Monolayer Cultures Of Cells Research Articles

Assessment of equine intestinal epithelial junctional complexes and barrier permeability using a monolayer culture system

Gastrointestinal disease is a leading cause of death in mature horses. A lack of in vitro modeling has impeded the development of novel therapeutics. The objectives of this study were to develop and further characterize a small intestinal monolayer cell culture derived from equine jejunum including establishing normal measurements of intestinal permeability and restitution. Three-dimensional enteroids, derived from postmortem sampling of equine jejunum, were utilized to develop confluent epithelial monolayers. The presence of differentiated intestinal epithelial cell types and tight junctions were confirmed using histology, reverse transcription PCR (RT-PCR), RNAscope, protein immunofluorescence and transmission electron microscopy. Transepithelial resistance (TER) and macromolecule flux were assessed as measurements of paracellular and transcellular permeability. Scratch assays were utilized to model and assess intestinal restitution. Monolayer cell cultures reached 100% confluency by ~5–7 days. Equine jejunum monolayers were confirmed as epithelial in origin, with identification of differentiated intestinal epithelial cell types and evidence of tight junction proteins. Function of the intestinal barrier was supported by acquisition of physiologically normal TER values (179.9 ± 33.7 ohms*cm2) and limited macromolecule flux (22 ± 8.8% at 60 min). Additionally, following a scratch wound, epithelial cell monolayers migrated to close gap defects within 24 h. In conclusion, this study describes the development of a novel intestinal epithelial monolayer cell culture for equine jejunum, and provides evidence of intestinal epithelial cell differentiation, formation of physiologically relevant barrier function and use as a model of intestinal restitution to test potential therapeutics for equine colic.

One-Step Soft Agar Enrichment and Isolation of Human Lung Bacteria Inhibiting the Germination of Aspergillus fumigatus Conidia

Fungi of the genus Aspergillus are widespread in the environment, where they produce large quantities of airborne conidia. Inhalation of Aspergillus spp. conidia in immunocompromised individuals can cause a wide spectrum of diseases, ranging from hypersensitivity responses to lethal invasive infections. Upon deposition in the lung epithelial surface, conidia encounter and interact with complex microbial communities that constitute the lung microbiota. The lung microbiota has been suggested to influence the establishment and growth of Aspergillus spp. in the human airways. However, the mechanisms underlying this interaction have not yet been sufficiently investigated. In this study, we aimed to enrich and isolate bacterial strains capable of inhibiting the germination and growth of A. fumigatus conidia from bronchoalveolar lavage fluid (BALF) samples of lung transplant recipients using a novel enrichment method. This method is based on a soft agar overlay plate assay in which bacteria are directly in contact with conidia, allowing inhibition to be readily observed during enrichment. We isolated a total of five clonal bacterial strains with identical genotypic fingerprints, as shown by random amplified polymorphic DNA PCR (RAPD–PCR). All strains were identified as Pseudomonas aeruginosa (strains b1–b5). The strains were able to inhibit the germination and growth of Aspergillus fumigatus in a soft agar confrontation assay, as well as in a germination multiplate assay. Moreover, when compared with ten P. aeruginosa strains isolated from expectoration through standard methods, no significant differences in inhibitory potential were observed. Additionally, we showed inhibition of A. fumigatus growth on Calu-3 cell culture monolayers. However, the isolated P. aeruginosa strains were shown to cause significant damage to the cell monolayers. Overall, although P. aeruginosa is a known opportunistic lung pathogen and antagonist of A. fumigatus, we validated this novel one-step enrichment approach for the isolation of bacterial strains antagonistic to A. fumigatus from BALF samples as a proof-of-concept. This opens up a new venue for the targeted enrichment of antagonistic bacterial strains against specific fungal pathogens.

7888 Mitotane Induces Different Modes Of Cell Death In Treatment Resistant And Sensitive Models Of Adrenocortical Carcinoma

Abstract Disclosure: S. Feely: None. N. Mullen: None. C. Hong: None. C. Hantel: None. W.E. Rainey: None. M.C. Dennedy: None. Adrenocortical carcinoma (ACC) is a rare aggressive cancer with poor survival. Adjuvant mitotane is the only approved drug for treatment of ACC. It improves survival but its use is limited by poor tolerability and drug resistance. For metastatic ACC, combination chemotherapy, using mitotane and etoposide, doxorubicin, and cisplatin improves survival, but efficacy is limited. Better understating the cytotoxic mechanisms of mitotane offers potential to develop improved therapeutic options for ACC. To investigate the underlying mechanisms in vitro, human ACC cells (HAC15 and H295R), in monolayer cell culture were treated with increasing concentrations of mitotane. Cell death was evaluated at 6 and 24 hours using flow cytometry analysis of Annexin V and Sytox Blue. This was complemented by Incucyte ® Live-Cell Analysis System imaging of annexin V and Sytox Blue staining. Expression of the markers of apoptosis, cleaved caspase 3, and necroptosis, phosphorylated MLKL and RIPK1 was measured at 6 and 24 hours. Mitotane significantly reduced the viability of H295R cells at 6 and 24 hours at the therapeutic concentration of 50uM (p>0.0001), thereby demonstrating treatment-sensitivity. In HAC15 cells, a supratherapeutic dose of 200uM was needed to significantly reduce viability (p>0.05) at 24 hours, indicating resistance to mitotane treatment at therapeutic concentrations. There was increasing Annexin V fluorescence with increasing mitotane concentration in H295R and HAC15 cells ar 6 and 24 hours without higher coinciding staining with Sytox Blue. Video analysis demonstrated these findings. Cleaved caspase 3 expression was associated with mitotane-induced cell death in H295R cells at 6 and 24 hours, indicating apoptosis. HAC15 cells demonstrated cleaved caspase 3 expression only at the highest concentration of mitotane exposure (600uM) at 6 hours, without appreciable expression at 24 hours. Phosphorylated-MLKL and RIPK1 expression was seen in controls and all mitotane doses in both cell lines at both timepoints, indicating necroptosis. In this study, H295R cells, representing a treatment-sensitive model of Mitotane undergo both apoptotic and necroptotic cell death at 6 and 24 hours of mitotane exposure. In contrast, HAC15 cells, demonstrated relative treatment resistance to mitotane, and undergo necroptotic cell death only. These results highlight the importance of understanding and targeting non-apoptotic and necroptotic cell death pathways, particularly when evaluating mechanisms of treatment resistance in ACC. Given the difference in the mechanism of cell death in treatment-sensitive and treatment-resistant cells, further exploration of necroptosis is necessary to better understand how ACC cells may manifest treatment resistance. Presentation: 6/1/2024

Tumor Cell Spatial Organization Directs EGFR/RAS/RAF Pathway Primary Therapy Resistance through YAP Signaling.

Non-small cell lung cancers (NSCLC) harboring common mutations in EGFR and KRAS characteristically respond transiently to targeted therapies against those mutations, but invariably, tumors recur and progress. Resistance often emerges through mutations in the therapeutic target or activation of alternative signaling pathways. Mechanisms of acute tumor cell resistance to initial EGFR (EGFRi) or KRASG12C (G12Ci) pathway inhibition remain poorly understood. Our study reveals that acute response to EGFR/RAS/RAF-pathway inhibition is spatial and culture context specific. In vivo, EGFR mutant tumor xenografts shrink by > 90% following acute EGFRi therapy, and residual tumor cells are associated with dense stroma and have increased nuclear YAP. Interestingly, in vitro EGFRi induced cell cycle arrest in NSCLC cells grown in monolayer, while 3D spheroids preferentially die upon inhibitor treatment. We find differential YAP nuclear localization and activity, driven by the distinct culture conditions, as a common resistance mechanism for selective EGFR/KRAS/BRAF pathway therapies. Forced expression of the YAPS127A mutant partially protects cells from EGFR-mediated cell death in spheroid culture. These studies identify YAP activation in monolayer culture as a non-genetic mechanism of acute EGFR/KRAS/BRAF therapy resistance, highlighting that monolayer vs spheroid cell culture systems can model distinct stages of patient cancer progression.

From 2D to 3D In Vitro World: Sonodynamically-Induced Prooxidant Proapoptotic Effects of C60-Berberine Nanocomplex on Cancer Cells.

The primary objective of this research targeted the biochemical effects of SDT on human cervix carcinoma (HeLa) and mouse Lewis lung carcinoma (LLC) cells grown in 2D monolayer and 3D spheroid cell culture. HeLa and LLC monolayers and spheroids were treated with a 20 µM C60-Ber for 24 h, followed by irradiation with 1 MHz, 1 W/cm2 US. To evaluate the efficacy of the proposed treatment on cancer cells, assessments of cell viability, caspase 3/7 activity, ATP levels, and ROS levels were conducted. Our results revealed that US irradiation alone had negligible effects on LLC and HeLa cancer cells. However, both monolayers and spheroids irradiated with US in the presence of the C60-Ber exhibited a significant decrease in viability (32% and 37%) and ATP levels (42% and 64%), along with a notable increase in ROS levels (398% and 396%) and caspase 3/7 activity (437% and 246%), for HeLa monolayers and spheroids, respectively. Similar tendencies were observed with LLC cells. In addition, the anticancer effects of C60-Ber surpassed those of C60, Ber, or their mixture (C60 + Ber) in both cell lines. The detected intensified ROS generation and ATP level drop point to mitochondria dysfunction, while increased caspase 3/7 activity points on the apoptotic pathway induction. The combination of 1 W/cm2 US with C60-Ber showcased a promising platform for synergistic sonodynamic chemotherapy for cancer treatment.

Sequencing-based study of neural induction of human dental pulp stem cells.

Techniques for triggering neural differentiation of embryonic and induced pluripotent stem cells into neural stem cells and neurons have been established. However, neural induction of mesenchymal stem cells, including dental pulp stem cells (DPSCs), has been assessed primarily based on neural-related gene regulation, and detailed studies into the characteristics and differentiation status of cells are lacking. Therefore, this study was aimed at evaluating the cellular components and differentiation pathways of neural lineage cells obtained via neural induction of human DPSCs. Human DPSCs were induced to neural cells in monolayer culture and examined for gene expression and mechanisms underlying differentiation using microarray-based ingenuity pathway analysis. In addition, the neural lineage cells were subjected to single-cell RNA sequencing (scRNA-seq) to classify cell populations based on gene expression profiles and to elucidate their differentiation pathways. Ingenuity pathway analysis revealed that genes exhibiting marked overexpression, post-neuronal induction, such as FABP7 and ZIC1, were associated with neurogenesis. Furthermore, in canonical pathway analysis, axon guidance signals demonstrated maximum activation. The scRNA-seq and cell type annotations revealed the presence of neural progenitor cells, astrocytes, neurons, and a small number of non-neural lineage cells. Moreover, trajectory and pseudotime analyses demonstrated that the neural progenitor cells initially engendered neurons, which subsequently differentiated into astrocytes. This result indicates that the aforementioned neural induction strategy generated neural stem/progenitor cells from DPSCs, which might differentiate and proliferate to constitute neural lineage cells. Therefore, neural induction of DPSCs may present an alternative approach to pluripotent stem cell-based therapeutic interventions for nervous system disorders.

Assessing Receptor Activation in 2D and 3D Cultured Hepatocytes: Responses to a Single Compound and a Complex Mixture.

Responding to global standards and legislative updates in Canada, including Bill S-5 (2023), toxicity testing is shifting towards more ethical, in vitro methods. Traditional two-dimensional (2D) monolayer cell cultures, limited in replicating the complex in vivo environment, have prompted the development of more relevant three-dimensional (3D) spheroidal hepatocyte cultures. This study introduces the first 3D spheroid model for McA-RH7777 cells, assessing xenobiotic receptor activation, cellular signaling, and toxicity against dexamethasone and naphthenic acid (NA)-fraction components; NAFCs. Our findings reveal that 3D McA-RH7777 spheroids demonstrate enhanced sensitivity and more uniform dose-response patterns in gene expression related to xenobiotic metabolism (AhR and PPAR) for both single compounds and complex mixtures. Specifically, 3D cultures showed significant gene expression changes upon dexamethasone exposure and exhibited varying degrees of sensitivity and resistance to the apoptotic effects induced by NAFCs, in comparison to 2D cultures. The optimization of 3D culture conditions enhances the model's physiological relevance and enables the identification of genomic signatures under varied exposures. This study highlights the potential of 3D spheroid cultures in providing a more accurate representation of the liver's microenvironment and advancing our understanding of cellular mechanisms in toxicity testing.

Sodium Tungstate Promotes Neurite Outgrowth and Confers Neuroprotection in Neuro2a and SH-SY5Y Cells.

Sodium tungstate (Na2WO4) normalizes glucose metabolism in the liver and muscle, activating the Mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway. Because this pathway controls neuronal survival and differentiation, we investigated the effects of Na2WO4 in mouse Neuro2a and human SH-SY5Y neuroblastoma monolayer cell cultures. Na2WO4 promotes differentiation to cholinergic neurites via an increased G1/G0 cell cycle in response to the synergic activation of the Phosphatidylinositol 3-kinase (PI3K/Akt) and ERK1/2 signaling pathways. In Neuro2a cells, Na2WO4 increases protein synthesis by activating the mechanistic target of rapamycin (mTOR) and S6K kinases and GLUT3-mediated glucose uptake, providing the energy and protein synthesis needed for neurite outgrowth. Furthermore, Na2WO4 increased the expression of myocyte enhancer factor 2D (MEF2D), a member of a family of transcription factors involved in neuronal survival and plasticity, through a post-translational mechanism that increases its half-life. Site-directed mutations of residues involved in the sumoylation of the protein abrogated the positive effects of Na2WO4 on the MEF2D-dependent transcriptional activity. In addition, the neuroprotective effects of Na2WO4 were evaluated in the presence of advanced glycation end products (AGEs). AGEs diminished neurite differentiation owing to a reduction in the G1/G0 cell cycle, concomitant with lower expression of MEF2D and the GLUT3 transporter. These negative effects were corrected in both cell lines after incubation with Na2WO4. These findings support the role of Na2WO4 in neuronal plasticity, albeit further experiments using 3D cultures, and animal models will be needed to validate the therapeutic potential of the compound.

Contactless Single Cell Extraction from Monolayer Cell Culture Using A MEMS Acoustic Droplet Ejector.

To achieve a contactless and damage-less extraction of a single (or a few) human retinal pigment epithelial (RPE) cell(s) from a cell monolayer with acoustic droplet ejection. An acoustic droplet ejector based on a self-focusing acoustic transducer (SFAT) is designed, microfabricated, and placed on a precision movable stage that aligns it to the targeted cell(s) in a Petri dish. The device delivers 20.1 MHz focused ultrasound (FUS) with a focal diameter of 100 μm, which ejects droplets capable of extracting and transferring only the targeted cell(s) from a monolayer. The extraction and collection of 1-10 cells are successfully demonstrated. The number of ejected cells can be controlled by the FUS pulse width. As confirmed by fluorescence-based cell viability assays and re-culture experiments, the ejected cell(s) and remaining monolayer cells are intact without damage after cell ejection. Furthermore, real-time reverse transcription polymerase chain reaction (RT-PCR) tests for both housekeeping genes (GAPDH and β -actin) and RPE-specific genes (MITF, PEDF, and PMEL17) show no significant difference between the acoustically ejected cells and those collected manually with a micropipette. The proposed technology realizes a contactless, damage-free extraction of cells with high spatial resolution and precise control of the number of cells ejected, with a simple setup. This powerful technology not only enables efficient, high-precision cell extraction for quality check applications but also opens new avenues for other advanced biotechnologies such as bioprinting.

Characterization of estrogen receptor mutant breast cancer in 3D cell culture.

14 Background: Breast cancer is a leading cancer in women worldwide. Many primary breast cancers are estrogen receptor positive (ER+) and responsive to anti-estrogenic therapies. These tumors can mutate estrogen receptors to survive, allowing the tumors to become more triple-negative-like and therefore more dangerous. Triple-negative breast cancer (TNBC) has been particularly challenging to treat due to its lack of estrogen (ER), progesterone (PR), and human epidermal growth factor (HER2) receptors. Due to the lack of treatment options, TNBC has a poor prognosis and contributes to a significant percentage of breast cancer mortalities. These ER mutants act more like TNBC, resulting in worse clinical outcomes. Current research on these ER mutants has been conducted using two-dimensional (2D), monolayer cell culture, which does not translate effectively in animal models, and ultimately, humans. Three-dimensional (3D) cell culture, which allows for the formation of spheroids, mimics actual tumors and provides results more consistent with tumor treatment in vitro. Due to the lack of research on these ER mutants in 3D culture, they must be characterized to determine baseline gene expression and behavior. After characterization, identifying changes resulting from drug treatment will be possible. Methods: Parental, ER+ MCF7-Luc cells, and daughter D538G and Y537S mutants were seeded at a density of 3000 cells/well in a low-attachment, round-bottomed 96-well plate. 48 hours and 7 days post-seeding, newly-formed spheroids were imaged. Using ImageJ analysis software, diameter, area, perimeter, circularity, aspect ratio, roundness, and solidity were determined. After 7 days in culture, spheres were collected for RNA extraction. Next, cDNA was synthesized, and qRT-PCR was performed to assess gene expression differences. Results: The wild-type ER+ spheres have smaller diameters, spherocity values closer to one, and are more compact. They express different levels of EMT markers from the controls, indicating alterations to signaling pathways. These 3D cultures vary in expression from the 2D cultures of the same cell lines. Conclusions: MCF-7 ER+ breast cancer cells aggregate more readily than ER+ mutants. The ER must be involved in signaling that promotes aggregation, as reduced ER signaling decreases the ability for spheroid formation. This phenomenon and the differences in gene expression may explain why mutants tend to behave in a more triple-negative manner. Cells cultured in 3D express some genes to different extents, confirming the importance of 3D culture for identifying future therapies – cells behave differently in different culturing contexts, 3D being more consistent with tumor behavior. Therefore, characterizing ER+ mutants prior to drug treatment studies is crucial to understanding how compounds affect cancer cells, as well as for identifying differences in various ER+ mutants for better treatment outcomes.

Generating Neural Progenitors from Embryonic Stem Cells in Serum-Free Monolayer Cultures

Generating Neural Progenitors from Embryonic Stem Cells in Serum-Free Monolayer Cultures

3D Culture Analysis of Cancer Cell Adherence to Ex Vivo Lung Microexplants.

Ex vivo 3D culture of human tissue explants addresses many limitations of traditional monolayer cell culture techniques, namely the lack of cellular heterogeneity and absence of 3D intercellular spatial relationships, but presents challenges with regard to repeatability owing to the difficulty of acquiring multiple tissue samples from the same donor. In this study, we used a cryopreserved bank of human lung microexplants, ∼1 mm3 fragments of peripheral lung from donors undergoing lung resection surgery, and a liquid-like solid 3D culture matrix to describe a method for the analysis of non-small-cell lung cancer adhesion to human lung tissue. H226 (squamous cell carcinoma), H441 (lung adenocarcinoma), and H460 (large cell carcinoma) cell lines were cocultured with lung microexplants. Confocal fluorescence microscopy was used to visualize the adherence of each cell line to lung microexplants. Adherent cancer cells were quantified following filtration of nonadherent cells, digestion of cultured microexplants, and flow cytometry. This method was used to evaluate the role of integrins in cancer cell adherence. A statistically significant decrease in the adherence of H460 cells to lung microexplants was observed when anti-integrins were administered to H460 cells before coculture with lung microexplants.

HPLC method for the determination of thymoquinone in growth cell medium.

Preclinical drug testing requires in vitro and in vivo assessments that are vital for studying drug pharmacokinetics and toxicity. Distinct factors that play an important role in drug screening, such as hydrophobicity, solubility of the substance and serum protein binding can be challenging by inducing result inconsistencies. Hence, establishing accurate methods to quantify drug concentrations in cell cultures becomes pivotal for reliable and reproducible results important for in vivo dosing predictions. This research focuses on developing an optimized analytical approach via high-pressure liquid chromatography (HPLC) to determine thymoquinone (TQ) levels in monolayer cell cultures. The method's validation adheres to the International Council for Harmonisation (ICH) guideline M10, ensuring its acceptance and applicability. Using an HPLC system with a Diode Array Detector (DAD), the study fine-tuned various parameters to achieve an efficient separation of TQ. Validation covered specificity, sensitivity, matrix effects, linearity, precision, and accuracy, alongside assessing TQ stability in RPMI-1640 medium. The HPLC method exhibited remarkable TQ specificity, free from interfering peaks at the analyte retention. Sensitivity analysis at the lower limit of quantification (LLOQ) revealed 5.68% %CV and 98.37% % mean accuracy. Matrix effect evaluation showcased accuracy within 85-115%. Linearity spanned in the concentration range of 2-10 μM with a correlation coefficient (r2) of 0.9993. Precision and accuracy were aligned with acceptance criteria. The proposed method was found to be greener in terms of usage of persistent, bioaccumulative, and toxic chemicals and solvents, corrosive samples, and waste production. The developed HPLC-DAD method emerges as specific, accurate, sensitive, and reliable for TQ determination in cell cultures. It ensures robust TQ quantification, enhancing precise in vitro assessments and dependable dosing predictions for in vivo studies. Further research is advocated to investigate TQ's stability across diverse environmental conditions.

Silmitasertib (CX-4945) Disrupts ERα/HSP90 Interaction and Drives Proteolysis through the Disruption of CK2β Function in Breast Cancer Cells.

Aberrant estrogen receptor (ERα) signaling mediates detrimental effects of tamoxifen including drug resistance and endometrial hyperplasia. ERα36, an alternative isoform of ERα, contributes to these effects. We have demonstrated that CK2 modulates ERα expression and function in breast cancer (BCa). Here, we assess if CX-4945 (CX), a clinical stage CK2 inhibitor, can disrupt ERα66 and ERα36 signaling in BCa. Using live cell imaging, we assessed the antiproliferative effects of CX in tamoxifen-sensitive and tamoxifen-resistant BCa cells in monolayer and/or spheroid cultures. CX-induced alterations in ERα66 and ERα36 mRNA and protein expression were assessed by RT-PCR and immunoblot. Co-immunoprecipitation was performed to determine the differential interaction of ERα isoforms with HSP90 and CK2 upon CX exposure. CX caused concentration-dependent decreases in proliferation in tamoxifen-sensitive MCF-7 and tamoxifen-resistant MCF-7 Tam1 cells and significantly repressed spheroid growth in 3D models. Additionally, CX caused dramatic decreases in endogenous or exogenously expressed ERα66 and ERα36 protein. Silencing of CK2β, the regulatory subunit of CK2, resulted in destabilization and decreased proliferation, similar to CX. Co-immunoprecipitation demonstrated that ERα66/36 show CK2 dependance for interaction with molecular chaperone HSP90. Our findings show that CK2 functions regulate the protein stability of ERα66 and ERα36 through a mechanism that is dependent on CK2β subunit and HSP90 chaperone function. CX may be a component of a novel therapeutic strategy that targets both tamoxifen-sensitive and tamoxifen-resistant BCa, providing an additional tool to treat ERα-positive BCa.

Nanostructured Implant-Tissue Interface Assessment Using a Three-Dimensional Gingival Tissue Equivalent.

Improved soft tissue integration (STI) around dental implants is key for implant success. The formation of an early and long-lasting transmucosal seal around the implant abutment might help to prevent peri-implantitis, one of the major causes of late implant failure. In natural teeth, collagen fibers are firmly inserted and fixed in the cementum of the tooth and emerge perpendicular to the gingival tissue. In contrast, around dental implants, collagen fibers run predominantly parallel to the implant surface, allowing bacterial migration into the peri-implant interface that might lead to peri-implantitis. Previous studies have shown that nanostructured Ti surfaces improve gingival cell response in monolayer cell cultures. Here, we aimed at evaluating the implant-tissue interface using a 3D gingival tissue equivalent (GTE). First, we evaluated the GTE response to a nanostructured (NN) and machined Ti surface after the stimulation with Porphyromonas gingivalis lipopolysaccharide (LPS), to simulate peri-implantitis conditions. Thus, GTE viability, through MTT assay, the release of metalloproteinase-1 (MMP1) and its inhibitor (TIMP1) through ELISA, and the gene expression of extracellular matrix turnover genes by real-time RT-PCR were analyzed. Second, GTE-implant interaction was characterized by serial block face scanning electron microscopy, and collagen-1 orientation at the tissue-implant interface was analyzed by immunofluorescence. While a similar GTE response to LPS stimulation was found for both implant surfaces, a higher proportion of collagen oriented perpendicular to the implant was observed on the NN implant surface. Thus, our results indicate that the nanostructuration of titanium dental implant abutments could allow the correct orientation of collagen fibers and greater soft tissue sealing, while keeping biocompatibility levels and LPS response comparable.

Far-Ultraviolet Light at 222 nm Affects Membrane Integrity in Monolayered DLD1 Colon Cancer Cells.

222 nm far-ultraviolet (F-UV) light has a bactericidal effect similar to deep-ultraviolet (D-UV) light of about a 260 nm wavelength. The cytotoxic effect of 222 nm F-UV has not been fully investigated. DLD-1 cells were cultured in a monolayer and irradiated with 222 nm F-UV or 254 nm D-UV. The cytotoxicity of the two different wavelengths of UV light was compared. Changes in cell morphology after F-UV irradiation were observed by time-lapse imaging. Differences in the staining images of DNA-binding agents Syto9 and propidium iodide (PI) and the amount of cyclobutane pyrimidine dimer (CPD) were examined after UV irradiation. F-UV was cytotoxic to the monolayer culture of DLD-1 cells in a radiant energy-dependent manner. When radiant energy was set to 30 mJ/cm2, F-UV and D-UV showed comparable cytotoxicity. DLD-1 cells began to expand immediately after 222 nm F-UV light irradiation, and many cells incorporated PI; in contrast, PI uptake was at a low level after D-UV irradiation. The amount of CPD, an indicator of DNA damage, was higher in cells irradiated with D-UV than in cells irradiated with F-UV. This study proved that D-UV induced apoptosis from DNA damage, whereas F-UV affected membrane integrity in monolayer cells.

X-ray Attenuating Vesicles with Neutrophil Extracellular Trap (NET) Specificity: Synthesis and Testing in a Model System.

X-ray attenuating contrast agents for imaging thrombi directly during endovascular thrombectomy (EVT) are urgently needed for shortening the wait time for treatment and for reducing the chances of blood clot fragmentation. Neutrophil extracellular traps (NETs) are a product of an innate immune system response by which neutrophils release decondensed chromatin strands decorated with granule and cytosolic proteins, including neutrophil elastase and citrullinated histone H3 (CitH3). NETs are frequently found within fibrous thrombi in pathology and represent a promising target for thrombi-specific imaging agents due to their common occurrence in human cerebrovascular thrombi. We designed and tested 200 nm lipid vesicles (LV) formulated in the presence of a combination of hydrophilic and hydrophobic computed tomography (CT) contrast agents with resultant efficacy of X-ray attenuation corresponding to 312 ± 54 mg/mL iodine. The LV incorporated trans-cyclooctene-terminated pegylated distearoylphosphatidylethanolamine (TCO-PEG-DSPE) for rapid conjugation of methyltetrazine(mTz)-modified monoclonal immunoglobulin G (IgG) with anti-citH3 binding specificity. By using differential fluorescent labeling of the antibody and lipid components, we determined that 80 ± 3% of mTz-linked IgG coprecipitated with the LV after conjugation in contrast to 0.1-0.2% of control IgG. The engineered NET-specific LV were tested in vitro using differentiated human HL60 promyeloblasts (dHL60) as a standard model of NETing neutrophils. Using fibrin meshwork-incorporated dHL60 as well as monolayer cell cultures, we determined that anti-citH3 LV showed specific and high-affinity binding to dHL60 cells, which were stimulated to undergo NETosis. This work suggests the high promise of NET-specific agents in providing thrombus-specific imaging contrast during EVT.

Probing polarization response of monolayer cell cultures with entangled photon pairs

Probing polarization response of monolayer cell cultures with entangled photon pairs

Multiaction Pt(IV) Prodrugs Releasing Cisplatin and Dasatinib Are Potent Anticancer and Anti-Invasive Agents Displaying Synergism between the Two Drugs.

Herein, we describe the general design, synthesis, characterization, and biological activity of new multitargeting Pt(IV) prodrugs that combine antitumor cisplatin and dasatinib, a potent inhibitor of Src kinase. These prodrugs exhibit impressive antiproliferative and anti-invasive activities in tumor cell lines in both two-dimensional (2D) monolayers of cell cultures and three-dimensional (3D) spheroids. We show that the cisplatin moiety and dasatinib in the investigated Pt(IV) complexes are both involved in the mechanism of action in MCF7 breast cancer cells and act synergistically. Thus, combining dasatinib and cisplatin into one molecule, compared to using individual components in a mix, may bring several advantages, such as significantly higher activity in cancer cell lines and higher selectivity for tumor cells. Most importantly, Pt(IV)-dasatinib complexes hold significant promise for potential anticancer therapies by targeting epithelial-mesenchymal transition, thus preventing the spread and metastasis of tumors, a value unachievable by a simple combination of both individual components.

Two-Dimensional and Spheroid-Based Three-Dimensional Cell Culture Systems: Implications for Drug Discovery in Cancer

The monolayer (two-dimensional or 2D) cell culture, while widely used, lacks fidelity in replicating vital cell interactions seen in vivo, leading to a shift toward three-dimensional (3D) models. Although monolayers offer simplicity and cost-effectiveness, spheroids mimic cellular environments better. This is due to its nutrient gradients, which influence drug penetration and provide a more accurate reflection of clinical scenarios than monolayers. Consequently, 3D models are crucial in drug development, especially for anti-cancer therapeutics, enabling the screening of cell cycle inhibitors and combination therapies vital for heterogeneous tumor populations. Inhibiting processes like migration and invasion often require drugs targeting the cytoskeleton, which can exhibit dual functionality with cell cycle inhibitors. Therapeutic approaches with promising anti-cancer potential often exhibit reduced efficacy in 3D cell culture compared to their performance in monolayer settings, primarily due to the heightened complexity inherent in this system. In the face of this scenario, this review aims to survey existing knowledge on compounds utilized in both 2D and 3D cell cultures, assessing their responses across different culture types and discerning the implications for drug screening, particularly those impacting the cell cycle and cytoskeletal dynamics.