br manufacturer s standard operating procedures
manufacturer’s standard operating procedures.
2.6.2. Determination of cell concentration and viability
Previously frozen Brequinar were thawed and pipetted into sterile test Colloids and Surfaces B: Biointerfaces 177 (2019) 160–168
tubes containing cell culture medium. The total volume was centrifuged at 800 rpm for 3 min and the supernatant discarded. The cell sediment was re-suspended in cell culture medium (3–5 mL). Cell counting was undertaken by the trypan blue assay. Briefly, the cell suspension (10 μL) was added to trypan blue (20 μL) and the combination was introduced into the chamber of a specialized cell counting slide. The concentration of cells was determined employing an automated cell counter (TC10™ Automated cell counter, BioRad Laboratories Inc., Hercules, CA, USA). For each analytical experiment, cells of the same passage number were employed with all analyses undertaken in triplicate. Moreover, only cells demonstrating ≥95% viability after freezing were used for ana-lyses.
2.6.3. Assessing cellular uptake of nano-lipobubbles through fluorescence microscopy
Cells were seeded at a density of 1 × 105 cells/well in sterile, 6-well cell culture plates and incubated at 37 °C in a humidified incubator with 95%O2 and 5%CO2. Fluorescently labeled CHO- and DSPE-NLBs were prepared as previously described, with the addition of SRB during the emulsification process. Following attachment of the cells over a 24-hour period, cells were treated with SRB-labeled CHO and DSPE-NLBs as well as cell culture medium (1:1; 2 mL), under sterile incubation conditions as described above. Following the incubation period, excess media and the NLB suspension were removed and the cells were washed twice with PBS (pH 7.4; 37 °C). PBS (pH 7.4; 37 °C; 1 mL) was added to each well to prevent dehydration of the cells during analysis. The fluorescent nature of SRB enabled qualitative determination of cellular uptake of the CHO-and DSPE-NLBs through the application of fluorescence microscopy. Fluorescent images were super-imposed onto bright field contrast images to highlight the presence of fluorescently labeled CHO- and DSPE-NLBs within the A2780 ovarian cancer cells.
2.6.4. Characterizing the cytotoxicity of formulated nano-lipobubbles Cytotoxicity studies are employed to establish the degree of toxicity
of the formulated NLB-DDS to the tumor cell line. In the present study toxicity of the formulation to the A2780 ovarian cancer cell was in-vestigated, with the objective of maximizing toxicity and cell death. The cytotoxicity of formulations cannot be accurately defined without the use of experimental controls. Hence, in addition to the formulated coated CHO and DSPE-NLBs, the cytotoxic activity of the controls outlined were also investigated. The determination of cytotoxicity was assessed quantitatively employing flow cytometry, as well as analysis of real-time cell growth and cytotoxicity data employing the xCELLigence™ Real-Time Cell Analyzer (RTCA) (Roche Applied Science, Penzberg, Upper Bavaria, Germany and ACEA Biosciences Inc., San Diego, CA, USA). Moreover, counting of cells employing the automated cell counter previously described before and after treatment, in order to determine and adjust the concentration for specific analytical proce-dures, provided a solid indication of cell viability. r> 2.6.5. Evaluating cytotoxicity employing flow cytometry
Following the determination of post-freezing cell viability, cells were seeded at a density of 5 × 105 cells per well in sterile 6-well cell culture plates and incubated at 37 °C in a humidified incubator with 95%O2 and 5%CO2. When regular microscopic evaluation revealed cells had reached ˜80% confluence, treatment of cells was undertaken in triplicate and maintained in the humidified incubator as previously described. The saturation solubility of CPT in DMSO is 10 mg/mL, which was subsequently diluted 25-fold in PBS and then 2-fold in cell culture medium to negate the toxic effect of DMSO on cells. The final concentration of CPT with which the cells were treated was thus 0.20 mg/mL. Suspensions of uncoated CHO and DSPE-NLBs formula-tions were prepared with a CPT concentration of 0.4 mg/mL. A 2- fold dilution with cell culture medium thus produced preparations with CPT concentration of 0.20 mg/mL. Placebo suspensions were prepared at concentrations equal to the overall concentrations of the CHO- and
DSPE-NLBs. The maximum concentration of SB in DMSO that could be obtained was 15 mg/mL, which was subsequently diluted 25-fold in PBS (pH 7.4; 37 °C) and 2-fold in cell culture medium to create a final solution of 0.3 mg/mL.