A novel approach to determine the critical survival threshold of cellular oxygen within spheroids via integrating live/dead cell imaging with oxygen modeling.

Defining the oxygen level that induces cell death within 3-D tissues is vital for understanding tissue hypoxia; however, obtaining accurate measurements has been technically challenging. In this study, we introduce a noninvasive, high-throughput methodology to quantify critical survival partial oxygen pressure (pO2) with high spatial resolution within spheroids by using a combination of controlled hypoxic conditions, semiautomated live/dead cell imaging, and computational oxygen modeling. The oxygen-permeable, micropyramid patterned culture plates created a precisely controlled oxygen condition around the individual spheroid. Live/dead cell imaging provided the geometric information of the live/dead boundary within spheroids. Finally, computational oxygen modeling calculated the pO2 at the live/dead boundary within spheroids. As proof of concept, we determined the critical survival pO2 in two types of spheroids: isolated primary pancreatic islets and tumor-derived pseudoislets (2.43 ± 0.08 vs. 0.84 ± 0.04 mmHg), indicating higher hypoxia tolerance in pseudoislets due to their tumorigenic origin. We also applied this method for evaluating graft survival in cell transplantations for diabetes therapy, where hypoxia is a critical barrier to successful transplantation outcomes; thus, designing oxygenation strategies is required. Based on the elucidated critical survival pO2, 100% viability could be maintained in a typically sized primary islet under the tissue pO2 above 14.5 mmHg. This work presents a valuable tool that is potentially instrumental for fundamental hypoxia research. It offers insights into physiological responses to hypoxia among different cell types and may refine translational research in cell therapies.NEW & NOTEWORTHY Our study introduces an innovative combinatory approach for noninvasively determining the critical survival oxygen level of cells within small cell spheroids, which replicates a 3-D tissue environment, by seamlessly integrating three pivotal techniques: cell death induction under controlled oxygen conditions, semiautomated imaging that precisely identifies live/dead cells, and computational modeling of oxygen distribution. Notably, our method ensures high-throughput analysis applicable to various cell types, offering a versatile solution for researchers in diverse fields.

Firefly Rats: Illuminating the Scientific Community in Transplantation Research.

Fireflies produce light through luciferase-catalyzed reactions involving luciferin, oxygen, and adenosine triphosphate, distinct from other luminescent organisms. This unique feature has revolutionized molecular biology and physiology, serving as a valuable tool for cellular research. Luciferase-based bioluminescent imaging enabled the creation of transgenic animals, such as Firefly Rats. Firefly Rats, created in 2006, ubiquitously express luciferase and have become a critical asset in scientific investigations. These rats have significantly contributed to transplantation and tissue engineering studies. Their low immunogenicity reduces graft rejection risk, making them ideal for long-term tracking of organ/tissue/cellular engraftments. Importantly, in the islet transplantation setting, the ubiquitous luciferase expression in these rats does not alter islet morphology or function, ensuring accurate assessments of engrafted islets. Firefly Rats have illuminated the path of transplantation research worldwide for over a decade and continue accelerating scientific advancements in many fields.

Low-Temperature Direct Growth of Nanocrystalline Multilayer Graphene on Silver with Long-Term Surface Passivation.

A wide variety of transition metals, including copper and gold, have been successfully used as substrates for graphene growth. On the other hand, it has been challenging to grow graphene on silver, so realistic applications by combining graphene and silver for improved electrode stability and enhanced surface plasmon resonance in organic light-emitting diodes and biosensing have not been realized to date. Here, we demonstrate the surface passivation of silver through the single-step rapid growth of nanocrystalline multilayer graphene on silver via low-temperature plasma-enhanced chemical vapor deposition (PECVD). The effect of the growth time on the graphene quality and the underlying silver characteristics is investigated by Raman spectroscopy, X-ray diffraction, atomic force microscopy, X-ray photoelectron spectroscopy (XPS), and cross-sectional annular dark-field scanning transmission electron microscopy (ADF-STEM). These results reveal nanocrystalline graphene structures with turbostratic layer stacking. Based on the XPS and ADF-STEM results, a PECVD growth mechanism of graphene on silver is proposed. The multilayer graphene also provides excellent long-term protection of the underlying silver surface from oxidation after 5 months of air exposure. This development thus paves the way toward realizing technological applications based on graphene-protected silver surfaces and electrodes as well as hybrid graphene-silver plasmonics.

Micropyramid-patterned, oxygen-permeable bottomed dish for high density culture of pancreatic islets.

The need for maintaining cell-spheroid viability and function within high-density cultures is unmet for various clinical and experimental applications, including cell therapies. One immediate application is for transplantation of pancreatic islets, a clinically recognized treatment option to cure type 1 diabetes; islets are isolated from a donor for subsequent culture prior to transplantation. However, high seeding conditions cause unsolicited fusion of multiple spheroids, thereby limiting oxygen diffusion to induce hypoxic cell death. Here we introduce a culture dish incorporating a micropyramid-patterned surface to prevent the unsolicited fusion and oxygen-permeable bottom for optimal oxygen environment. A 400µm-thick, oxygen-permeable polydimethylsiloxane sheet topped with micropyramid pattern of 400µm-base and 200µm-height was fabricated to apply to the 24-well plate format. The micropyramid pattern separated the individual pancreatic islets to prevent the fusion of multiple islets. This platform supported the high oxygen demand of islets at high seeding density at 260 islet equivalents cm-2, a 2-3-fold higher seeding density compared to the conventional islet culture used in a preparation for the clinical islet transplantations, demonstrating improved islet morphology, metabolism and function in a 4 d-culture. Transplantation of these islets into immunodeficient diabetic mice exhibited significantly improved engraftment to achieve euglycemia compared to islets cultured in the conventional culture wells. Collectively, this simple design modification allows for high-density cultures of three-dimensional cell spheroids to improve the viability and function for an array of investigational and clinical replacement tissues.

A wearable eddy current based pulmonary function sensor for continuous non-contact point-of-care monitoring during the COVID-19 pandemic.

Pulmonary function testing (PFT) allows for quantitative analysis of lung function. However, as a result of the coronavirus disease 2019 (COVID-19) pandemic, a majority of international medical societies have postponed PFTs in an effort to mitigate disease transmission, complicating the continuity of care in high-risk patients diagnosed with COVID-19 or preexisting lung pathologies. Here, we describe the development of a non-contact wearable pulmonary sensor for pulmonary waveform analysis, pulmonary volume quantification, and crude thoracic imaging using the eddy current (EC) phenomenon. Statistical regression analysis is performed to confirm the predictive validity of the sensor, and all data are continuously and digitally stored with a sampling rate of 6,660 samples/second. Wearable pulmonary function sensors may facilitate rapid point-of-care monitoring for high-risk individuals, especially during the COVID-19 pandemic, and easily interface with patient hospital records or telehealth services.

Noninvasive transcranial classification of stroke using a portable eddy current damping sensor.

Existing paradigms for stroke diagnosis typically involve computed tomography (CT) imaging to classify ischemic versus hemorrhagic stroke variants, as treatment for these subtypes varies widely. Delays in diagnosis and transport of unstable patients may worsen neurological status. To address these issues, we describe the development of a rapid, portable, and accurate eddy current damping (ECD) stroke sensor. Copper wire was wound to create large (11.4 cm), medium (4.5 cm), and small (1.5 cm) solenoid coils with varying diameters, with each connected to an inductance-to-digital converter. Eight human participants were recruited between December 15, 2019 and March 15, 2020, including two hemorrhagic stroke, two ischemic stroke, one subarachnoid hemorrhage, and three control participants. Observers were blinded to lesion type and location. A head cap with 8 horizontal scanning paths was placed on the patient. The sensor was tangentially rotated across each row on the patient's head circumferentially. Consent, positioning, and scanning with the sensor took roughly 15 min from start to end for each participant and all scanning took place at the patient bedside. The ECD sensor accurately classified and imaged each of the varying stroke types in each patient. The sensor additionally detected ischemic and hemorrhagic lesions located deep inside the brain, and its range is selectively tunable during sensor design and fabrication.

Novel effect and the mechanistic insights of fruiting body extract of medicinal fungus Antrodia cinnamomea against T47D breast cancer.

INTRODUCTION: Tamoxifen, an anti-oestrogenic drug for estrogen receptor positive (ER+) breast cancer, was observed to stimulate tumor growth or drug resistance in patients. Antrodia cinnamomea (AC), a precious medicinal fungus has been traditionally used as a folk remedy for cancers in Asian countries. The objective of this study was to investigate the bioefficacy and the underlying molecular mechanisms of the AC fruiting bodies extracts (AC-3E) against human ER+ T47D breast cancer cells, and compare the effect with that of tamoxifen. METHODS: Cell proliferation, migration, TUNEL assay, western blotting, time-lapse confocal microscopy analyses, chorioallantoic membrane assay, and a xenograft BALB/c nude mouse system were used in this study. Chemical fingerprinting of AC-3E was established using LC-MS. RESULTS: AC-3E attenuated T47D breast cancer cell activity by deregulating the PI3K/Akt/mTOR signaling pathway and key cell-cycle mediators, and inducing apoptosis. AC-3E also effectively inhibited tube-like structures of endothelial cells, blood vessel branching and microvessel formation ex vivo and in vivo. Significant preventive and therapeutic effects against T47D mammary tumor growth of AC-3E was observed comparable or superior to tamoxifen treatment in xenograft BALB/c nude mice. Dehydroeburicoic acid (2) was characterized as the main chemical constituent in AC-3E against breast cancer. CONCLUSION: This study suggests that AC-3E extracts can be employed as a double-barreled approach to treat human ER+ breast cancer by attacking both cancer cells and tumor-associated blood vessel cells.