Single-Cell MTT: A Simple and Sensitive Assay for Determining the Viability and Metabolic Activity of Polyploid Giant Cancer Cells (PGCCs).

Solid tumors and tumor-derived cell lines commonly contain highly enlarged (giant) cancer cells that enter a state of transient dormancy (active sleep) after they are formed, but retain viability, secrete growth promoting factors, and exhibit the ability to generate rapidly proliferating progeny with stem cell-like properties. Giant cells with a highly enlarged nucleus or multiple nuclei are often called polyploid giant cancer cells (PGCCs). Although PGCCs constitute only a subset of cells within a solid tumor/tumor-derived cell line, their frequency can increase markedly following exposure to ionizing radiation or chemotherapeutic drugs. In this chapter we outline a simple and yet highly sensitive cell-based assay, called single-cell MTT, that we have optimized for determining the viability and metabolic activity of PGCCs before and after exposure to anticancer agents. The assay measures the ability of individual PGCCs to convert the MTT tetrazolium salt to its water insoluble formazan metabolite. In addition to evaluating PGCCs, this assay is also a powerful tool for determining the viability and metabolic activity of cancer cells undergoing premature senescence following treatment with anticancer agents, as well as for distinguishing dead cancer cells and dying cells (e.g., exhibiting features of apoptosis, ferroptosis, etc.) that have the potential to resume proliferation through a process called anastasis.

Re-evaluation and modification of dehydrogenase activity tests in assessing microbial activity for wastewater treatment plant operation.

Reliable and cost-effective methods for monitoring microbial activity are critical for process control in wastewater treatment plants. The dehydrogenase activity (DHA) test has been recognized as an efficient measure of biological activity due to its simplicity and broad applicability. Nevertheless, the existing DHA test methods suffer from imperfections and are difficult to implement as routine monitoring techniques. In this work, an accurate and cost-effective modified DHA approach was developed and the procedure for the DHA test was critically evaluated with respect to the standard construction, sample pretreatment, incubation and extraction conditions. The feasibility of the modified DHA test was demonstrated by comparison with the oxygen uptake rate and adenosine triphosphate in a sequencing batch reactor. The sensitivities of the two typical tetrazolium salts to toxicant inhibition by heavy metals and antibiotics were compared, revealing that 2,3,5-triphenyltetrazolium chloride (TTC) exhibited a higher sensitivity. Furthermore, the sensitivity mechanism of the two DHA tests was elucidated through electrochemical experiments, theoretical analysis and molecular simulations. Both tetrazolium salts were found to be effective artificial electron acceptors due to their low redox potentials. Molecular docking simulations revealed that TTC could outperform other tetrazolium salts in accepting electrons and hydrogens from dehydrogenase. Overall, the modified DHA approach presents an accurate and cost-effective way to measure microbial activity, making it a practical tool for wastewater treatment plants.

Pyrazoline derivatives as promising novel antischistosomal agents.

Praziquantel is the only available drug to treat schistosomiasis, a parasitic disease that currently infects more than 240 million people globally. Due to increasing concerns about resistance and inadequate efficacy there is a need for new therapeutics. In this study, a series of 17 pyrazolines (15-31) and three pyrazoles (32-34) were synthesized and evaluated for their antiparasitic properties against ex vivo adult Schistosoma mansoni worms. Of the 20 compounds tested, six had a 50% effective concentration (EC50) below 30 µM. Our best hit, pyrazoline 22, showed promising activity against adult schistosomes, with an EC50 < 10 µM. Additionally, compound 22 had low cytotoxicity, with selectivity index of 21.6 and 32.2 for monkey and human cell lines, respectively. All active pyrazolines demonstrated a negative effect on schistosome fecundity, with a marked reduction in the number of eggs. Structure-activity relationship analysis showed that the presence of the non-aromatic heterocycle and N-substitution are fundamental to the antischistosomal properties. Pharmacokinetics, drug-likeness and medicinal chemistry friendliness studies were performed, and predicted values demonstrated an excellent drug-likeness profile for pyrazolines as well as an adherence to major pharmaceutical companies' filters. Collectively, this study demonstrates that pyrazoline derivatives are promising scaffolds in the discovery of novel antischistosomal agents.

Comparative assessment of antimicrobial, antiradical and cytotoxic activities of cannabidiol and its propyl analogue cannabidivarin.

Cannabidiol and cannabidivarin are phytocannabinoids produced by Cannabis indica and Cannabis sativa. Cannabidiol has been studied more extensively than its propyl analogue cannabidivarin. Therefore, we performed a battery of in vitro biological assays to compare the cytotoxic, antiradical and antibacterial activities of both cannabinoids. Potential mitochondrial metabolism alterations, DNA synthesis inhibition, and plasma membrane damage were studied by MTT assay, BrdU-ELISA and LDH assay of cancer and normal human cells exposed to cannabinoids. ABTS and DPPH assays were performed to observe the effects of the cannabinoids on free radicals. Microbial susceptibility tests were performed to study the activity of the cannabinoids in two bacterial species implicated in human infections, Escherichia coli and Staphylococcus aureus. The results showed that the cannabinoids induced medium levels of cytotoxicity in cancer and normal cells at concentrations ranging from 15.80 to 48.63 and from 31.89 to 151.70 µM, respectively, after 72 h of exposure. Cannabinoids did not exhibit a strong antioxidant capacity in scavenging ABTS or DPPH radicals. No evident differences were observed between the two cannabinoids in antimicrobial activity, except with respect to S. aureus, which showed greater susceptibility to cannabidiol than to cannabidivarin after 72 h of exposure.

Biocompatible graphene-zirconia nanocomposite as a cyto-safe immunosensor for the rapid detection of carcinoembryonic antigen.

Graphene-based materials have gained remarkable attention in numerous disciplines owing to their unique electrochemical properties. Out of various hybridized nanocomposites, graphene-zirconia nanocomposite (GZ) was distinctive due to its biocompatibility. Zirconia nanoparticles serve as spacers that reduce the stacking of graphene and improve the electrochemical performance of the material. Considering that lungs and skin suffer the greatest exposure to nanoparticles, this study aimed to evaluate the cytotoxicity of the as-synthesized GZ nanocomposites on MRC5 (lung cells) and HaCaT (skin cells) via morphological observation and cell viability assay using 3-(4,5 dimethylthiazol-2-yl)-(2,5-diphenyltetrazolium bromide) tetrazolium (MTT). GZ-treated cells showed a comparable proliferation rate and morphology with untreated cells under microscopic evaluation. Based on MTT results, the IC50 values of GZ were > 500 µg/ml for MRC5 and HaCaT cells. The excellent biocompatibility was the supremacy of GZ over other nanocomposites applied as electrode materials in biosensors. GZ was functionalized with biolinker for the detection of carcinoembryonic antigen (CEA). The proposed immunosensor exhibited good responses towards CEA detection, with a 4.25 pg/ml LOD and correlation coefficient of R2 = 0.99 within a linear working range from 0.01 to 10 ng/ml. The performance of the immunosensor to detect CEA present in human serum was also evaluated. Good recovery of CEA was found, suggesting that the proposed immunosensor possess a high affinity to CEA even in a complex biological matrix, rendering it a promising sensing platform for real sample analysis and open a new way for the detection of cancer-associated proteins.

MP-SeNPs; A Promising Cytokines Suppressor in Benzo[a]pyrene-Induced Mammal Tissue Injury in Rats.

<b>Background and Objective:</b> <i>Moringa peregrina</i> (family Moringaceae) is a common flowering plant found in the Arabian Peninsula, Horn of Africa and Southern Sinai, Egypt. The purpose of this study was to investigate the protective activity of MP-SeNPs against BaP-induced mammal tissue injury in rats. <b>Materials and Methods:</b> MP-SeNPs were prepared and characterized in terms of particle size and zeta potential. Furthermore, the IC₅₀ of MP-SeNPs against the Mcf7 breast carcinoma cell line and LD₅₀ was evaluated. Adult albino rats weighing approximately 187±10 g was used to assess the lung protective activity of MP-SeNPs (28.7 and 71.75 mg kg¹ b.wt.) against BaP-induced mammal tissue injury in rats. <b>Results:</b> The mean particle size of MP-SeNPs was 134.69±8.24 nm with negative zeta potential of +26.04 with the observed shapes of nano particle was spherical. Also, IC₅₀ of MP-SeNPs against Mcf7 breast carcinoma cell line = 89.57 µg mL¹ and LD₅₀ equals and 1435 mg kg¹ b.wt., respectively. The daily oral administration of MP-SeNPs at concentrations of 28.7 and 71.75 mg kg¹ b.wt. for 30 days to rats treated with BaP (20 mg kg¹ b.wt.) resulted in a significant improvement of IL-2, IL-6 and IL-10. Oral administration of MP-SeNPs, on the other hand, increased the levels of SOD, GPx, TNF-α, iNOs and GSH as well as decreased the level of MDA in mammal tissue of BaP-treated rats. Furthermore, MP-SeNPs almost normalized these effects in mammal tissue histoarchitecture and MRI examination. <b>Conclusion:</b> The biochemical, histological and MRI findings incurrent study demonstrated that MP-SeNPs have protective activity against BaP-induced mammal tissue injury in rats.

Antimutagenic, Cytoprotective and Antioxidant Properties of Ficus deltoidea Aqueous Extract In Vitro.

Ficus deltoidea var. deltoidea is used as traditional medicine for diabetes, inflammation, and nociception. However, the antimutagenic potential and cytoprotective effects of this plant remain unknown. In this study, the mutagenic and antimutagenic activities of F. deltoidea aqueous extract (FDD) on both Salmonella typhimurium TA 98 and TA 100 strains were assessed using Salmonella mutagenicity assay (Ames test). Then, the cytoprotective potential of FDD on menadione-induced oxidative stress was determined in a V79 mouse lung fibroblast cell line. The ferric-reducing antioxidant power (FRAP) assay was conducted to evaluate FDD antioxidant capacity. Results showed that FDD (up to 50 mg/mL) did not exhibit a mutagenic effect on either TA 98 or TA 100 strains. Notably, FDD decreased the revertant colony count induced by 2-aminoanthracene in both strains in the presence of metabolic activation (p < 0.05). Additionally, pretreatment of FDD (50 and 100 µg/mL) demonstrated remarkable protection against menadione-induced oxidative stress in V79 cells significantly by decreasing superoxide anion level (p < 0.05). FDD at all concentrations tested (12.5-100 µg/mL) exhibited antioxidant power, suggesting the cytoprotective effect of FDD could be partly attributed to its antioxidant properties. This report highlights that F. deltoidea may provide a chemopreventive effect on mutagenic and oxidative stress inducers.

Piperlongumine Analogs Promote A549 Cell Apoptosis through Enhancing ROS Generation.

Chemotherapeutic agents, which contain the Michael acceptor, are potent anticancer molecules by promoting intracellular reactive oxygen species (ROS) generation. In this study, we synthesized a panel of PL (piperlongumine) analogs with chlorine attaching at C2 and an electron-withdrawing/electron-donating group attaching to the aromatic ring. The results displayed that the strong electrophilicity group at the C2-C3 double bond of PL analogs plays an important role in the cytotoxicity whereas the electric effect of substituents, which attached to the aromatic ring, partly contributed to the anticancer activity. Moreover, the protein containing sulfydryl or seleno, such as TrxR, could be irreversibly inhibited by the C2-C3 double bond of PL analogs, and boost intracellular ROS generation. Then, the ROS accumulation could disrupt the redox balance, induce lipid peroxidation, lead to the loss of MMP (Mitochondrial Membrane Potential), and ultimately result in cell cycle arrest and A549 cell line death. In conclusion, PL analogs could induce in vitro cancer apoptosis through the inhibition of TrxR and ROS accumulation.

SN38 loaded nanostructured lipid carriers (NLCs); preparation and in vitro evaluations against glioblastoma.

SN38 is the active metabolite of irinotecan with 1000-fold greater cytotoxicity compared to the parent drug. Despite the potential, its application as a drug is still seriously limited due to its stability concerns and low solubility in acceptable pharmaceutical solvents. To address these drawbacks here nanostructured lipid carrier (NLC) containing SN38 was prepared and its cytotoxicity against U87MG glioblastoma cell line was investigated. The formulations were prepared using hot ultrasonication and solvent evaporation/emulsification methods. NLCs with a mean size of 140 nm and particle size distribution (PDI) of 0.25 were obtained. The average loading efficiency was 9.5% and its entrapment efficiency was 81%. In order to obtain an accurate determination of released amount of SN38 a novel medium and extraction method was designed, which lead to an appropriate in vitro release profile of the drug from the prepared NLCs. The MTT test results revealed the significant higher cytotoxicity of NLCs on U87MG human glioblastoma cell line compared with the free drug. The confocal microscopy images confirmed the proper penetration of the nanostructures into the cells within the first 4 h. Consequently, the results indicated promising potentials of the prepared NLCs as a novel treatment for glioblastoma.

Bio-corrosion impacts on mechanical integrity of ZM21 Mg for orthopaedic implant application processed by equal channel angular pressing.

The mechanical integrity of rolled ZM21 Mg was improved by equal channel angular pressing (ECAP) to function as a potential biodegradable bone screw implant. Electron backscattered diffraction (EBSD) revealed deformed grains of 45 µm observed in rolled ZM21 Mg. They were transformed to equiaxed fine grains of 5.4 µm after 4th pass ECAP. The yield strength of rolled and ECAPed ZM21 Mg alloys were comparable. In contrast, 4th pass ZM21 Mg exhibited relatively higher elongation when compared to rolled sample. The mechanical properties of rolled and ECAPed ZM21 Mg were dependant on both grain refinement and crystallographic texture. The rolled and 4th pass ECAPed tensile samples exhibited nonlinear deterioration of mechanical properties when tested after 7, 14, 21 and 28 days immersion in Hank's solution. The evaluation signifies that regardless their processing condition, ZM21 Mg alloys are suitable for surgical areas that requires high mechanical strength. In addition, the 4th pass ECAP samples were viable to MG-63 cells proving themselves to be promising candidates for future in vivo studies.

Automated antimicrobial susceptibility testing of slow-growing Pseudomonas aeruginosa strains in the presence of tetrazolium salt WST-1.

Slow growing, mucoid isolates of Pseudomonas aeruginosa require adaptation of the protocol used for automated antimicrobial susceptibility testing (AST). In the present study we used a water soluble tetrazolium salt WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate) in combination with menadione for possibly improving AST of slow growing and biofilm-forming P. aeruginosa isolates from cystic fibrosis (CF) patients. WST-1 and menadione addition ensures sensitive detection of microbial growth increase in the presence of antibiotics that may remain undetected with the automated VITEK® 2 method. We observed that 32.8% of P. aeruginosa isolates from CF and bronchiectasis patients produced an elevated absorbance signal intensity thereby increasing the sensitivity while maintaining the accuracy of VITEK 2. Our study merits future investigation with other slow growing pathogenic bacterial species.

Nobiletin promotes the pyroptosis of breast cancer via regulation of miR-200b/JAZF1 axis.

Nobiletin is a polymethoxylated flavone present in citrus fruits, which has been reported to have inhibitory effects on tumorigenesis of cancers. However, the biological function of nobiletin in breast cancer (BC) is largely unknown. To investigate the effect of nobiletin on growth of BC cells, the cell viability of BC was measured by MTT assay. In addition, gene and protein expressions were detected by qRT-PCR and western blot, respectively. The apoptosis and pyroptosis of BC cells were tested by flow cytometry. Finally, the correlation between miR-200b and JAZF1 was detected by dual luciferase report. The data indicated that nobiletin inhibited the proliferation of BC cells in a dose-dependent manner. Moreover, miR-200b mimics-induced pyroptosis of BC cells was further increased by nobiletin. Meanwhile, JAZF1 was found to be the target of miR-200b. Moreover, nobiletin induced apoptosis and pyroptosis of BC cells via miR-200b/JAZF1/NF-κB axis. In conclusion, nobiletin inhibited the tumorigenesis of BC via regulation of miR-200b/JAZF1 axis. Thus, nobiletin might serve as a new agent for the treatment of BC.

Magnetic cobalt oxide nanosheets: green synthesis and in vitro cytotoxicity.

Cobalt oxide nanoparticles were prepared via green chemistry route and fully characterized by Field Emission Scanning Electron Microscope (FESEM), Energy-dispersive X-ray spectroscopy (EDAX), X-ray diffraction (XRD), High-resolution transmission electron microscopy (HRTEM) and Transmission electron microscopy (TEM) analyses; the CoO and Co3O4 nanoparticles, in sheet-shaped cobalt oxide form, ensued simultaneously in one step. The varying concentrations of NPs were analyzed via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test on the cancer cell line (U87) which revealed that with increasing concentration of cobalt oxide nanoparticles, the survival rate of U87 tumor cells decreases; IC50 of nanoparticles being ~ 55 µg/ml-1.

Microwave-Assisted Rapid Green Synthesis of Gold Nanoparticles Using Seed Extract of Trachyspermum ammi: ROS Mediated Biofilm Inhibition and Anticancer Activity.

Green synthesis of metal nanoparticles using plant extracts as capping and reducing agents for the biomedical applications has received considerable attention. Moreover, emergence and spread of multidrug resistance among bacterial pathogens has become a major health concern and lookout for novel alternative effective drugs has gained momentum. In current study, we synthesized gold nanoparticles using the seed extract of Trachyspermum ammi (TA-AuNPs), assessed its efficacy against drug resistant biofilms of Listeria monocytogenes and Serratia marcescens, and evaluated its anticancer potential against HepG2 cancer cell lines. Microwave-assisted green synthesis of gold nanoparticles was carried out and characterization was done using UV-vis spectroscopy, X-ray diffraction (XRD), transmission electron microscopy (TEM), and dynamic light scattering (DLS). Most nanoparticles were observed as spherical and spheroidal with few anisotropies with an average crystalline size of 16.63 nm. Synthesized TA-AuNPs demonstrated significant biofilm inhibitory activity against L. monocytogenes (73%) as well as S. marcescens (81%). Exopolysaccharide (EPS), motility, and CSH, key elements that facilitate the formation and maintenance of biofilm were also inhibited significantly at the tested sub-minimum inhibitory concentrations (sub-MICs). Further, TA-AuNPs effectively obliterated preformed mature biofilms of S. marcescens and L. monocytogenes by 64% and 58%, respectively. Induction of intracellular ROS production in TA-AuNPs treated bacterial cells could be the plausible mechanism for the reduced biofilm formation in test pathogens. Administration of TA-AuNPs resulted in the arrest of cellular proliferation in a concentration-dependent manner. TA-AuNPs decrease the intracellular GSH in HepG2 cancer cell lines, cells become more prone to ROS generation, hence induce apoptosis. Thus, this work proposes a new eco-friendly and rapid approach for fabricating NPs which can be exploited for multifarious biomedical applications.

Apigenin suppresses proliferation, invasion, and epithelial-mesenchymal transition of cervical carcinoma cells by regulation of miR-152/BRD4 axis.

The present study aimed to investigate the role of apigenin and the molecular mechanism of miR-152-5p and bromodomain containing 4 (BRD4) in the proliferation, invasion, and epithelial-mesenchymal transition (EMT) of cervical carcinoma cells. Quantitative real-time PCR was used to detect the transfection efficiency and the expression of miR-152-5p and BRD4. Western blotting was conducted to evaluate the protein level of BRD4, E-cadherin, N-cadherin, and MMP9. Luciferase reporter assay was performed to confirm whether miR-152-5p bound to BRD4. MTT and Transwell invasion assay were applied to determine the cell proliferation and invasion, respectively. MiR-152-5p was downregulated and BRD4 was upregulated in cervical carcinoma tissue. Besides, miR-152-5p could directly bind to BRD4 in Hela and CaSki cells. In addition, apigenin inhibited proliferation, invasion, and EMT of Hela and CaSki cells by regulating miR-152-5p/BRD4 axis. Apigenin suppresses proliferation, invasion, and induced EMT of cervical carcinoma cells by regulation of miR-152-5p/BRD4 axis.

Near-infrared fluorogenic receptor for selective detection of cysteine in blood serum and living cells.

A novel near-infrared fluorescent probe, namely propane-2,2-diylbis(2-((E)-2-(benzo[d]thiazol-2-yl)-2-cyanovinyl)-4,1-phenylene) diacrylate (BTA), was synthesized for selective detection of cysteine over other biologically significant amino acids. Upon addition of cysteine, the probe BTA displays a dramatic increase in fluorescence intensity at 715 nm along with a fast response time (4 min). The limit of detection (LOD) was calculated as 0.12 µM. In addition, the synthesized probe BTA was effectively utilized for the recognition of cysteine in blood serum and living cells.

MiR-182 inhibits proliferation, migration, invasion and inflammation of endometrial stromal cells through deactivation of NF-κB signaling pathway in endometriosis.

Endometriosis affects about 10-15% women for reproductive age, but it is not currently curable and the underlying etiology for this disease is still not clear. In the present study, functions and mechanisms of miR-182 and RELA in endometriosis were investigated. BAY 11-7082 was used to block NF-κB pathway. qRT-PCR, ELISA and western blot assays were employed to evaluate the expressions of miR-182 and RELA, inflammatory factors and epithelial-mesenchymal transition (EMT)-related markers, and activation of NF-κB pathway. MTT, wound healing or Transwell assays were used to evaluate the cell proliferation, migration and invasion capacities. Bioinformatic and dual-luciferase reporter assays were carried out to analyze the interaction between miR-182 and RELA. MiR-182 expression was decreased, while RELA was increased as developed from normal to eutopic and ectopic status, which was accompanied by upregulated inflammatory factors and EMT-related proteins. RELA was directly targeted by miR-182 in human endometrial stromal cells. Overexpression of RELA increased inflammation-associated and EMT-related markers expression, while miR-182 upregulation decreased the expression of these genes in a dose-dependent manner, which finally attenuated the proliferation, migration and invasion capacities of endometrial stromal cells through deactivation of NF-κB signaling pathway. Moreover, co-overexpression of RELA reversed the above effects induced by miR-182. In a word, miR-182 directly targeted RELA and inhibited proliferation, migration, invasion, EMT and inflammation of endometrial stromal cells through deactivation of NF-κB signaling pathway in endometriosis. These results provide new insights into the interaction between miR-182 and NF-κB pathway and their potential as therapeutic targets for treatment of endometriosis.

LncRNA TP73-AS1 regulates miR-495 expression to promote migration and invasion of nasopharyngeal carcinoma cells through junctional adhesion molecule A.

The main obstacle to the treatment of nasopharyngeal carcinoma (NPC) is metastasis. Long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) are highly involved in the progression of NPC. In this study, we aimed to explore the regulatory role of lncRNA P73 antisense RNA 1 T (TP73-AS1) and miR-495 in migration and invasion of NPC cells. The expression levels of TP73-AS1, miR-495, and junctional adhesion molecule A (JAM-A) in NPC tissue samples and cell lines were examined by quantitative real-time PCR (qRT-PCR) and/or Western blot. NPC cells were transfected with vectors overexpressing TP73-AS1, short hairpin RNA (shRNA) against TP73-AS1, shRNA against JAM-A, miR-495 mimics, miR-495 inhibitor, and their corresponding negative controls as designated. The MTT assay, cell migration assay, and transwell assay were performed to detect cell viability, migration, and invasion, respectively. Dual-luciferase reporter assay was performed to confirm the binding of TP73-AS1 and miR-495, and miR-495 and JAM-A. TP73-AS1 and JAM-A were significantly upregulated while miR-495 was markedly downregulated in NPC tissues and cell lines compared to normal controls. The overexpression of TP73-AS1 promoted migration and invasion of NPC cell line CNE-2. TP73-AS1 targeted miR-495 and negatively regulated its expression. TP73-AS1 upregulated the expression of JAM-A through miR-495. TP73-AS1 mediated migration and invasion of CNE-2 cells via upregulating JAM-A. LncRNA TP73-AS1, miR-495, and JAM-A are involved in migration and invasion of NPC cells. The TP73-AS1/miR-495/JAM-A axis may serve as a therapeutic target for the treatment of NPC.

Characterization and cytocompatibility of 3D porous biomimetic scaffold derived from rabbit nucleus pulposus tissue in vitro.

Intervertebral disc (IVD) degeneration is one of the most important causes of lower back pain. Tissue engineering provides a new method for the experimental treatment of degenerative disc diseases. This study aims to develop a natural, acellular, 3D interconnected porous scaffold derived from the extracellular matrix (ECM) of nucleus pulposus. The nucleus pulposus (NP) was decellularized by sequential detergent-nuclease methods, including physical crushing, freeze-drying and cross-linking. These 3D porous scaffolds were fabricated with a high porosity of (81.28 ± 4.10)%, an ideal pore size with appropriate mechanical properties. Rabbit bone marrow mesenchymal stem cells (rBMSCs) were seeded and cultured on the scaffolds. And the mechanical tests showed the compressive elastic modulus of the scaffolds cultured for 4 weeks reached 0.12 MPa, which was better than that of the scaffolds cultured for 2 weeks (0.07 MPa) and that of the control group (0.04 MPa). Scanning electron microscopy (SEM), histological assays, molecular biology assays revealed that the scaffolds could provide an appropriate microstructure and environment for the adhesion, proliferation, migration and secretion of seeded cells in vitro. As assays like histology, immunohistochemistry and the real-time qRT-PCR showed, NP-like tissues were preliminarily formed. In conclusion, the 3D porous scaffold derived from NP ECM is a potential biomaterial for the regeneration of NP tissues. A natural, acellular, 3D interconnected porous scaffold derived from the extracellular matrix (ECM) of nucleus pulposus was developed by sequential detergent-nuclease and freeze-drying method, which can reduce the damage of protein activity to the minimum. It is very similar to the composition and internal environment of the natural nucleus pulposus, because it derived from the natural nucleus pulposus. Scanning electron microscopy (SEM), histological assays, molecular biology assays revealed that the scaffolds could provide an appropriate microstructure and environment for the adhesion, proliferation, migration, and secretion of seeded cells in vitro.

Paper-supported co-culture system for dynamic investigations of the lung-tropic migration of breast cancer cells.

Tumor tropism metastasis is a multi-step process that involves interactions between tumor cells and the microenvironment. Due to the limitations of experimental techniques, current studies are not able to gain insight into the dynamic process of such tropism migration. To overcome this issue, we developed a paper-supported co-culture system for dynamic investigations of the lung-tropic migration of breast cancer cells. This co-culture system contains a tumor layer, a recruitment layer, and several invasion layers between these two parts. The tumor and recruitment layers are impregnated with breast cancer cells and lung cells, respectively. Stacking these layers forms a co-culture device that comprises interactions between breast cancer and lung, destacking such a device represents cancer cells at different stages of the migration process. Thus, the paper-supported co-culture system offers the possibility of investigating migration from temporal and spatial aspects. Invasion assays using the co-culture system showed that breast cancer cells induced lung fibroblasts to convert to cancer-associated fibroblasts (CAFs), and the CAFs, in turn, recruited breast cancer cells. During migration, the local invasion of the cancer cells is a collective behavior, while the long-distance migration comes from individual cell behaviors. Breast cancer cells experienced repetitive processes of migration and propagation, accompanied by epithelial-mesenchymal and mesenchymal-epithelial transitions, and changes in stemness and drug resistance. Based on these results, the lung-tropic migration of breast cancer is interpreted as a process of bilateral interaction with the local and host-organ microenvironment. The developed paper-supported co-culture system offers the possibility of dynamically investigating tropism migration under the pre-metastatic niche, thus providing an advantageous tool for studying tumor metastasis.