Conventional Cell Culture Systems Research Articles

IPSC-derived lung and lung cancer organoid model to evaluate cisplatin encapsulated autologous iPSC-derived mesenchymal stromal cell-isolated extracellular vesicles

BackgroundLung cancer remains a leading cause of cancer-related mortality globally. Although recent therapeutic advancements have provided targeted treatment approaches, the development of resistance and systemic toxicity remain primary concerns. Extracellular vesicles (EVs), especially those derived from mesenchymal stromal cells (MSC), have gained attention as promising drug delivery systems, offering biocompatibility and minimal immune responses. Recognizing the limitations of conventional 2D cell culture systems in mimicking the tumor microenvironment, this study aims to describe a proof-of-principle approach for using patient-specific organoid models for both lung cancer and normal lung tissue and the feasibility of employing autologous EVs derived from induced pluripotent stem cell (iPSC)-MSC in personalized medicine approaches.MethodsFirst, we reprogrammed healthy fibroblasts into iPSC. Next, we differentiated patient-derived iPSC into branching lung organoids (BLO) and generated patient-matched lung cancer organoids (LCO) from patient-derived tumor tissue. We show a streamlined process of MSC differentiation from iPSC and EV isolation from iPSC-MSC, encapsulated with 0.07 µg/mL of cytotoxic agent cisplatin and applied to both organoid models. Cytotoxicity of cisplatin and cisplatin-loaded EVs was recorded with LDH and CCK8 tests.ResultsFibroblast-derived iPSC showed a normal karyotype, pluripotency staining, and trilineage differentiation. iPSC-derived BLO showed expression of lung markers, like TMPRSS2 and MUC5A while patient-matched LCO showed expression of Napsin and CK5. Next, we compared the effects of iPSC-MSC derived EVs loaded with cisplatin against empty EVs and cisplatin alone in lung cancer organoid and healthy lung organoid models. As expected, we found a cytotoxic effect when LCO were treated with 20 µg/mL cisplatin. Treatment of LCO and BLO with empty EVs resulted in a cytotoxic effect after 24 h. However, EVs loaded with 0.07 µg/mL cisplatin failed to induce any cytotoxic effect in both organoid models.ConclusionWe report on a proof-of-principle pipeline towards using autologous or allogeneic iPSC-MSC EVs as drug delivery tests for lung cancer in future. However, due to the time and labor-intensive processes, we conclude that this pipeline might not be feasible for personalized approaches at the moment.

Comparing the BAD Protein Interactomes in 2D and 3D Cell Culture Using Proximity Labeling.

Protein-protein interaction studies using proximity labeling techniques, such as biotin ligase-based BioID, have become integral in understanding cellular processes. Most studies utilize conventional 2D cell culture systems, potentially missing important differences in protein behavior found in 3D tissues. In this study, we investigated the protein-protein interactions of a protein, Bcl-2 Agonist of cell death (BAD), and compared conventional 2D culture conditions to a 3D system, wherein cells were embedded within a 3D extracellular matrix (ECM) mimic. Using BAD fused to the engineered biotin ligase miniTurbo (BirA*), we identified both overlapping and distinct BAD interactomes under 2D and 3D conditions. The known BAD binding proteins 14-3-3 isoforms and Bcl-XL interacted with BAD in both 2D and 3D. Of the 131 BAD-interactors identified, 56% were specific to 2D, 14% were specific to 3D, and 30% were common to both conditions. Interaction network analysis demonstrated differential associations between 2D and 3D interactomes, emphasizing the impact of the culture conditions on protein interactions. The 2D-3D overlap interactome encapsulated the apoptotic program, which is a well-known role of BAD. The 3D unique pathways were enriched in ECM signaling, suggestive of hitherto unknown functions for BAD. Thus, exploring protein-protein interactions in 3D provides novel clues into cell behavior. This exciting approach has the potential to bridge the knowledge gap between tractable 2D cell culture and organoid-like 3D systems.

Improving the Detection and Understanding of Infectious Human Norovirus in Food and Water Matrices: A Review of Methods and Emerging Models.

Human norovirus (HuNoV) is a leading global cause of viral gastroenteritis, contributing to numerous outbreaks and illnesses annually. However, conventional cell culture systems cannot support the cultivation of infectious HuNoV, making its detection and study in food and water matrices particularly challenging. Recent advancements in HuNoV research, including the emergence of models such as human intestinal enteroids (HIEs) and zebrafish larvae/embryo, have significantly enhanced our understanding of HuNoV pathogenesis. This review provides an overview of current methods employed for HuNoV detection in food and water, along with their associated limitations. Furthermore, it explores the potential applications of the HIE and zebrafish larvae/embryo models in detecting infectious HuNoV within food and water matrices. Finally, this review also highlights the need for further optimization and exploration of these models and detection methods to improve our understanding of HuNoV and its presence in different matrices, ultimately contributing to improved intervention strategies and public health outcomes.

Extracellular vesicles secreted by 3D tumor organoids are enriched for immune regulatory signaling biomolecules compared to conventional 2D glioblastoma cell systems.

Newer 3D culturing approaches are a promising way to better mimic the in vivo tumor microenvironment and to study the interactions between the heterogeneous cell populations of glioblastoma multiforme. Like many other tumors, glioblastoma uses extracellular vesicles as an intercellular communication system to prepare surrounding tissue for invasive tumor growth. However, little is known about the effects of 3D culture on extracellular vesicles. The aim of this study was to comprehensively characterize extracellular vesicles in 3D organoid models and compare them to conventional 2D cell culture systems. Primary glioblastoma cells were cultured as 2D and 3D organoid models. Extracellular vesicles were obtained by precipitation and immunoaffinity, with the latter allowing targeted isolation of the CD9/CD63/CD81 vesicle subpopulation. Comprehensive vesicle characterization was performed and miRNA expression profiles were generated by smallRNA-sequencing. In silico analysis of differentially regulated miRNAs was performed to identify mRNA targets and corresponding signaling pathways. The tumor cell media and extracellular vesicle proteome were analyzed by high-resolution mass spectrometry. We observed an increased concentration of extracellular vesicles in 3D organoid cultures. Differential gene expression analysis further revealed the regulation of twelve miRNAs in 3D tumor organoid cultures (with nine miRNAs down and three miRNAs upregulated). MiR-23a-3p, known to be involved in glioblastoma invasion, was significantly increased in 3D. MiR-7-5p, which counteracts glioblastoma malignancy, was significantly decreased. Moreover, we identified four miRNAs (miR-323a-3p, miR-382-5p, miR-370-3p, miR-134-5p) located within the DLK1-DIO3 domain, a cancer-associated genomic region, suggesting a possible importance of this region in glioblastoma progression. Overrepresentation analysis identified alterations of extracellular vesicle cargo in 3D organoids, including representation of several miRNA targets and proteins primarily implicated in the immune response. Our results show that 3D glioblastoma organoid models secrete extracellular vesicles with an altered cargo compared to corresponding conventional 2D cultures. Extracellular vesicles from 3D cultures were found to contain signaling molecules associated with the immune regulatory signaling pathways and as such could potentially change the surrounding microenvironment towards tumor progression and immunosuppressive conditions. These findings suggest the use of 3D glioblastoma models for further clinical biomarker studies as well as investigation of new therapeutic options.

Effect of gut microbiome-derived metabolites and extracellular vesicles on hepatocyte functions in a gut-liver axis chip

Metabolism, is a complex process involving the gut and the liver tissue, is difficult to be reproduced in vitro with conventional single cell culture systems. To tackle this challenge, we developed a gut-liver-axis chip consisting of the gut epithelial cell chamber and three-dimensional (3D) uniform-sized liver spheroid chamber. Two cell culture chamber compartments were separated with a porous membrane to prevent microorganisms from passing through the chamber. When the hepG2 spheroids cultured with microbiota-derived metabolites, we observed the changes in the physiological function of hepG2 spheroids, showing that the albumin and urea secretion activity of liver spheroids was significantly enhanced. Additionally, the functional validation of hepG2 spheroids treated with microbiota-derived exosome was evaluated that the treatment of the microbiota-derived exosome significantly enhanced albumin and urea in hepG2 spheroids in a gut-liver axis chip. Therefore, this gut-liver axis chip could be a potentially powerful co-culture platform to study the interaction of microbiota and host cells.Graphical

Abstract 4230: Modeling the cDC1 ex vivo and in vitro: Development and comparison of a conventional dendritic cell culture system for industry

Abstract Conventional dendritic cells (cDC) are innate immune cells specialized in antigen sampling and subsequent cross-presentation to immune cells and are critical for an effective anti-tumor immune response. While crucial to immunity against intracellular pathogens, viruses, and tumors, cDC type I (cDC1) are a relatively rare population and constitute about 0.05% of cells in the blood. Using these rare cells in screening assays can be a challenge, as very small proportions are found in peripheral human blood and since they are difficult to isolate with high viability and purity. Existing methods to generate acceptable numbers of cDC1s require a feeder cell line, which is not accessible to many laboratories. Using a systematic approach, we developed a scalable differentiation system to generate a mixed culture containing cDC1 cells from CD34+ hematopoietic stem cells isolated from cord blood that does not require feeder cells. This culture of cells has been phenotypically and functionally characterized to express high levels of CD141 and Clec9a, as well as responsiveness to various activating stimuli including poly (I:C). Cells derived from this culture method express similar levels of CD141 and Clec9a compared to those observed in peripheral blood of healthy donors. Our effort to find a less laborious, more efficient system includes comparison to cDC1 responses from an in vitro differentiation system to cDC1s isolated from humanized mouse bone marrow, with evaluation of both surface marker phenotype and responsiveness to activating stimuli. We also compared the responses of these cells to monocyte derived dendritic cells that express CD141 and Clec9a. These data demonstrate that the HSC-derived cDC1 cultures provide a comparison of unique dendritic cells that may be utilized for screening assays in an industry setting. Citation Format: Laura L. Goodfield, Johanna Lahdenranta, Heather Scott, Tamera Ashworth, Lia Luus, Anna F. Licht, Kevin McDonnell, Phil Brandish, Nicholas Keen. Modeling the cDC1 ex vivo and in vitro: Development and comparison of a conventional dendritic cell culture system for industry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4230.

Abstract 6018: Expansion of mouse mammary epithelial stem cells in serum-free organoid growth medium

Abstract Growing mammary epithelial cells as organoids in a three-dimensional (3D) cell culture environment represents a more physiological model system than conventional 2D adherent cell culture systems for studying many different aspects of mammary epithelial cell biology. We have developed MammoCult™ Organoid Growth Medium (Mouse), a serum- and phenol red-free medium for the long-term propagation of organoids from mouse mammary tissue. To initiate the cultures, mouse mammary glands are resected and enzymatically dissociated sequentially in Gentle Collagenase/Hyaluronidase, trypsin, and dispase/DNase I to generate a single-cell suspension, and 4.5 x 103 of the liberated cells are then embedded in Corning® Matrigel® and maintained in MammoCult™ Organoid Growth Medium (Mouse). Alternatively, freshly dissociated mammary cells can be expanded in adherent culture in EpiCult™ Plus Medium prior to seeding into Matrigel®. Approximately 8.4 ± 0.6% (mean ± SEM; n=10) of the freshly dissociated cells seeded in the Corning® Matrigel® will proliferate and generate organoids that are typically > 200 µm in diameter and have a highly branched morphology, although organoids with a cystic morphology are also observed. Immunostaining of these branched organoids reveals that they are composed of a polarized epithelium made up of keratin (K) 8-expressing luminal cells and K5/14-expressing basal cells (n=7). Organoids upregulate transcripts for caseins (Csn1s1, Csn2), whey acidic protein (Wap), and lactoferrin (Ltf) upon stimulation with prolactin, hydrocortisone, and insulin (n=4). These multilineage organoids can be established from multiple inbred mouse strains, including C57Bl6/J, FVB, BALB/c, and CD-1. Organoids can be passaged every 10 - 14 days as small fragments at a 1:3 to 1:4 split ratio, and can be maintained for a minimum of 10 passages while still maintaining multilineage potential and a normal karyotype. These results demonstrate that MammoCult™ Organoid Growth Medium (Mouse) efficiently generates and expands mammary epithelial cell-derived organoids and offers researchers a novel tool for in vitro mammary studies. Citation Format: David Rowbotham, Samantha Rothwell, Allen C. Eaves, Sharon A. Louis, John Stingl. Expansion of mouse mammary epithelial stem cells in serum-free organoid growth medium [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6018.

Gemini surfactant-based nanoparticles T-box1 gene delivery as a novel approach to promote epithelial stem cells differentiation and dental enamel formation.

Enamel is the highest mineralized tissue in the body protecting teeth from external stimuli, infections, and injuries. Enamel lacks the ability to self-repair due to the absence of enamel-producing cells in the erupted teeth. Here, we reported a novel approach to promote enamel-like tissue formation via the delivery of a key ameloblast inducer, T-box1 gene, into a rat dental epithelial stem cell line, HAT-7, using non-viral gene delivery systems based on cationic lipids. We comparatively assessed the lipoplexes prepared from glycyl-lysine-modified gemini surfactants and commercially available 1,2-dioleoyl-3-trimethylammonium-propane lipids at three nitrogen-to phosphate (N/P) ratios of 2.5, 5 and 10. Our findings revealed that physico-chemical characteristics and biological activities of the gemini surfactant-based lipoplexes with a N/P ratio of 5 provide the most optimal outcomes among those examined. HAT-7 cells were transfected with T-box1 gene using the optimal formulation then cultured in conventional 2D cell culture systems. Ameloblast differentiation, mineralization, bio-enamel interface and structure were assessed at different time points over 28days. Our results showed that our gemini transfection system provides superior gene expression compared to the benchmark agent, while keeping low cytotoxicity levels. T-box1-transfected HAT-7 cells strongly expressed markers of secretory and maturation stages of the ameloblasts, deposited minerals, and produced enamel-like crystals when compared to control cells. Taken together, our gemini surfactant-based T-box1 gene delivery system is effective to accelerate and guide ameloblastic differentiation of dental epithelial stem cells and promote enamel-like tissue formation. This study would represent a significant advance towards the tissue engineering and regeneration of dental enamel.

Testis on a chip—a microfluidic three-dimensional culture system for the development of spermatogenesis in-vitro

This research presents a novel testis-on-a-chip (ToC) platform. Testicular cells are enzymatically isolated from the seminiferous tubules of sexually immature mice, seeded in a methylcellulose gel and cultured in a microfluidic chip. The unique design sandwiches the soft methylcellulose between stiffer agar support gels. The cells develop into spheroids continuing to proliferate and differentiate. After seven weeks of culture the cells have over 95% viability. Confocal microscopy of the developed spheroids reveals a structure containing the various stages of spermatogenesis up to and including meiosis II: premeiotic, meiotic and post-meiotic germ cells. The spheroid structure also contains the supporting Sertoli and peritubular cells. The responsiveness of the system to the addition of testosterone and retinoic acid to the culture medium during the experiment was also investigated. As a benchmark, the ToC is compared to a conventional three-dimensional methylcellulose cell culture system in a well plate. Analysis via fluorescence-activated cell sorting shows more haploid cells in the chip as compared to the plates. Immunofluorescence staining after seven weeks of culture shows more differentiated cells in the chip as compared to the well plate. This demonstrates the feasibility of our platform as well as its advantages. This research opens new horizons for the study and realization of spermatogenesis in-vitro. It can also enable the implementation of microfluidic technologies in future therapeutic strategies for pre-pubertal male fertility preservation and adults with maturation arrest. Lastly, it can serve as a platform for drug and toxin testing.

Emergent Differential Organization of Airway Smooth Muscle Cells on Concave and Convex Tubular Surface.

Airway smooth muscle cells (ASMCs) exist in a form of helical winding bundles within the bronchial airway wall. Such tubular tissue provides cells with considerable curvature as a physical constraint, which is widely thought as an important determinant of cell behaviors. However, this process is difficult to mimic in the conventional planar cell culture system. Here, we report a method to develop chips with cell-scale tubular (concave and convex) surfaces from fused deposition modeling 3D printing to explore how ASMCs adapt to the cylindrical curvature for morphogenesis and function. Results showed that ASMCs self-organized into two distinctively different patterns of orientation on the concave and convex surfaces, eventually aligning either invariably perpendicular to the cylinder axis on the concave surface or curvature-dependently angled on the convex surface. Such oriented alignments of the ASMCs were maintained even when the cells were in dynamic movement during migration and spreading along the tubular surfaces. Furthermore, the ASMCs underwent a phenotype transition on the tubular (both concave and convex) surfaces, significantly reducing contractility as compared to ASMCs cultured on a flat surface, which was reflected in the changes of proliferation, migration and gene expression of contractile biomarkers. Taken together, our study revealed a curvature-induced pattern formation and functional modulation of ASMCs in vitro, which is not only important to better understanding airway smooth muscle pathophysiology, but may also be useful in the development of new techniques for airway disease diagnosis and therapy such as engineering airway tissues or organoids.

Critical Conditions for Studying Interleukin-11 Signaling In Vitro and Avoiding Experimental Artefacts.

Interleukin (IL) 11 is a member of the IL6 family of cytokines which require the ubiquitous gp130 receptor to activate canonical (JAK/STAT) and non-canonical (e.g., ERK) signaling pathways. The IL11 cytokine is upregulated in a number of fibro-inflammatory diseases and cancer, where it binds the cognate IL11 receptor alpha subunit (IL11RA) to form a hexameric IL11:IL11RA:gp130 signaling complex. The specific IL11RA receptor is highly expressed on cells of the stromal and parenchymal niche but expressed at low levels on immune cells, highly passaged cells, or transformed cell lines. Consequently, primary cells such as hepatic stellate cells, fibroblasts, and hepatocytes are ideal experimental systems to study IL11 signaling in vitro. In contrast to immortalized cell lines, primary cells better display relevant cellular physiology and pathobiology. This collection of protocols details experimental and culturing conditions for primary cells that preserve meaningful cellular states and physiological responses ex vivo in conventional 2D cell culture systems. Readouts of cellular activity are chosen carefully to capture the non-canonical, post-transcriptional activity of IL11 signaling. Our data suggest that cell type, cell culture conditions, passage number, concentrations of stimuli, timing, and other factors have major implications for studies of IL11 signaling. In vitro experiments with primary cell material need to be planned and executed with great caution. Otherwise, physiologically relevant mechanisms may become dysfunctional and reproducible experimental artefacts can obscure our view of true cytokine biology. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Expansion of primary human hepatic stellate cells (HSCs) and human renal proximal tubular epithelial cells (HRPTEpiCs) Basic Protocol 2: Expansion of primary human lung fibroblasts (HLFs) Alternate Protocol 1: Isolation and expansion of primary mouse lung fibroblasts Support Protocol 1: Freezing and thawing of primary cells Support Protocol 2: Operetta high-content imaging-based phenotyping Support Protocol 3: Colorimetric assay of solubilized collagen Support Protocol 4: Quantification of fibrosis marker secretion Support Protocol 5: Western blotting studies of IL11 signaling in HSCs, HLFs, and HRPTEpiCs Basic Protocol 3: IL11 stimulation of primary human hepatocytes Alternate Protocol 2: IL11 stimulation of primary mouse hepatocytes Support Protocol 6: Alanine transaminase (ALT) secretion by human and mouse hepatocytes.

Brain-on-a-Chip: Characterizing the next generation of advanced in vitro platforms for modeling the central nervous system.

The complexity of the human brain creates significant, almost insurmountable challenges for neurological drug development. Advanced in vitro platforms are increasingly enabling researchers to overcome these challenges, by mimicking key features of the brain's composition and functionality. Many of these platforms are called “Brains-on-a-Chip”—a term that was originally used to refer to microfluidics-based systems containing miniature engineered tissues, but that has since expanded to describe a vast range of in vitro central nervous system (CNS) modeling approaches. This Perspective seeks to refine the definition of a Brain-on-a-Chip for the next generation of in vitro platforms, identifying criteria that determine which systems should qualify. These criteria reflect the extent to which a given platform overcomes the challenges unique to in vitro CNS modeling (e.g., recapitulation of the brain's microenvironment; inclusion of critical subunits, such as the blood–brain barrier) and thereby provides meaningful added value over conventional cell culture systems. The paper further outlines practical considerations for the development and implementation of Brain-on-a-Chip platforms and concludes with a vision for where these technologies may be heading.

Translating nanoparticle dosimetry from conventional in vitro systems to occupational inhalation exposures

As encouraged by Toxicity Testing in the 21st Century, researchers increasingly apply high-throughput in vitro approaches to identify and characterize nanoparticle hazards, including conventional aqueous cell culture systems to assess respiratory hazards. Translating nanoparticle dose from conventional toxicity testing systems to relevant human exposures remains a major challenge for assessing occupational risk of nanoparticle exposures. Here, we explored existing computational tools and data available to translate nanoparticle dose metrics from cellular test systems to inhalation exposures of silver nanoparticles in humans. We used the Multiple-Path Particle Dosimetry (MPPD) Model to predict nanoparticle deposition of humans exposed to 20 and 110 nm silver nanoparticles at 0.9 μg/m3 over an 8 h period, the proposed National Institute of Occupational Safety and Health (NIOSH) recommended exposure limit (REL). MPPD predicts 8.1 and 3.7 μg of silver deposited in an 8 h period for 20 and 110 nm nanoparticles, respectively, with 20 nm particles displaying nearly 11-fold higher total surface area deposited. Peak deposited nanoparticle concentrations occurred more proximal in the pulmonary tract compared to mass deposition patterns (generation 4 vs. generations 20–21, respectively) due to regional differences in lung lining fluid volumes. Assuming 0.4% nanoparticle dissolution by mass measured in previous studies predicted peak concentrations of silver ions in cells of 1.06 and 0.89 μg/mL for 20 and 110 nm particles, respectively. Both predicted concentrations are below the measured toxic threshold of 1.7 μg/mL of silver ions in cells from in vitro assessments. Assuming 4% dissolution by mass predicted 10-fold higher silver concentrations in tissues, peaking at 10.6 and 8.9 μg/mL, for 20 and 110 nm nanoparticles respectively, exceeding the observed in vitro toxic threshold and highlighting the importance and sensitivity of dissolution rates. Overall, this approach offers a framework for extrapolating nanotoxicity results from in vitro cell culture systems to human exposures. Aligning appropriate dose metrics from in vitro and in vivo hazard characterizations and human pulmonary doses from occupational exposures are critical components for successful nanoparticle risk assessment and worker protection providing guidance for designing future in vitro studies aimed at relevant human exposures.

Microfluidic and Organ-on-a-Chip Approaches to Investigate Cellular and Microenvironmental Contributions to Cardiovascular Function and Pathology.

Over the past decade, advances in microfabrication and biomaterials have facilitated the development of microfluidic tissue and organ models to address challenges with conventional animal and cell culture systems. These systems have largely been developed for human disease modeling and preclinical drug development and have been increasingly used to understand cellular and molecular mechanisms, particularly in the cardiovascular system where the characteristic mechanics and architecture are difficult to recapitulate in traditional systems. Here, we review recent microfluidic approaches to model the cardiovascular system and novel insights provided by these systems. Key features of microfluidic approaches include the ability to pattern cells and extracellular matrix (ECM) at cellular length scales and the ability to use patient-derived cells. We focus the review on approaches that have leveraged these features to explore the relationship between genetic mutations and the microenvironment in cardiovascular disease progression. Additionally, we discuss limitations and benefits of the various approaches, and conclude by considering the role further advances in microfabrication technology and biochemistry techniques play in establishing microfluidic cardiovascular disease models as central tools for understanding biological mechanisms and for developing interventional strategies.

Iron (III)-Quercetin Complex: Synthesis, Physicochemical Characterization, and MRI Cell Tracking toward Potential Applications in Regenerative Medicine.

In cell therapy, contrast agents T1 and T2 are both needed for the labeling and tracking of transplanted stem cells over extended periods of time through magnetic resonance imaging (MRI). Importantly, the metal-quercetin complex via coordination chemistry has been studied extensively for biomedical applications, such as anticancer therapies and imaging probes. Herein, we report on the synthesis, characterization, and labeling of the iron (III)-quercetin complex, “IronQ,” in circulating proangiogenic cells (CACs) and also explore tracking via the use of a clinical 1.5 Tesla (T) MRI scanner. Moreover, IronQ had a paramagnetic T1 positive contrast agent property with a saturation magnetization of 0.155 emu/g at 1.0 T and longitudinal relaxivity (r1) values of 2.29 and 3.70 mM−1s−1 at 1.5 T for water and human plasma, respectively. Surprisingly, IronQ was able to promote CAC growth in conventional cell culture systems without the addition of specific growth factors. Increasing dosages of IronQ from 0 to 200 μg/mL led to higher CAC uptake, and maximum labeling time was achieved in 10 days. The accumulated IronQ in CACs was measured by two methodologies, an inductively coupled plasma optical emission spectrometry (ICP-EOS) and T1-weighted MRI. In our research, we confirmed that IronQ has excellent dual functions with the use of an imaging probe for MRI. IronQ can also act as a stimulating agent by favoring circulating proangiogenic cell differentiation. Optimistically, IronQ is considered beneficial for alternative labeling and in the tracking of circulation proangiogenic cells and/or other stem cells in applications of cell therapy through noninvasive magnetic resonance imaging in both preclinical and clinical settings.

A Microfluidic Probe Integrated Device for Spatiotemporal 3D Chemical Stimulation in Cells.

Numerous in vitro studies have been conducted in conventional static cell culture systems. However, most of the results represent an average response from a population of cells regardless of their local microenvironment. A microfluidic probe is a non-contact technology that has been widely used to perform local chemical stimulation within a restricted space, providing elaborated modulation and analysis of cellular responses within the microenvironment. Although microfluidic probes developed earlier have various potential applications, the two-dimensional structure can compromise their functionality and flexibility for practical use. In this study, we developed a three-dimensional microfluidic probe integrated device equipped with vertically oriented microchannels to overcome crucial challenges and tested the potential utility of the device in biological research. We demonstrated that the device tightly regulated spatial diffusion of a fluorescent molecule, and the flow profile predicted by simulation replicated the experimental results. Additionally, the device modulated the physiological Ca2+ response of cells within the restricted area by altering the local and temporal concentrations of biomolecules such as ATP. The novel device developed in this study may provide various applications for biological studies and contribute to further understanding of molecular mechanisms underlying cellular physiology.

A Custom Multiphoton Microscopy Platform for Live Imaging of Mouse Cornea and Conjunctiva.

Conventional histological analysis and cell culture systems are insufficient to simulate in vivo physiological and pathological dynamics completely. Multiphoton microscopy (MPM) has become one of the most popular imaging modalities for biomedical study at cellular levels in vivo, advantages include high resolution, deep tissue penetration and minimal phototoxicity. We have designed an MPM imaging platform with a customized mouse eye holder and a stereotaxic stage for imaging ocular surface in vivo. Dual fluorescent protein reporter mouse enables visualization of cell nuclei, cell membranes, nerve fibers, and capillaries within the ocular surface. In addition to multiphoton fluorescence signals, acquiring second harmonic generation (SHG) simultaneously allows for the characterization of collagenous stromal architecture. This platform can be employed for intravital imaging with accurate positioning across the entire ocular surface, including cornea and conjunctiva.

Three-Dimensional Modeling of Avascular Tumor Growth in Both Static and Dynamic Culture Platforms.

Microfluidic cell culture platforms are ideal candidates for modeling the native tumor microenvironment because they can precisely reconstruct in vivo cellular behavior. Moreover, mathematical modeling of tumor growth can pave the way toward description and prediction of growth pattern as well as improving cancer treatment. In this study, a modified mathematical model based on concentration distribution is applied to tumor growth in both conventional static culture and dynamic microfluidic cell culture systems. Apoptosis and necrosis mechanisms are considered as the main inhibitory factors in the model, while tumor growth rate and nutrient consumption rate are modified in both quiescent and proliferative zones. We show that such modification can better predict the experimental results of tumor growth reported in the literature. Using numerical simulations, the effects of the concentrations of the nutrients as well as the initial tumor radius on the tumor growth are investigated and discussed. Furthermore, tumor growth is simulated by taking into account the dynamic perfusion into the proposed model. Subsequently, tumor growth kinetics in a three-dimensional (3D) microfluidic device containing a U-shaped barrier is numerically studied. For this case, the effect of the flow rate of culture medium on tumor growth is investigated as well. Finally, to evaluate the impact of the trap geometry on the tumor growth, a comparison is made between the tumor growth kinetics in two frequently used traps in microfluidic cell culture systems, i.e., the U-shaped barrier and microwell structures. The proposed model can provide insight into better predicting the growth and development of avascular tumor in both static and dynamic cell culture platforms.

Metabolic signature of extracellular vesicles depends on the cell culture conditions

ABSTRACTOne of the greatest bottlenecks in extracellular vesicle (EV) research is the production of sufficient material in a consistent and effective way using in vitro cell models. Although the production of EVs in bioreactors maximizes EV yield in comparison to conventional cell cultures, the impact of their cell growth conditions on EVs has not yet been established. In this study, we grew two prostate cancer cell lines, PC-3 and VCaP, in conventional cell culture dishes and in two-chamber bioreactors to elucidate how the growth environment affects the EV characteristics. Specifically, we wanted to investigate the growth condition-dependent differences by non-targeted metabolite profiling using liquid chromatography–mass spectrometry (LC–MS) analysis. EVs were also characterized by their morphology, size distribution, and EV protein marker expression, and the EV yields were quantified by NTA. The use of bioreactor increased the EV yield >100 times compared to the conventional cell culture system. Regarding morphology, size distribution and surface markers, only minor differences were observed between the bioreactor-derived EVs (BR-EVs) and the EVs obtained from cells grown in conventional cell cultures (C-EVs). In contrast, metabolomic analysis revealed statistically significant differences in both polar and non-polar metabolites when the BR-EVs were compared to the C-EVs. The results show that the growth conditions markedly affected the EV metabolite profiles and that metabolomics was a sensitive tool to study molecular differences of EVs. We conclude that the cell culture conditions of EV production should be standardized and carefully detailed in publications and care should be taken when EVs from different production platforms are compared with each other for systemic effects.

3D cell coculture tumor model: A promising approach for future cancer drug discovery

Abstract It is recognized that tumors do not exist and grow as isolated entities of neoplastic cells, but rather in continuous and bidirectional communication with adjacent stromal cells. Stromal cellular components, like fibroblasts, endothelial cells and multiple immune cells, have been proven to play critical roles in tumor development. In the past decade, a variety of in vitro three-dimensional (3D) tumor models were developed to replace conventional monolayer cell culture systems. 3D cell coculture between tumor and stromal cells has also gained increasing interest due to improved mimicry of in vivo tumor microenvironments. In this review, we first provide a general overview of the mechanisms for cancer development mediated by several key types of stromal cells, and their current applications in cell coculture. Next, a variety of current methodologies for 3D cell coculture are summarized in terms of cell contact methods, 3D scaffolds and culture devices. The potential of an integrated high throughput (HT) 3D cell coculture screening platform for cancer drug discovery is also discussed in this review.