Determining the physiological effects of microgravity on the human kidney is limited to relatively insensitive tests of biofluids (blood and urine) that do not return abnormal results until more than 50% of kidney function is lost. We have developed an “organ on chip” microphysiological model of the human kidney proximal tubule (PT-MPS) that can recapitulate many kidney functions and disease states and could play a critical role in determining mechanisms of early kidney dysfunction in microgravity. However, the ground-based PT-MPS system is incompatible with spaceflight as it requires a large pneumatic system coupled to a cell incubator for perfusion and intensive hand-on manipulation. Herein, we report the hardware engineering and performance of the Kidney Chip Perfusion Platform (KCPP), a small, advanced, semi-autonomous hardware platform to support kidney microphysiological model experiments in microgravity. The KCPP is composed of five components, the kidney MPS, the MPS housing and valve block, media cassettes, fixative cassettes, and the programable precision syringe pump. The system has been deployed twice to the ISSNL (aboard CRS-17 and CRS-22). From each set of ISSNL experiments and ground-based controls, we were able to recover PT-MPS effluent for biomarker analysis and RNA suitable for transcriptomics analysis demonstrating the usability and functionality of the KCPP.

Effects of Hypoxia and Reoxygenation on Metabolic Profiles of Cardiomyocytes.

In vitro cellular models provide valuable insights into the adaptive biochemical mechanisms triggered by cells to cope with the stress situation induced by hypoxia and reoxygenation cycles. The first biological data generated in studies based on this micrometric life-scale has the potential to provide us a global overview about the main biochemical phenomena presented in some reported preconditioning therapies in life-scale of higher dimensions. Thus, in this study, a cell incubator was designed and manufactured to produce a cellular model of heart hypoxia followed by reoxygenation (HfR) through consecutive repetitions of hypoxia-normoxia gas exchange. Samples of cellular extracts and culture media were obtained from non-proliferative cardiomyocytes (CMs) cultivated under challenging HfR (stressed CMs) and regular cultivation (unstressed CMs) in rounds of four days for each case. Metabolomic based on proton magnetic resonance spectroscopy (1H-MRS) was used as an analytical approach to identify and quantify the metabolomes of these samples, the endo- and exo-metabolome. Despite the stressed CMs presented over 90% higher cellular death rate compared to the unstressed CMs, the metabolic profiles indicates that the surviving cells up-regulate their amino acid metabolism either by active protein degradation or by the consumption of culture media components to increase coenzyme A-dependent metabolic pathways. This cell auto-regulation mechanism could be well characterized in the first two days when the difference smears off under once the metabolomes become similar. The metabolic adaptations of stressed CMs identified the relevance of the cyclic oxidation/reduction reactions of nicotinamide adenine dinucleotide phosphate molecules, NADP+/NADPH, and the increased tricarboxylic acid cycle activity in an environment overloaded with such a powerful antioxidant agent to survive an extreme HfR challenge. Thus, the combination of cellular models based on CMs, investigative methods, such as metabolomic and 1H-MRS, and the instrumental development of hypoxia incubator shown in this work were able to provide the first biochemical evidences behind therapies of gaseous exchanges paving the way to future assays.

Toroidal-spiral particles as a CAR-T cell delivery device for solid tumor immunotherapy

Chimeric antigen receptor (CAR) T cell therapy has resulted in positive effects on patients with hematologic malignancy but shows limited efficacy in solid tumor treatments due to insufficient trafficking and tumor infiltration, intensive CAR-T-related toxicities, and antigen escape. In this work, we developed and investigated a biodegradable and biocompatible polymeric toroidal-spiral particle (TSP) as a in vivo cell incubator and delivery device that can be implanted near tumor through a minimally invasive procedure or injected near or into solid tumors by using a biopsy needle. The main matrix structure of the millimeter-sized TSP is made from crosslinking of gelatin methacrylamine (GelMA) and poly (ethylene glycol) diacrylate (PEGDA) with a tunable degradation rate from a few days to months, providing appropriate mechanical properties and sustained release of co-encapsulated drugs and/or stimulation compounds. The toroidal-spiral layer of the particles, presenting an internal void volume for high-capacity cell loading and flexibility of co-encapsulating small and large molecular compounds with individually manipulated release schedules, is filled with collagen and suspended T cells. The TSPs promote cell proliferation, activation, and migration in the tumor micro-environment in a prolonged and sustained manner. In this study, the efficacy of mesothelin (MSLN) CAR-T cells released from the TSPs was tested in preclinical mouse tumor models. Compared to systemic and intratumoral injection, peritumoral delivery of MSLN CAR-T cells using the TSPs resulted in a superior antitumor effect. The TSPs made of FDA approved materials as an in vivo reactor may provide an option for efficiently local delivery of CAR-T cells to solid tumors for higher efficacy and lower toxicity, with a minimally invasive administration procedure.

Hyperpolarized 13C NMR for longitudinal in-cell metabolism using a mobile 3D cell culture system

Hyperpolarization with the dissolution dynamic nuclear polarization (dDNP) technique yields > 10,000-fold signal increases for NMR-active nuclei (e.g. 13C). Hyperpolarized 13C-labeled metabolic tracer molecules thus allow real-time observations of biochemical pathways in living cellular systems without interfering background. This methodology lends itself to the direct observation of altered intracellular reaction chemistry imparted for instance by drug treatment, infections, or other diseases. A reoccurring challenge for longitudinal cell studies of mammalian cells with NMR and dDNP-NMR is maintaining cell viability in the NMR spectrometer. 3D cell culture methods are increasing in popularity because they provide a physiologically more relevant environment compared to 2D cell cultures. Based on such strategies a mobile 3D culture system was devised. The clinical drug etoposide was used to treat cancer cells (HeLa) and the resulting altered metabolism was measured using hyperpolarized [1–13C]pyruvate. We show that sustaining the cell cultivation in cell incubators and only transferring the cells to the NMR spectrometer for the few minutes required for the dDNP-NMR measurements is an attractive alternative to cell maintenance in the NMR tube. High cell viability is sustained, and experimental throughput is many doubled.

Quartz-Enhanced Photoacoustic Spectroscopy-Conductance Spectroscopy for Gas Mixture Analysis.

A novel spectroscopic method, named quartz-enhanced photoacoustic spectroscopy-conductance spectroscopy (QEPAS-CS), was first developed for gas mixture analysis. In QEPAS-CS, the advantage of photoacoustic detection and conductance analysis was realized by a quartz tuning fork (QTF). Two-component gas analysis was done by photoacoustic detection and conductance detection. For an explicit application, natural spider silk was used as a water vapor transducer to modify the QTF, making a conductance sensing channel. A 2004 nm laser diode was used as an excitation source for a photoacoustic sensing channel. Such a QEPAS-CS sensor was used for H2O/CO2 gas mixture analysis in a cell incubator. This provides a solution to calibrate an infrared photoacoustic spectroscopy gas sensor. This example effectively confirms the capacity of multigas analysis by the QEPAS-CS sensor.

Fast and Precise Temperature Control for Axon Stretch Growth Bioreactor Based on Fuzzy PID Control.

A suitable environment is essential for successful long-term cell culturing in vitro. Too high or too low temperature will affect the growth of cells, so we need to maintain the constant temperature of the cell culture environment. Usually, cells are cultured in a cell incubator, and the constant temperature is provided by the cell incubator. Recently, we have developed a multi-channel axon stretch growth bioreactor for rapid acquisition of autologous nerve tissue. Since the motor and controller are placed in the incubator for a long time, the service life of the equipment will be shortened or even damaged due to high humidity and weak acid environment. In order to enable the axon stretch growth bioreactor to culture cells independently, we designed a constant temperature control system for the device. Firstly, the simulation results show that the fuzzy PID control reduces the overshoot and improves the traditional PID control with large overshoot and low control precision. Then, the two control algorithms were applied to the multi-channel axon stretch growth bioreactor by STM32F4 microcontroller. The experimental data show that the fuzzy PID control algorithm has good control effect and can meet the requirement of constant temperature of cell growth. Finally, nerve cells derived from human pluripotent stem cells were successfully cultured in a cell culture amplification chamber under a constant temperature environment provided by a fuzzy PID controller, and well-developed axons could be seen. In the future, we may transplant stretch growth axons into living organisms to repair nerve damage.

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.

Design of Biplanar Excitation Magnetic Field Coils With Small-Size and Large Uniform Area in Near-Zero Magnetic Cell Incubator

Low-frequency excitation magnetic field coils are potent tools for studying the effects of magnetic fields on cells. However, the existing excitation magnetic field coils are vulnerable to external magnetic fields and difficult to generate a high uniform excitation magnetic field with a small size. Thus, this study proposes a novel optimization design method combining the target field method (TFM), thickness mirror image method, and fruit fly optimization algorithm (FOA) to design a new type of bi-planar excitation magnetic field coil placed in a near-zero magnetic cell incubator. Based on the optimization design method, three bi-planar coils of <italic xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">B_x</i> , <italic xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">B_y</i> and <italic xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">B_z</i> with the paralleled arrangement in a near-zero magnetic cell incubator with the size of 365 mm × 336 mm × 200 mm were designed and measured. The coil constants of <italic xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">B_x</i> ( <italic xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">B_y</i> ) and <italic xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">B_z</i> are 8860 nT/A and 100640 nT/A, respectively. The non-uniformity of the <italic xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">B_x</i> ( <italic xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">B_y</i> ) and <italic xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">B_z</i> coils are less than 5% and 1% over the central volume of 80 × 80 × 80 mm <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">3</sup> , 100 × 100 × 100 mm <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">3</sup> , respectively, meeting the most requirement of working space and the amplitude of magnetic field for cell research.

Simple CO2 Regulator for Laboratory Cell Incubator from Available Components

Simple CO2 Regulator for Laboratory Cell Incubator from Available Components

Self-loading microfluidic platform with ultra-thin nanoporous membrane for organ-on-chip by wafer-level processing

Embedded porous membranes are a key element of various organ-on-chip systems. The widely used commercial polymer membranes impose limits with regard to chip integration and thinness. We report a microfluidic chip platform with the key element of a monolithically integrated, ultra-thin (700 nm) nanoporous membrane made of ultra-low-stress (&lt;35 MPa) SixNy for culturing and testing reconstructed tissue. The membrane is designed to support various in vitro tissues including co-cultures and to allow passage of molecules but not of cells. A digital laser write method was used to produce nanopores with deterministic but highly flexible positioning within the membrane. A thin layer of photoresist was exposed by accumulation of femtosecond pulses for local two-photon polymerization, which allowed nanopores as small as 350 nm in diameter to be generated through the membranes in a subsequent plasma etch process. The fabricated membranes were used to separate a microfluidic chip into two compartments, which are connected to the outside by microchannel structures. With unique side inlets for fluids, all cells are exposed to identical flow velocities and shear stresses. With the hydrophilic nature of chip materials the self-loading seeding is controlled bottom-up by capillary forces, which makes the seeding procedure homogeneous and less dependent on the operator. The chip is designed to allow fabrication by wafer-level MEMS manufacturing technologies without critical assembly steps, thereby promoting reproducibility and scale-up of fabrication. In order to establish a fully functional test system to be used in a lab incubator, a holder for the bare chip was designed and 3D-printed with additional elements for gravity driven pumping. In order to mimic physiological conditions, the holder was designed to provide not only media delivery but also appropriate shear stress to the tissue. To prove usability, murine microvascular endothelial cells (muMEC) were seeded on the membrane within the chip. Cell compatibility was confirmed after 3 days of dynamic cultivation using fluorescence live/dead assays. Cultivation proved to be reproducible and led to confluent layers with cells preferentially grown on nanoporous areas. The system can in future be cost effectively manufactured in larger quantities in MEMS foundries and can be used for a wide variety of in vitro tissues and test scenarios including pumpless operation within cell incubator cabinets.

Development of biocompatible superelastic materials and shape memory implants based on titanium nickelide for the creation of highly effective medical technologies

The paper presents fundamental and applied research in a new direction in science and technology, which has a world priority – medical materials and implants with shape memory. The main directions of the development of a new generation of more advanced biocompatible superelastic implantable materials and long-term shape memory implants based on titanium nickelide are formulated. The physical, biological principles of creating a new class of biocompatible superelastic materials based on titanium nickelide with a certain set of properties for various fields of medicine using technologies of induction melting, self-propagating high-temperature synthesis, and sintering are presented.Among the materials created, the development of alloys for long-term functioning in a living organism is of great importance: monolithic elastic, porous and ultra-thin porous-permeable materials, fabric and textile materials made of thin filaments based on titanium nickelide. The main medical and technical requirements for a new class of implants of the future have been identified. Criteria for the biomechanical compatibility of metallic materials under deformation conditions in aggressive tissue media have been determined. Conditions are formulated under which monolithic and porous materials exhibit properties that are optimal for implantation. A comprehensive study of the properties of cell incubators made of porous titanium nickelide was carried out, and the morphological characteristics and patterns of cell development in the porous structure of bulk biomaterials made of titanium nickelide were revealed.The main directions of creation of unique medical technologies based on the developed materials are characterized. The principles of application of biocompatible superelastic materials in various fields of medicine are described. Titanium nickelide-based materials and implants with shape memory implanted into the body are intended for a wide range of medical purposes in dentistry, traumatology, surgery, ophthalmology, vascular surgery, oncology and other fields of medicine as implants for osteosynthesis, endoprosthetics, replacement of tissue defects, creating anastomoses and support frames of organs.

3D time-lapse imaging of a mouse embryo using intensity diffraction tomography embedded inside a deep learning framework.

We present a compact 3D diffractive microscope that can be inserted directly in a cell incubator for long-term observation of developing organisms. Our setup is particularly simple and robust, since it does not include any moving parts and is compatible with commercial cell culture containers. It has been designed to image large specimens (>100×100×100µm3) with subcellular resolution. The sample's optical properties [refractive index (RI) and absorption] are reconstructed in 3D from intensity-only images recorded with different illumination angles produced by an LED array. The reconstruction is performed using the beam propagation method embedded inside a deep-learning network where the layers encode the optical properties of the object. This deep neural network is trained for a given multiangle intensity acquisition. After training, the weights of the neural network deliver the 3D distribution of the optical properties of the sample. The effect of spherical aberrations due to the sample holder/air interfaces are taken into account in the forward model. Using this approach, we performed time-lapse 3D imaging of preimplantation mouse embryos over six days. Images of embryos from a single cell (low-scattering regime) to the blastocyst stage (highly scattering regime) were successfully reconstructed. Due to its subcellular resolution, our system can provide quantitative information on the embryos' development and viability. Hence, this technology opens what we believe to be novel opportunities for 3D label-free live-cell imaging of whole embryos or organoids over long observation times.

A Versatile, Incubator-Compatible, Monolithic GaN Photonic Chipscope for Label-Free Monitoring of Live Cell Activities.

The ability to quantitatively monitor various cellular activities is critical for understanding their biological functions and the therapeutic response of cells to drugs. Unfortunately, existing approaches such as fluorescent staining and impedance‐based methods are often hindered by their multiple time‐consuming preparation steps, sophisticated labeling procedures, and complicated apparatus. The cost‐effective, monolithic gallium nitride (GaN) photonic chip has been demonstrated as an ultrasensitive and ultracompact optical refractometer in a previous work, but it has never been applied to cell studies. Here, for the first time, the so‐called GaN chipscope is proposed to quantitatively monitor the progression of different intracellular processes in a label‐free manner. Specifically, the GaN‐based monolithic chip enables not only a photoelectric readout of cellular/subcellular refractive index changes but also the direct imaging of cellular/subcellular ultrastructural features using a customized differential interference contrast (DIC) microscope. The miniaturized chipscope adopts an ultracompact design, which can be readily mounted with conventional cell culture dishes and placed inside standard cell incubators for real‐time observation of cell activities. As a proof‐of‐concept demonstration, its applications are explored in 1) cell adhesion dynamics monitoring, 2) drug screening, and 3) cell differentiation studies, highlighting its potential in broad fundamental cell biology studies as well as in clinical applications.

Neuroprotective potential of nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant response element signaling modulator cucurbitacin I upon glucose and oxygen deprivation/reperfusion (OGD/RP).

This study aimed to investigate the inhibitory effect and mechanism of Cucurbitacin I (Cu I) on apoptosis, oxidative stress, and mitophagy in PC12 cells with glucose and oxygen deprivation/reperfusion (OGD/RP) injury. OGD/RP cell injury model was established by gas anoxic cell incubator and glucose-free medium. The cells were divided into the control group, OGD/RP group, OGD/RP + Cu I group, and OGD/RP + Cu I + 2 µM nuclear factor erythroid 2-related factor 2 (Nrf2) inhibitor ML385 group. The results showed that apoptotic rate and reactive oxygen species (ROS) production were significantly increased in OGD/RP group, which were reversed by Cu I pretreatment. Meanwhile, western blot analysis proved that Cu I inhibited OGD/RP-induced mitophagy, manifested as the decreased expression of PTEN-induced kinase 1 (PINK1) and parkin RBR E3 ubiquitin-protein ligase (Parkin), and light chain 3 (LC3) Ⅱ∕LC3 I, as well as the increased expression of P62. Furthermore, immunofluorescence (IF) staining showed that Cu I reduced the co-localized puncta of LC3 with TOM20 in OGD/RP-induced PC12 cells. Similarly, transmission electron microscope finding is consistent with the IF results. Mechanically, after Cu I and OGD/RP treatments, nuclear Nrf2 expression and the levels of downstream target genes were significantly upregulated compared with OGD/RP alone treatment. Nrf2 inhibition reversed the protective effects of Cu I on OGD/RP-induced injury in PC12 cells. The present study provides evidence of the neuroprotective effect of Cu I unraveling its potential as a potential therapeutic candidate for the treatment of ischemic stroke.

An Endogenous miRNA-Initiated Hybridization Chain Reaction and Subsequent DNAzyme Activation for Cellular Theranostics

An Endogenous miRNA-Initiated Hybridization Chain Reaction and Subsequent DNAzyme Activation for Cellular Theranostics

Abstract 10535: Tissue-Incubation-Chamber Provides Enhanced Viability of Porcine Aortic Valve Tissue in Dynamic Long-Term Culture

Introduction: Aortic valve (AV) stenosis is caused by fibrosis and calcification of the three-layered valve tissue. Current in vitro and in vivo models are insufficient to reflect cell-cell or cell-matrix interaction and pathophysiological processes. Therefore a novel tissue incubation chamber (TIC) enabling dynamic AV-tissue culture in micro-physiological systems (MPS) was designed to improve emulation of the extreme in vivo conditions of the cardiac cycle. This pilot study aims to verify tissue viability to ensure applicability of the TIC in conjunction with a MPS in AV research. Methods: Porcine AV tissue of 3x5 mm is maintained in the TIC for 14 days within a conventional cell incubator under sterile conditions. A pump-chip able to generate a pulsatile flow rate of 13.4 μl/s (150 bpm) accomplishes average shear forces of 0.017 dyn/cm 2 . Two viability assays were performed to compare dynamic and static setups. AV samples were snap frozen, cryo sectioned and intracellular LDH activity in central tissue regions was visualized using a Tetrazolium-based dye. In addition, a Resazurin reduction assay (50-300 μM) was applied. Results: The LDH-viability stain reveals no significant reduction of viable tissue sections in dynamically incubated AV tissues (96.4 ± 1.3 %) but a significant decrease in the static counterparts (67.8 ± 4.7 %, n=3). The Resazurin assay on the other hand showed significant viability reduction in dynamic setup after 12 days (57 ± 5.4 %), whereas significant reduction was already detected in static culture after 8 days (57.5 ± 15.1 %, n=4). Due to the discrepancy of both viability assays, an inadequate diffusion rate based on structural AV tissue integrity was hypothesized with the need to adapt Resazurin assay concentration. A saturation curve conducted consecutively determined an optimal Resazurin concentration of 300μM to be used in upcoming AV tissue culture (n=4). Conclusion: Enhanced viability of porcine AV tissue following dynamic incubation in the TIC was achieved but further adaptation in terms of higher shear forces and a more physiological sample fixation is envisioned. Dynamically cultured AV tissue cultures in the novel TICs offer a relevant improvement for investigation of AV pathophysiology.