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The quantification of live and dead cells in a substrate is often an essential step in cell biology research. A staining protocol that acts differently on live and on dead cells is applied and the number of cells visible is counted using a microscope. Often this counting is done manually or only evaluated qualitatively. If the number of samples to...
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... First, a live/dead assay at different time-points was performed, confirming the biocompatibility of the functionalized biomaterials. From that, according to literature [46], the cell viability was then quantified ( Figure 5B). Second, the cells were stained using phalloidin/DAPI to get information of their cytoskeleton architectures when exposed to the different treated COL films ( Figure 5C). ...
Aim: Cell microenvironment contains a plethora of information that influences cell modulation. Indeed, the extracellular matrix plays a central role in tissue development. Reproducing the cell–extracellular matrix crosstalk able to recapitulate both physical and biochemical signals is crucial to obtain functional tissue models or regenerative strategies. Materials & methods: Here, a combined method is proposed to easily functionalize collagen surface films, tailoring morphological properties. Oxygen nonthermal plasma treatment and glyco-conjugation with chondroitin sulfate are used to modify surface properties. Results: It results in higher adhesion, proliferation and morphological organization of U87 glioblastoma cells. Conclusion: Our finding suggests new promising strategies for the development of collagen-based biomaterials, which can be employed for advanced in vitro models.
... Signal intensity was quantified using ImageJ software. Original RGB images were converted to 8-bit (grey scale) format, and tagged area intensities were expressed as mean value of pixel intensity within a range from 0 (dark) to 255 (white), as previously reported (Spaepen et al., 2011). ...
In this work, a 3D environment obtained using fibrin scaffold and two cell populations, such as bone marrow-derived mesenchymal stem cells (BM-MSCs), and primary skeletal muscle cells (SkMs), was assembled. Peripheral blood mononuclear cells (PBMCs) fraction obtained after blood filtration with HemaTrate ® filter was then added to the 3D culture system to explore their influence on myogenesis. The best cell ratio into a 3D fibrin hydrogel was 1:1 (BM-MSCs plus SkMs:PBMCs) when cultured in a perfusion bioreactor; indeed, excellent viability and myogenic event induction were observed. Myogenic genes were significantly overexpressed when cultured with PBMCs, such as MyoD1 of 118-fold at day 14 and Desmin 6-fold at day 21. Desmin and Myosin Heavy Chain were also detected at protein level by immunostaining along the culture. Moreover, the presence of PBMCs in 3D culture induced a significant downregulation of pro-inflammatory cytokine gene expression, such as IL6 . This smart biomimetic environment can be an excellent tool for investigation of cellular crosstalk and PBMC influence on myogenic processes.
... National Institutes of Health, Bethesda, MD, United States). Original images in RGB format were converted into an 8-bit (gray scale) format, and tagged areas were expressed as an average value of pixel intensity within a range from 0 (dark) to 255 (white) as better described elsewhere (Spaepen et al., 2011). ...
In this study, chondrogenic potentials of 3D high-density cultures of Bone Marrow (BM) and Wharton’s Jelly (WJ)-derived mesenchymal stromal cells (MSCs) was investigated by chondrogenesis- and cytokine-related gene expression over a 16-day culture period supplemented with human transforming growth factor (hTGF)-β1 at 10 ng/ml. In BM-MSC 3D models, a marked upregulation of chondrogenesis-related genes, such as SOX9, COL2A1, and ACAN (all p < 0.05) and formation of spherical pellets with structured type II collagen fibers were observed. Similarly, WJ-based high-density culture appeared higher in size and more regular in shape, with a significant overexpression of COL2A1 and ACAN (all p < 0.05) at day 16. Moreover, a similar upregulation trend was documented for IL-6 and IL-10 expression in both BM and WJ 3D systems. In conclusion, MSC-based high-density cultures can be considered a promising in vitro model of cartilage regeneration and tissue engineering. Moreover, our data support the use of WJ-MSCs as a valid alternative for chondrogenic commitment of stem cells in regenerative medicine.
... Quantitative evaluation of the SEM images was performed using two-dimensional (2D) fast Fourier transform (FFT), calculated through the directionality plugin in the FIJI software [26,36]. A total of 15 collagen scaffolds were analysed for the SEM characterisation. ...
The use of neural scaffolds with a highly defined microarchitecture, fabricated with standard techniques such as electrospinning and microfluidic spinning, requires surgery for their application to the site of injury. To circumvent the risk associated with aciurgy, new strategies for treatment are sought. This has led to an increase in the quantity of research into injectable hydrogels in recent years. However, little research has been conducted into controlling the building blocks within these injectable hydrogels to produce similar scaffolds with a highly defined microarchitecture. “Magnetic particle string” and biomimetic amphiphile self-assembly are some of the methods currently available to achieve this purpose. Here, we developed a “magnetic anchor” method to improve the orientation of collagen fibres within injectable 3D scaffolds. This procedure uses GMNP (gold magnetic nanoparticle) “anchors” capped with CMPs (collagen mimetic peptides) that “chain” them to collagen fibres. Through the application of a magnetic field during the gelling process, these collagen fibres are aligned accordingly. It was shown in this study that the application of CMP functionalised GMNPs in a magnetic field significantly improves the alignment of the collagen fibres, which, in turn, improves the orientation of PC12 neurites. The growth of these neurite extensions, which were shown to be significantly longer, was also improved. The PC12 cells grown in collagen scaffolds fabricated using the “magnetic anchor” method shows comparable cellular viability to that of the untreated collagen scaffolds. This capability of remote control of the alignment of fibres within injectable collagen scaffolds opens up new strategic avenues in the research for treating debilitating neural tissue pathologies.
... After 2 days and 7 days, the cells were stained with LIVE/DEAD V R ThermoFisher reagent and incubated for 30 min at room temperature before analysis with Leica TCS SP2 confocal microscope. Cell counting was performed via processing of confocal images using the software imageJ [50]. ...
Hydrogel wound dressings can play critical roles in wound healing protecting the wound from trauma or contamination and providing an ideal environment to support the growth of endogenous cells and promote wound closure. This work presents a self-assembling hydrogel dressing that can assist the wound repair process mimicking the hierarchical structure of skin extracellular matrix. To this aim, the co-assembly behaviour of a carboxylated variant of xyloglucan (CXG) with a peptide amphiphile (PA-H3) has been investigated to generate hierarchical constructs with tuneable molecular composition, structure, and properties. Transmission electron microscopy and circular dichroism at a low concentration shows that CXG and PA-H3 co-assemble into nanofibres by hydrophobic and electrostatic interactions and further aggregate into nanofibre bundles and networks. At a higher concentration, CXG and PA-H3 yield hydrogels that have been characterized for their morphology by scanning electron microscopy and for the mechanical properties by small-amplitude oscillatory shear rheological measurements and compression tests at different CXG/PA-H3 ratios. A preliminary biological evaluation has been carried out both in vitro with HaCat cells and in vivo in a mouse model.
... After 24 h from cell seeding, cells were stained with live/dead imaging as previously described [38] and imaged using a confocal imaging system (Carl Zeiss, Oberkochen, Germany). Live cell percentage was measured from Z-stack images as described elsewhere [39]. A 3D projection of cells was generated using ImageJ (3D Plugin, V3.96) for Pillar and Bullet substrates. ...
Three-dimensional (3D) microstructure arrays (MSAs) have been widely used in material science and biomedical applications by providing superhydrophobic surfaces, cell-interactive topography, and optical diffraction. These properties are tunable through the engineering of microstructure shapes, dimensions, tapering, and aspect ratios. However, the current fabrication methods are often too complex, expensive, or low-throughput. Here, we present a cost-effective approach to fabricating tapered 3D MSAs using dual-exposure lithography (DEL) and soft lithography. DEL used a strip-patterned film mask to expose the SU-8 photoresist twice. The mask was reoriented between exposures (90 • or 45 •), forming an array of dual-exposed areas. The intensity distribution from both exposures overlapped and created an array of 3D overcut micro-pockets in the unexposed regions. These micro-pockets were replicated to DEL-MSAs in polydimethylsiloxane (PDMS). The shape and dimension of DEL-MSAs were tuned by varying the DEL parameters (e.g., exposure energy, inter-exposure wait time, and the photomask reorientation angle). Further, we characterized various properties of our DEL-MSAs and studied the impact of their shape and dimension. All DEL-MSAs showed optical diffraction capability and increased hydrophobicity compared to plain PDMS surface. The hydrophobicity and diffraction angles were tunable based on the MSA shape and aspect ratio. Among the five MSAs fabricated, the two tallest DEL-MSAs demonstrated superhydrophobicity (contact angles >150 •). Further, these tallest structures also demonstrated patterning proteins (with 6-7 µm resolution), and mammalian cells, through microcontact printing and direct culturing, respectively. Our DEL method is simple, scalable, and cost-effective to fabricate structure-tunable microstructures for anti-wetting, optical-, and bio-applications.
... Twenty minutes after staining with 1 M calcein and ethidium homodimer, fluorescence visualization was performed using epifluorescence microscope EVOS FL cell imaging system (Life Technologies, Eugene, OR, USA) equipped with a 40x objective, GFP and RFP filter cubes. Quantification of live and dead cells was analyzed in 3 aleatory fields using ImageJ (National Institutes of Health, Bethesda, MD) software according to recommendations [23]. ...
Bioactive glasses (BG) applications include tissue engineering for bone regeneration, coating for implants, and scaffolds for wound healing. BG can be conjugated to ions like silver, which might add some antimicrobial properties to this biomaterial. The immunomodulatory activity of ion-doped bioactive glasses particles was not investigated before. The aim of this work was to evaluate the cytotoxic and immunomodulatory effect of BG and silver-doped bioactive glass (BGAg) in human peripheral blood cells. BG and BGAg samples belonging to the system 58SiO 2• (36-x)CaO · 6P 2 O 5· xAg 2 O, where x = 0 and 1 mol%, respectively, were synthesized via sol–gel method and characterized. Cytotoxicity, modulation of cytokine production (TNF- α , IL-1 β , IL-6, IL-4, and IL-10), and oxidative stress response were investigated in human polymorphonuclear cells (PMNs) and peripheral blood mononuclear cells (PBMCs) cultures. Cell viability in the presence of BG or BGAg was concentration-dependent. In addition, BGAg presented higher PBMCs toxicity (LC50 = 0.005%) when compared to BG (LC50 = 0.106%). Interestingly, interleukin4 was produced by PBMCs in response to BG and BGAg in absence of phytohemagglutinin (PHA) and did not modulate PHA-induced cytokine levels. Subtoxic concentrations (0.031% for BG and 0.0008% for BGAg) did not change other cytokines in PBMCs nor reactive oxygen species (ROS) production by PMN. However, BG and BGAg particles decreased zymosan-induced ROS levels in PMN. Although ion incorporation increased BG cytotoxicity, the bioactive glass particles demonstrated a in vitro anti-inflammatory potencial. Future studies are needed to clarify the scavenger potential of the BG/BGAg particles/scaffolds as well as elucidate the effect of the anti-inflammatory potential in modulating tissue growth in vivo .
... Finally, also VIS on its own, did not perform that well. The reason for the lower viability compared to the control sample in this case is not directly clear, but is only evident through combination of the results with the second test we conducted; the Live/Dead assay (Spaepen et al., 2011). Namely, the lowered viabilities are most likely the result of the fibroblasts growing onto the VIS fibers that are soaked in the growth media (Fu et al., 2013). ...
Despite a range of advanced wound dressings that can facilitate wound healing, there are still no clinically used dressings for effective local pain management. The latter was the main motivation of the present study. We designed a novel wound dressing with three layers. A macro-porous polyethylene terephthalate (PET) mesh with incorporated lidocaine, a fast-acting local anesthetic, was chosen as the layer in direct contact with the skin. Fast release from this layer enables an immediate pain relieving effect, caused by dressing changes. For the second and third layer, alginate and viscose were chosen respectively. A potential long-lasting pain relieving effect was achieved through incorporation of a nonsteroidal anti-inflammatory drug diclofenac into both layers. The chosen dressing structure enables also an unhindered absorption of the wound exudate, which is possible through the macro-porous PET into the alginate layer. Alginate additionally maintains a moist wound environment. Our novel wound dressing was systematically tested in regard of the structural (contact angle measurements, IR spectroscopy, SEM), functional (water retention, air permeability) properties and its biocompatibility (Live/Dead and MTT assays) towards human skin fibroblasts. Combined results confirmed the suitability of the chosen wound dressing composition for a faster and painless wound treatment.
... Calcein AM stains live cells based on the esterase activity in the cell cytoplasm, which then produces an intense uniform green fluorescence. Ethidium Homodimer 1 is attached to the DNA of cells with compromised membrane, which results in a red staining of the nucleus of dead cells (Spaepen et al., 2011). The commonly used method for evaluating the number of dead/live cells in the resulting fluorescent images is manual counting (MC). ...
Fluorescent-based live/dead labelling combined with fluorescent microscopy is one of the widely used and reliable methods for assessment of cell viability. This method is, however, not quantitative. Many image-processing methods have been proposed for cell quantification in an image. Among all these methods, several of them are capable of quantifying the number of cells in high-resolution images with closely packed cells. However, no method has addressed the quantification of the number of cells in low-resolution images containing closely packed cells with variable sizes. This paper presents a novel method for automatic quantification of live/dead cells in 2D fluorescent low-resolution images containing closely packed cells with variable sizes using a mean shift-based gradient flow tracking. Accuracy and performance of the method was tested on growth plate confocal images. Experimental results show that our algorithm has a better performance in comparison to other methods used in similar detection conditions.
