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Nanofiber Electrospun Membrane Based on Biodegradable Polymers for Biomedical and Tissue Engineering Application
Published in Ahmad Fauzi Ismail, Nidal Hilal, Juhana Jaafar, Chris J. Wright, Nanofiber Membranes for Medical, Environmental, and Energy Applications, 2019
Lim Mim Mim, Naznin Sultana, Hasrinah Hasbullah, Madzlan Aziz
In this study the number of viable cells attached to the nanofiber membrane was also counted by using a dye exclusion method and trypan blue (Lim et al. 2015). Trypan blue is a dye that can differentiate between living and dead cells. It selectively colors dead cells in blue and living cells will refract brightly. Figure 3.3 shows the result of the viable cell count. Tissue culture plate wells with only cells were used as control. No dead cell were observed, but the growth of cells on each sample was different. According to the growth curve, cells started to attach after 4 h and were well attached after 24 h. Cells were in the proliferation state at 72 h. With increasing time, the number of cells on each sample increased, demonstrating cell attachment occurs successfully and cells have proliferated. The high porosity of both nanofiber membranes provides an optimum growth environment for cells by giving more spaces for nutrient and metabolic waste exchange. All of the data were statistically significant.
In vitro Evaluation
Published in Raj Bawa, János Szebeni, Thomas J. Webster, Gerald F. Audette, Immune Aspects of Biopharmaceuticals and Nanomedicines, 2019
Summarizing cytotoxicity assessment techniques in vitro, a combination of at least two techniques is recommended when performing a safety profile of nanomaterials in vitro to have complete and reliable results. Although in the vast majority of studies, only the MTT (or the alternative MTS) test is used, as described above, there are some specific cases in which it does not give reliable results. For this reason, it is recommended to perform the MTT but in combination with another technique. Among the other three assays described in this chapter, trypan blue is used in most studies only for the qualitative assessment of the cells when passaging cultures, due to its easy and rapid performance, to ensure that the number of cells counted are alive. Neutral red is less used. Therefore, for comparison with previous studies, it is recommended to test the MTT together with the LDH.
Nanotoxicology
Published in Pradipta Ranjan Rauta, Yugal Kishore Mohanta, Debasis Nayak, Nanotechnology in Biology and Medicine, 2019
Pavani Gonnabathula, Nageswararao Mekala, Praveen Kumar Relangi
Dyeing of the cells with suitable coloring agents like neutral red and trypan blue helps in differentiating living and dead cells. Neutral red forms hydrophobic bonds in lysosomal matrix, and nanoparticle accumulation weakens the surface of the cell, which leads to reducing uptake levels of the dye; thus, the dead cells can be differentiated. In a study, silver nanoparticle exposure lead to an instability in the endosome-lysosome system (Miranda et al., 2017). Trypan blue is an exclusion test: it enters the dead cells and is excluded from living cells. Integrity of the cell membrane can be assessed in the presence of zinc nanoparticles; significant cytotoxicity has been observed (Kononenko et al., 2017).
Gold nanorods conjugated upconversion nanoparticles nanocomposites for simultaneous bioimaging, local temperature sensing and photothermal therapy of OML-1 oral cancer cells
Published in International Journal of Smart and Nano Materials, 2021
Duc Tu Vu, Thanh Thu Vu-Le, Van Nghia Nguyen, Quoc Minh Le, Churng-Ren Chris Wang, Lai-Kwan Chau, Tzyy-Schiuan Yang, Michael W. Y. Chan, Cheng-I Lee, Chu-Chi Ting, Jiunn-Yuan Lin, Hung-Chih Kan, Chia Chen Hsu
For the photothermal ablation test, a continuous-wavelength (CW) 976 nm wavelength NIR laser was used to expose the sample due to the large transmittance of biotissues at this wavelength and the overlap of the excitation laser wavelength with the longitudinal absorption band of AuNRs used in this work. Typically, the OML-1 oral cancer cells were loaded in a 96 well-plate (1000 cells per well), and approximately 100 μg/mL of hybrid nanocomposites (AuNR@Silica-UCNPs) were added for 4 h. After the treatment, the target cells were irradiated by a 976 nm NIR laser with 0.3 W/cm2 intensity for 1 min. To examine cell viability, trypan blue was used to stain dead tissues or cells into blue by incubating with the cell dishes for 3 min. After staining, the cell images were examined using an optical microscope.
Fabrication and characterization of hydroxypropyl guar-poly (vinyl alcohol)-nano hydroxyapatite composite hydrogels for bone tissue engineering
Published in Journal of Biomaterials Science, Polymer Edition, 2018
Anil Parameswaran-Thankam, Qudes Al-Anbaky, Zeiyad Al-karakooly, Ambar B. RanguMagar, Bijay P. Chhetri, Nawab Ali, Anindya Ghosh
Cell viability was also determined using Trypan blue dye exclusion assay. Briefly, MC3T3 cells were seeded at density of 1.5 x 104 cells per mL per well on the scaffolds (PVA, HPG/PVA, and HPG/PVA/n-HA) separately placed in 24-well tissue culture plates and allowed to grow for indicated time periods as described above. Following cell growth, the scaffolds were removed and placed into separate wells and rinsed with PBS. In order to detach the cells from the scaffolds, 50 µL Trypsin-EDTA was added and cells incubated for 2-3 min and then mixed with 50 µL of fresh MEM medium. The cell suspension was collected and the medium was removed following centrifugation (500g, 3 min). The pellet of MC3T3 cells was resuspended in 100 µL of the fresh medium. An aliquot of 90 μL of the cell suspension was transferred to an Eppendorf tube and mixed with 10 μL of 0.4% (w/v) Trypan blue dye. An aliquot of 10 μL of this mixture was loaded onto a hemocytometer for cell counting using a light microscope (40 x magnification). Cells stained with Trypan blue were counted as dead cells whereas cells without the Trypan blue stain were counted as live cells. A total of 200-300 cells were counted from each sample to determine statistical significance. The cell viability data was presented as % of the control values obtained from untreated cells. All determinations were performed in triplicate experiments repeated independently at least three times.
The investigation of in vitro dark cytotoxicity and photodynamic therapy effect of a 2,6-dibromo-3,5-distyryl BODIPY dye encapsulated in Pluronic® F-127 micelles
Published in Journal of Coordination Chemistry, 2018
Nthabeleng Molupe, Balaji Babu, David O. Oluwole, Earl Prinsloo, John Mack, Tebello Nyokong
The in vitro dark cytotoxicity studies were performed using MCF-7 cells that were cultured using DMEM containing 4.5 g L−1 glucose with l-glutamine and phenol red. The medium was supplemented with 100 unit mL−1 penicillin–100 µg mL−1 streptomycin–amphotericin B and 10% (v/v) heat-inactivated FCS to obtain the cultured medium (supplemented DMEM). All experiments were conducted in triplicate [10, 20]. The cells were grown in T25 cm2 vented flasks (Porvair®) and incubated at 37 °C and 5% CO2. When the cells reached 100% confluence (after 72 h of incubation), they were subcultured by standard trypsinization into T75 cm2 vented flasks. The viable cells were counted with trypan blue dye exclusion assay (0.4% trypan blue solution) using a hemocytometer. The cells were subsequently seeded into 96-well tissue culture plates (Porvair®) at a 10,000 cells well−1 cell density in cultured/supplemented DMEM containing phenol red. The cells were further incubated at the previously mentioned conditions for 24 h to promote attachment to the 96 wells. The attached cells were washed with 100 µL DPBS followed by drug administration. The drug formulations comprised 100 µL supplemented DMEM containing BODIPY 1 and 1-Pmicelles at a range of different concentrations (5, 10, 20, 40, and 80 µg mL−1).