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A simple layer-stacking technique to generate biomolecular and mechanical gradients in photocrosslinkable hydrogels
摘要： Physicochemical and biological gradients are desirable features for hydrogels to enhance their relevance to biological environments for three-dimensional (3D) cell culture. Therefore, simple and efficient techniques to generate chemical, physical and biological gradients within hydrogels are highly desirable. This work demonstrates a technique to generate biomolecular and mechanical gradients in photocrosslinkable hydrogels by stacking and crosslinking prehydrogel solution in a layer by layer manner. Partial crosslinking of the hydrogel allows mixing of prehydrogel solution with the previous hydrogel layer, which makes a smooth gradient profile, rather than discrete layers. This technique enables the generation of concentration gradients of bovine serum albumin in both gelatin methacryloyl (GelMA) and poly(ethylene glycol) diacrylate hydrogels, as well as mechanical gradients across a hydrogel containing varying gel concentrations. Fluorescence microscopy, mechanical testing, and scanning electron microscopy show that the gradient profiles can be controlled by changing both the volume and concentration of each layer as well as intensity of UV exposure. GelMA hydrogel gradients with different Young’s moduli were successfully used to culture human fibroblasts. The fibroblasts migrated along the gradient axis and showed different morphologies. In general, the proposed technique provides a rapid and simple approach to design and fabricate 3D hydrogel gradients for in vitro biological studies and potentially for in vivo tissue engineering applications.
关键词： 3D cell culture,Gelatin methacryloyl,Photocrosslinkable hydrogel,Poly(ethylene glycol) diacrylate,Gradient
更新于2025-11-21 11:24:58
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Isolation and Culture of Primary Mouse Retinal Pigment Epithelial (RPE) Cells with Rho-Kinase and TGFβR-1/ALK5 Inhibitor
摘要： Primary RPE cells could be a reliable model for representing in vivo status of RPE compared with cell lines. We present a protocol for in vitro isolation and culture of primary RPE cells from C57BL mice. We used C57BL mice ages 7 days to 4 months. The RPE layer was separated from the neural retina layer by digestion with 2% Dispase for 45 min and scraped off from the choroid after 25-min incubation in 37°C. Collected RPE sheets were gently pipetted up into smaller sheets. RPE sheets were transferred into well plates and cultured in vitro for 2 weeks. To inhibit epithelial-mesenchymal transition (EMT) of RPE cells, we used Y27632 and Repsox to treat cultured primary RPE cells. RPE cells isolated from C57BL mice maintained pigmented and hexagonal morphology in culture. However, long-term in vitro culture lead to the periphery cells of a RPE sheet becoming mesenchymal-like cells. In contrast to the control group, Y27632 and Repsox, which are inhibitors of Rho-kinase or TGFbR-1/ALK5, promoted primary RPE cells to maintain epithelial-like morphology and eventually become confluent. RPE cells isolated from C57BL mice could be a powerful cell model to study the biological function of RPE. Especially, C57BL mice with different defective genetic background resulting in ocular diseases, would expand the genome type of RPE cells. The method presented here could be an efficient and applicable technique to obtain large numbers of primary RPE cells that maintain some characteristics of in vivo RPE.
关键词： Primary Cell Culture,Mice,Retinal Pigment Epithelium,Inbred C57BL
更新于2025-09-23 15:23:52
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Assessing UV Inactivation of Adenovirus 41 Using Integrated Cell Culture Real-Time qPCR/RT-qPCR
摘要： Enteric adenoviruses are among most UV-resistant viruses in water. Cytopathic effects (CPE)-based cell culture TCID50 assay as a conventional virus assessment approach has major drawbacks for enteric adenovirus since it is selective on cell lines and takes longer time to show CPE. Integrated cell culture real-time quantitative PCR (ICC-qPCR) and reverse transcriptase (RT)-qPCR were applied in this study, in comparison with TCID50, to assess UV inactivation of adenovirus type 41 (Ad41) in water. Adenovirus type 41 was exposed to UV doses of 40, 80, 160, and 320 mJ/cm2 using a collimated beam apparatus. There was no significant difference of inactivation at conducted UV doses between measurements using TCID50 assay and ICC-RT-qPCR. Both assays fitted the Chick–Watson model at 95% confidence level. The inactivation measured by ICC-qPCR did not fit the Chick–Watson model. In summary, ICC-RT-qPCR is the most appropriate alternate to CPE-based assay for assessing UV inactivation of enteric adenoviruses. Water Environ. Res., 89, 323 (2017).
关键词： cytopathic effects,cell culture,real-time quantitative PCR/RT-PCR,adenovirus type 41,water,UV inactivation
更新于2025-09-23 15:22:29
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Lens-free microscopy for 3D + time acquisitions of 3D cell culture
摘要： Thanks to a novel three-dimensional imaging platform based on lens-free microscopy, it is possible to perform multi-angle acquisitions and holographic reconstructions of 3D cell cultures directly into the incubator. Being able of reconstructing volumes as large as ~5 mm3 over a period of time covering several days, allows us to observe a broad range of migration strategies only present in 3D environment, whether it is single cell migration, collective migrations of cells and dispersal of cells. In addition we are able to distinguish new interesting phenomena, e.g. large-scale cell-to-matrix interactions (>1 mm), fusion of cell clusters into large aggregate (~10,000 μm2) and conversely, total dissociation of cell clusters into clumps of migrating cells. This work on a novel 3D + time lens-free microscopy technique thus expands the repertoire of phenomena that can be studied within 3D cell cultures.
关键词： cell migration,holographic reconstruction,extracellular matrix,3D cell culture,lens-free microscopy
更新于2025-09-23 15:21:01
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A novel strategy for the synthesis of gold nanoparticles with Catharanthus roseus cell suspension culture
摘要： The purpose of this research was to develop a green method to synthesise gold nanoparticles (Au NPs) with cell suspension cultures of Catharanthus roseus. Reaction mixtures containing cell-free culture filtrate of C. roseus and gold III ions, which was initially pale yellow, turned wine red after 24-h incubation due to Au NP formation. UV–Vis spectra of the reaction mixtures showed a characteristic absorption peak at 546 nm. The biosynthesised NPs were predominantly spherical with 2–20 nm size; their crystalline nature was revealed by X-ray diffraction patterns. FTIR analysis indicated that Au NPs were capped by amide characteristic of plant proteins, which makes them apt for various applications.
关键词： Nanoparticle,Green chemistry,Cell culture,Gold,Catharanthus roseus
更新于2025-09-23 15:19:57
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Non-Invasive Quantification of the Growth of Cancer Cell Colonies by a Portable Optical Coherence Tomography
摘要： Investigation of tumor development is essential in cancer research. In the laboratory, living cell culture is a standard bio-technology for studying cellular response under tested conditions to predict in vivo cellular response. In particular, the colony formation assay has become a standard experiment for characterizing the tumor development in vitro. However, quantification of the growth of cell colonies under a microscope is difficult because they are suspended in a three-dimensional environment. Thus, optical coherence tomography (OCT) imaging was develop in this study to monitor the growth of cell colonies. Cancer cell line of Huh 7 was used and the cells were applied on a layer of agarose hydrogel, i.e., a non-adherent surface. Then, cell colonies were gradually formed on the surface. The OCT technique was used to scan the cell colonies every day to obtain quantitative data for describing their growth. The results revealed the average volume increased with time due to the formation of cell colonies day-by-day. Additionally, the distribution of cell colony volume was analyzed to show the detailed information of the growth of the cell colonies. In summary, the OCT provides a non-invasive quantification technique for monitoring the growth of the cell colonies. From the OCT images, objective and precise information is obtained for higher prediction of the in vivo tumor development.
关键词： cell culture,cell colonies,tumor spheroids,optical coherence tomography
更新于2025-09-19 17:15:36
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Cytotoxicity assays to evaluate tannery effluents treated by photoelectrooxidation
摘要： The advanced oxidation process (AOP) is used to increase the treatment efficiency of effluents however, it is necessary to compare the toxicity of treated and untreated effluents to evaluate if the decontamination process does not cause any biological harm. Cultured cells have been previously used to assess the genotoxic and cytotoxic potential of various compounds. Hence, the aim of this work was to assess the applicability of cytotoxicity assays to evaluate the toxicity related to the AOP treatment. Samples of an industrial effluent were collected after their treatment by a conventional method. Cytotoxicity of standard and AOP treated effluents was assessed in CRIB and HEp-2 cell line using the MTT and neutral red assays. We observed decrease at cell viability in the both assays (50% MTT and 13% NRU) when cells were exposed to the AOP treatment in the highest concentration. Thus, cytotoxic assays in cultured cells can be explored as an useful method to evaluate toxicity as well as to optimize effluents treatment process.
关键词： cell culture,effluent,photoelectrooxidation,tannery,cytotoxicity
更新于2025-09-19 17:15:36
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Continuous Live-Cell Culture Monitoring by Compact Lensless LED Microscopes
摘要： A compact lensless microscope comprising a custom-made LED engine and a CMOS imaging sensor has been developed for live-cell culture imaging inside a cell incubator environment. The imaging technique is based on digital inline-holographic microscopy, while the image reconstruction is carried out by angular spectrum approach with a custom written software. The system was tested with various biological samples including immortalized mouse astrocyte cells inside a petri dish. Besides the imaging possibility, the capability of automated cell counting and tracking could be demonstrated. By using image sensors capable of video frame rate, time series of cell movement can be captured.
关键词： lensless holographic microscopy,cell imaging,LED,CMOS image sensor,cell culture,cell counting
更新于2025-09-16 10:30:52
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Photodynamic therapy in 2D and 3D human cervical carcinoma cell cultures employing LED light sources emitting at different wavelengths
摘要： Light of different wavelengths can be used to obtain a more profitable outcome of photodynamic therapy (PDT), according to the absorption bands of the photosensitizer (PS). Low-grade cervical intraepithelial neoplasias (CINs) are superficial lesions that can be treated with light of shorter wavelength than red because a large light penetration depth in tissue is not necessary. We report a comparative investigation performed to evaluate the efficacy of light-emitting diodes (LEDs) of different wavelengths in the photodynamic treatment applied to both 2D and 3D HeLa cell spheroid cultures. The spheroids are utilized as a PDT dosage model, and cell viability is evaluated at different sections of the spheroids by confocal microscopy. Cells incubated with m-tetrahydroxyphenyl chlorin are illuminated with LED systems working in the low fluence range, emitting in the violet (390 – 415 nm), blue (440-470 nm), red (620 – 645 nm) and deep red (640 – 670 nm) regions of the light spectrum at various exposures times (tI) comprised between 0.5 and 30 min. PDT experiments performed on both 2D and 3D cell cultures indicate that the PDT treatment outcome is more efficient with violet light followed by red light. Dynamic data from the front displacement velocity of large 2D-quasi-radial colonies generated from cell spheroids adhered to the Petri dish bottom as well as the evolution of the 3D growth give further insight about the effect of PDT at each condition. Results from 3D cultures indicate that the penetration of the violet light is appropriate to kill HeLa cells several layers below, showing cell damage and death not only in the outer rim of the illuminated spheroids, where a PS accumulation exists, but also in the more internal region. Results indicate that violet LED light could be useful to treat CINs involving superficial dysplasia.
关键词： LEDs,cervical intraepithelial neoplasias,Photodynamic therapy,m-THPC,2D and 3D HeLa cell culture
更新于2025-09-11 14:15:04
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Generation of Dispersed Presomitic Mesoderm Cell Cultures for Imaging of the Zebrafish Segmentation Clock in Single Cells
摘要： Segmentation is a periodic and sequential morphogenetic process in vertebrates. This rhythmic formation of blocks of tissue called somites along the body axis is evidence of a genetic oscillator patterning the developing embryo. In zebrafish, the intracellular clock driving segmentation is comprised of members of the Her/Hes transcription factor family organized into negative feedback loops. We have recently generated transgenic fluorescent reporter lines for the cyclic gene her1 that recapitulate the spatio-temporal pattern of oscillations in the presomitic mesoderm (PSM). Using these lines, we developed an in vitro culture system that allows real-time analysis of segmentation clock oscillations within single, isolated PSM cells. By removing PSM tissue from transgenic embryos and then dispersing cells from oscillating regions onto glass-bottom dishes, we generated cultures suitable for time-lapse imaging of fluorescence signal from individual clock cells. This approach provides an experimental and conceptual framework for direct manipulation of the segmentation clock with unprecedented single-cell resolution, allowing its cell-autonomous and tissue-level properties to be distinguished and dissected.
关键词： Developmental Biology,Somitogenesis,Biological Clocks,Primary Cell Culture,Primary Culture,Zebrafish,Oscillator,Fluorescence,In Vitro,Time-lapse Imaging
更新于2025-09-10 09:29:36