High-Throughput Analysis of 3D Cell Culture Oxygen Consumption Using Sensor Arrays: A Novel Platform for Hypoxia/Normoxia Research

Microfluidic devices can be used to simulate complex microhabitats where the concentration gradients of oxygen, nutrients, or other chemical stimulants need to be dynamically controlled. Though oxygen gradients can be achieved by flowing an oxygenated fluid through a microchannel and allowing the oxygen to diffuse through a polymer membrane/hydrogel to the cell culture area, this configuration does not allow the precise control either spatially or temporally [1]. The diverse microbial community of the termite gut is an example of a naturally occurring microhabitat that experiences steep oxygen gradients [2]. These oxygen gradients are really necessary for the formation of spatially-distinct microniches for the protists, bacteria, and archea inhabiting the gut, while the microniches allow microbial symbionts to co-exist with each other and the termite host while breaking down various carbon sources into useful products such as acetate. In this presentation, we will detail a new method to generate, control, and model the oxygen gradients within a microfluidic habitat. A novel microfabrication method was developed to photopattern gold electrodes on the vertical side walls of the microchannels for oxygen generation, as shown in Figure 1 [3]. Finite element simulations were conducted to model oxygen production rates under varied geometries, operating currents, and flow rates. Additionally, in-plane hydrogel barriers were constructed between the channels containing the electrodes and the cell culture chambers, to maintain the stable oxygen gradient and also dampen any sudden changes to the system that could harm cells. With these recent advances in microfabrication and modeling techniques, the micro-scale physical and chemical features of a termite gut (and other communities) can be synthetically recreated in microfluidic devices. [1] Brune, A., D. Emerson, and J.A. Breznak, The Termite Gut Microflora as an Oxygen Sink: Microelectrode Determination of Oxygen and pH Gradients in Guts of Lower and Higher Termites. Appl Environ Microbiol, 1995. 61, 2681-2687. [2] Brune, A., Termite Guts: The World's Smallest Bioreactors. Trends Biotechnol., 1998. 16, 16-21. [3] Kadilak, A. L., Liu, Y., Shrestha, S., Bernard, J. R., Mustain, W. E., and Shor, L. M., Selective Deposition of Chemically-bonded Gold Electrodes onto PDMS Microchannel Side Walls. J. Electroanal. Chem. , 2014. 727, 141-147. Figure 1

Recent microfluidic advancements in oxygen gradients have greatly promoted controllable oxygen-sensitive cellular investigations at microscale resolution. However, multi-gradient integration in a single microfluidic device for tissue-mimicking cell investigation is not yet well established. In this study, we describe a method that can generate oxygen and chemical concentration gradients in a single microfluidic device via the formation of an oxygen gradient in a chamber and a chemical concentration gradient between adjacent chambers. The oxygen gradient dynamics were systematically investigated, and were quantitatively controlled using simple exchange between the aerial oxygen and the oxygen-free conditions in the gas-permeable polydimethylsiloxane channel. Meanwhile, the chemical gradient dynamics was generated using a special channel-branched device. For potential medical applications of the established oxygen and chemical concentration gradients, a tumor cell therapy assessment was performed using two antitumor drugs (tirapazamine and bleomycin) and two tumor cell lines (human lung adenocarcinoma A549 cells and human cervical carcinoma HeLa cells). The results of the proof-of-concept experiment indicate the dose-dependent antitumor effect of the drugs and hypoxia-induced cytotoxicity of tirapazamine. We demonstrate that the integration of oxygen and chemical concentration gradients in a single device can be applied to investigating oxygen- and chemical-sensitive cell events, which can also be valuable in the development of multi-gradient generating procedures and specific drug screening.

Novel emerging cell and organoid systems for the study of drug metabolism and toxicity in humans.

A laser-induced fluorescence (LIF) technique is employed for visualizing a thin two-dimensional (2D) dissolved oxygen concentration field and measuring local oxygen concentration gradients near the surface of an oxygen bubble in water containing surfactant (Triton X-100, SigmaAldrich, St Louis, MO, USA)). The fluorescence of pyrene butyric acid (PBA) is induced by a planar pulse of nitrogen laser light. Oxygen transferring from the bubble to the deoxygenated water quenches the fluorescence of the PBA. Images of the fluorescence fields are captured by a UV-intensified CCD camera. The intensity of fluorescence quenching at each image pixel is used to measure dissolved oxygen concentration in a 2D field. Images of bubbles are obtained at 200 ppm, 100 ppm, and 50 ppm Triton X-100-containing water and in ultra clean deionized water. Higher surfactant concentrations decrease local and average concentration gradients of oxygen at the bubble surface. The ensemble means of dissolved oxygen concentration boundary layer thicknesses of 0.160 mm, 0.130 mm, and 0.072 mm, for the images of bubbles obtained at 200 ppm, 100 ppm, and 50 ppm Triton X-100-containing water, respectively. Local concentration boundary layer thickness increases from the top to the bottom along the bubble surface. A series of images of the bubble flow fields are analyzed to measure the oxygen concentration gradients in water in the presence of surfactant. The images captured in clean water are not fully resolvable because of their poor resolution. The formation of the attached wake in the fluorescence field images at the bottom of the bubbles in clean water tends to be promoted by increasing oblateness owing to the presence of surfactant at the surface.

In this work we introduce a new series of ratiometric oxygen sensors based on phosphorescent cyclometalated iridium centers partnered with organic coumarin fluorophores. Three different cyclometalating ligands and two different pyridyl-containing coumarin types were used to prepare six target complexes with tunable excited-state energies. Three of the complexes display dual emission, with fluorescence arising from the coumarin ligand, and phosphorescence from either the cyclometalated iridium center or the coumarin. These dual-emitting complexes function as ratiometric oxygen sensors, with the phosphorescence quenched under O2 while fluorescence is unaffected. The use of blue-fluorescent coumarins results in good signal resolution between fluorescence and phosphorescence. Moreover, the sensitivity and dynamic range, measured with Stern–Volmer analysis, can be tuned two orders of magnitude by virtue of our ability to synthetically control the triplet excited-state ordering. The complex with cyclometalated iridium 3MLCT phosphorescence operates under hyperoxic conditions, whereas the two complexes with coumarin-centered phosphorescence are sensitive to very low levels of O2 and function as hypoxic sensors.

Oxygen concentration plays a crucial role in (3D) cell culture. However, the oxygen content in vitro is usually not comparable to the in vivo situation, which is partly due to the fact that most experiments are performed under ambient atmosphere supplemented with 5% CO2, which can lead to hyperoxia. Cultivation under physiological conditions is necessary, but also fails to have suitable measurement methods, especially in 3D cell culture. Current oxygen measurement methods rely on global oxygen measurements (dish or well) and can only be performed in 2D cultures. In this paper, we describe a system that allows the determination of oxygen in 3D cell culture, especially in the microenvironment of single spheroids/organoids. For this purpose, microthermoforming was used to generate microcavity arrays from oxygen-sensitive polymer films. In these oxygen-sensitive microcavity arrays (sensor arrays), spheroids cannot only be generated but also cultivated further. In initial experiments we could show that the system is able to perform mitochondrial stress tests in spheroid cultures to characterize mitochondrial respiration in 3D. Thus, with the help of sensor arrays, it is possible to determine oxygen label-free and in real-time in the immediate microenvironment of spheroid cultures for the first time.

A 3D cell culture is an artificially created environment in which cells are permitted to grow/interact with their surroundings in all three dimensions. Derived from 3D cell culture, organoids are generally small-scale constructs of cells that are fabricated in the laboratory to serve as 3D representations of in vivo tissues and organs. Due to regulatory, economic and societal issues concerning the use of animals in scientific research, it seems clear that the use of 3D cell culture and organoids in for example early stage studies of drug efficacy and toxicity will increase. The combination of such 3D tissue models with mass spectrometry imaging provides a label-free methodology for the study of drug absorption/penetration, drug efficacy/toxicity, and drug biotransformation. In this article, some of the successes achieved to date and challenges to be overcome before this methodology is more widely adopted are discussed.

3D Cell culture is an alternative to animal use in many drug development and toxicity studies. The 3D cell culture can mimic and reproduce the original tissue microenvironment, morphology, and mechanical and physiological characteristics, to provide a more realistic and reliable response as compared to two-dimensional cultures. 3D cell culture encapsulated in alginate beads is a very simple and relatively inexpensive tool that is easy to handle and to maintain. The alginate beads function as a scaffold that imprisons cells and allows 3D cell growth, to generate spheroids that can have greater genic expression and cell-cell communication as a nano or microtissue. The HepG2 cell line is a human hepatocellular carcinoma cell derivative. HepG2 cells preserve several of the characteristics of hepatocytes and are therefore often used in toxicity studies. Here, we describe HepG2 cell encapsulation in alginate beads and analyze the resulting spheroids formed within the alginate beads by immunocytochemistry, by staining a certain structure with a specific antibody coupled with a fluorophore. This method preserves the beads and enables cell analysis by confocal microscopy.

Three-dimensional (3D) cell culture techniques represent a transformative advancement in biomedical research, particularly in drug discovery and development. By more closely replicating the physiological and microenvironmental conditions of in vivo tissues, 3D cell cultures enable more accurate assessments of drug efficacy, toxicity, and therapeutic potential compared to traditional two-dimensional (2D) cultures. These systems not only provide a more realistic model for preclinical testing but also allow for the study of complex cell-cell and cell-matrix interactions, which are often overlooked in 2D systems. This review provides a comprehensive examination of studies utilizing spheroids and organoids in 3D culture systems for drug screening and development. Furthermore, it highlights the critical role of these models in uncovering novel therapeutic targets, understanding disease mechanisms, and optimizing drug delivery strategies. Key challenges, such as scalability, standardization, and integration with high-throughput screening platforms, are also discussed. In conclusion, 3D cell culture techniques hold immense promise for revolutionizing the drug discovery pipeline, offering a more predictive and ethical approach to preclinical research while bridging the gap between laboratory findings and clinical outcomes.

Cell migration plays a pivotal role in various physiological and pathological processes including angiogenesis, cancer metastasis, wound healing, inflammation, and embryogenesis. For cell migration in vitro , the conventional cell migration assays like the Boyden chamber and wound-healing assays are unable to meet the requirements of high-throughput, and are incapable of integrating complex environmental factors to comprehensively consider multi-parameters factors, such as cell matrix and concentration gradient, particularly those effecting cell migration. In contrast, microfluidic chips have the potential to take these challenges by allowing for precise and simultaneous control of multiple environmental factors mimicking in vivo environment of cells. Microfluidic devices successfully produce accurately controllable fluid and stable concentration gradient quickly, and manipulate gradients spatially and temporally. In addition, microfluidic devices enables accurate and reliable cell migration assay on real-time observation with limited amounts of reagents. It has exhibited tremendous potential due to its miniaturization, integration, high-throughput and high-precision. Owing to these particular advantages, over the past few years, a number of microfluidic chips combining two-dimensional (2D) and three-dimensional (3D) cell cultures have already been widely used in cell migration assays, demonstrating that microfluidic technology has significant implications for cell biology and cell-based assays. The microfluidic chips for cell migration assay using 2D platform could be classified into wound-healing and non-wound assays. Wound edges could be attained though a laminar flow of trypsin solution or removing solid barrier. After wound formed, cell migration could be detected and monitored using electric cell-substrate impedance sensing. Non-wound assays include chemotaxis and electrotaxis. The in vivo microenvironment is characterized by a 3D scaffold and multiple cell types. Therefore, increasing microfluidic devices with a 3D cell culture system have been developed for cell chemotaxis. These chips embed cells in a structure manufactured using agarose, collagen and hydrogel that mimics the extracellular matrix (ECM) of structural proteins found in real and living tissues. Various microfluidic devices with a 3D cell culture model can present cell-matrix and cell-cell interactions and provide reliable analysis platform for cell migration. Briefly, 2D and 3D microfludic chip-based models for concentration gradients, chemotaxis, electrotaxis and cells interactions have beneficial effects on cell migration assays. As a breakthrough to conventional methods, those microfluidic chip-based cell migration assays really promote the advanced progress in the area of cell biology and biomedicine. In this review, we describe the characterization of cell migration assays in microfluidic systems, highlight latest advances of the microfluidic device for researching in cell migration, focusing on both 2D and 3D cell cultures, and discuss advantages and disadvantages of this rapidly developed analysis technology.

The process of evaluating the efficacy and toxicity of drugs is important in the production of new drugs to treat diseases. Testing in humans is the most accurate method, but there are technical and ethical limitations. To overcome these limitations, various models have been developed in which responses to various external stimuli can be observed to help guide future trials. In particular, three-dimensional (3D) cell culture has a great advantage in simulating the physical and biological functions of tissues in the human body. This article reviews the biomaterials currently used to improve cellular functions in 3D culture and the contributions of 3D culture to cancer research, stem cell culture and drug and toxicity screening.

Drug toxicity is closely related to both clinical drug safety and new drug development. Therefore, it is vital to understand the mechanisms of drug toxicity fully and to use appropriate research models with advanced technologies. Zebrafish has become an important vertebrate animal model for high-throughput drug screening and toxicity assessment. At the same time, zebrafish has an intact biological complexity, reflecting the whole organism's toxicity, which gives it an advantage over other high-throughput models in toxicity studies. Despite the gradual increase in toxicity studies utilizing zebrafish, a comprehensive and systematic review of the underlying mechanisms and new techniques is still lacking. This review aims to analyze common toxicity mechanisms in zebrafish models, such as oxidative stress, endoplasmic reticulum stress, inflammation, and apoptosis, and macroscopic changes in biological processes like lipid metabolism disorders and neurotransmitter expression abnormalities. It also introduces new technologies applied in toxicity assessment, such as gene editing, novel fluorescence imaging technology, 3D imaging technology, and novel automated technology for high-throughput screening, such as fish capsules. In addition, it also summarizes the advantages and disadvantages of the model. By doing so, it will provide new suggestions for the development and improvement of the model, make it better serve the toxicity study of clinical drugs and provide a more comprehensive perspective for drug toxicity study, thus promoting the development of the field of drug toxicity study.

Oxygen is vital to the function of all tissues including the liver and lack of oxygen, that is, hypoxia can result in both acute and chronic injuries to the liver in vivo and ex vivo. Furthermore, a permanent oxygen gradient is naturally present along the liver sinusoid, which plays a role in the metabolic zonation and the pathophysiology of liver diseases. Accordingly, here, we introduce an in vitro microfluidic platform capable of actively creating a series of oxygen concentrations on a single continuous microtissue, ranging from normoxia to severe hypoxia. This range approximately captures both the physiologically relevant oxygen gradient generated from the portal vein to the central vein in the liver, and the severe hypoxia occurring in ischemia and liver diseases. Primary rat hepatocytes cultured in this microfluidic platform were exposed to an oxygen gradient of 0.3-6.9%. The establishment of an ascending hypoxia gradient in hepatocytes was confirmed in response to the decreasing oxygen supply. The hepatocyte viability in this platform decreased to approximately 80% along the hypoxia gradient. Simultaneously, a progressive increase in accumulation of reactive oxygen species and expression of hypoxia-inducible factor 1α was observed with increasing hypoxia. These results demonstrate the induction of distinct metabolic and genetic responses in hepatocytes upon exposure to an oxygen (/hypoxia) gradient. This progressive hypoxia-on-a-chip platform can be used to study the role of oxygen and hypoxia-associated molecules in modeling healthy and injured liver tissues. Its use can be further expanded to the study of other hypoxic tissues such as tumors as well as the investigation of drug toxicity and efficacy under oxygen-limited conditions.

Three-dimensional (3D) cultivation platforms allow the creation of cell models, which more closely resemble in vivo-like cell behavior. Therefore, 3D cell culture platforms have started to replace conventional two-dimensional (2D) cultivation techniques in many fields. Besides the advantages of 3D culture, there are also some challenges: cultivation in 3D often results in an inhomogeneous microenvironment and therefore unique cultivation conditions for each cell inside the construct. As a result, the analysis and precise control over the singular cell state is limited in 3D. In this work, we address these challenges by exploring ways to monitor oxygen concentrations in gelatin methacryloyl (GelMA) 3D hydrogel culture at the cellular level using hypoxia reporter cells and deep within the construct using a non-invasive optical oxygen sensing spot. We could show that the appearance of oxygen limitations is more prominent in softer GelMA-hydrogels, which enable better cell spreading. Beyond demonstrating novel or space-resolved techniques of visualizing oxygen availability in hydrogel constructs, we also describe a method to create a stable and controlled oxygen gradient throughout the construct using a 3D printed flow-through chamber.

Numerous cancer cell models exist used to investigate disease mechanisms and to screen potential cancer therapeutics. Roughly 90% of promising preclinical drugs fail to result in efficacious human treatments. Traditional two-dimensional (2D) tissue culture models lack realistic complexity, while animal models are expensive, time consuming, and too frequently fail to reflect human tumor biology. Recently, three-dimensional (3D) cell culture models are a new method to generate new drug candidates before moving to expensive and time-consuming animal models. We have developed a biochemically defined hydrogel platform formed by mixing various polymers with chemical crosslinkers with enhanced functionality. The hydrogel system employs one of two types of backbone polymers: a synthetic non-degradable polyvinyl alcohol (PVA) or an enzymatically-degradable dextran. Both polymers are functionalized either with fast or slow thiol-reactive groups. Crosslinkers consist of either PEG non-cell-degradable or a CD cell-degradable crosslinkers containing peptide sequences that create cleavage sites for matrix metalloproteases (MMPs) that allow cell migration. The technology provides mechanical and biochemical cues to investigate both morphological and physiological properties of cells in a 3D environment. The hydrogel allows precise control over hydrogel stiffness, gelation speed, cell migration and allows cell recovery for downstream applications. Here, we demonstrate the utility of this hydrogel platform to grow epithelial, fibroblast and tumor cells in 3D cell cultures with high cell viabilities and functionalities. Furthermore, we have constructed a 3D tumor/stromal cell co-culture using the hydrogel to model the dynamic tumor cell microenvironment. This technology will allow the creation of more accurate 3D cancer cell models for basic research and drug discovery applications. Note: This abstract was not presented at the meeting. Citation Format: Hsu-Kun Wang, Vi Chu, Nick Asbrock. Development of a novel tunable synthetic 3D hydrogel platform for the study of tumor and stromal cell interactions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5173.