For cell culture, MRC-5 (Medical Research Council cell strain 5), which is a fibroblast cell line, was purchased from the Korean Cell Line Bank (Korean Cell Line Research Foundation, Republic of Korea). DMEM (Gibco®, Thermo Fisher Scientific, USA) was used as a basal medium to culture MRC-5 cells. Fetal bovine serum (FBS; Gibco®, Thermo Fisher Scientific, USA) and Antibiotic-Antimycotic (Anti-Anti, 100X; Gibco®, Thermo Fisher Scientific, USA) were used as supplements. For NB generation in DMEM, highly-purity nitrogen gas (99.999%; Shinyoung Gas Co., Republic of Korea) was used.
2.2. Preparation of cell medium with N-NBs
In this study, a gas-liquid mixing (agitation) method with a linear actuator was used19 to generate a large number of N-NBs in DMEM in a short time. A sterilized disposable conical tube was dipped in DMEM vertically, and nitrogen gas was supplied to fill half of the tube. The tube was then capped and sealed with parafilm to prevent the influx of contaminants such as particles, oil, and microbes, which can negatively affect the cell culture. The sealed tube was mounted onto an actuator and agitated with the set cycle (117 strokes/min). After N-NB generation in DMEM, the cell medium with N-NBs was prepared by mixing the DMEM containing N-NBs with 10% FBS and 1% Anti-Anti.
2.3. Measurements of concentration and size of N-NBs in DMEM
For the analysis of N-NBs in DMEM, a nanoparticle tracking analysis (NTA) method was used with a NTA instrument (NanoSight LM10-HSBFT14; Malvern, UK), which is widely used in the NB research field20,21. It is a NB visualization technique that provides size, count and concentration measurements. The NTA instrument was equipped with a charge-coupled device (CCD) camera to capture the dispersed light and a red laser light source with a wavelength of 642 nm to excite fluorescence. The DMEM containing N-NBs was placed in the sample chamber, which had a volume of 0.3 mL. With laser light illumination, the N-NBs appeared individually as fast-moving dispersed dots of light (white dots) under Brownian motion, which were automatically tracked and captured by the CCD camera. Subsequently, the NTA image analysis program (i.e., the NTA software) determined the concentration and size of the N-NBs in DMEM. In addition, changes in the concentration and size of the N-NBs in DMEM was observed for 48 h to ensure the presence and stability of the N-NBs during the cell culture process.
2.4. Cell culture experiment
We used MRC-5 cells between passage 10 and 15 in the experiment. The cells were cultured in an incubator at 37°C with 5% CO2 and 95% humidity using a culture medium containing DMEM supplemented with 10% FBS and 1% Anti-Anti. There was a slight difference in the cell culture process due to the difference in the analysis method as follows.
First, cell proliferation was analyzed by image analysis. To obtain reliable data, cell culture experiments were performed 5 times independently. To attach the cells, we loaded 1 mL of non-NB culture medium including MRC-5 cells (5 × 103 / mL) in 24-well plates. After 24 h of cell attachment, NucBlue® Live ReadyProbes™ reagent (NucBlue; Invitrogen™, Thermo Fisher Scientific, USA) was added (1 drop per well) and incubated at 37°C for 30–40 min to stain the cells. Images at the center of the well were taken using an inverted fluorescence microscope (CKX41; Olympus, Japan). Then, half of the 24 well was replaced with the culture medium with N-NBs (experimental group), and the remaining half of the 24well was replaced with the fresh culture medium without N-NBs (control group). After incubation for 48 h, NucBlue staining was performed, and images of the cells were taken. To count the number of cells per image, image analysis was performed using imageJ. (US National Institutes of Health, USA)
Second, cell proliferation was analyzed by flowcytometry. The cells were stained using CellTrace™ carboxyfluorescein succinimidyl ester (CFSE) Cell Proliferation Kit (Invitrogen™, Thermo Fisher Scientific, USA). CFSE dye was diluted in dimethyl sulfoxide (DMSO) at a concentration of 5 mM to make a stock solution, and the stock solution was diluted in PBS at a ratio of 1000:1 to prepare a PBS-dye solution. The cells were stained by adding 1 mL of the PBS-dye solution to 1 × 106 cells in suspension and incubated at 37°C for 20 min. After adding the culture medium without N-NBs to the stained cell solution (5×) with incubation for 5 min, the cell was pelleted using centrifuge, and the supernatant was removed. The stained cells were seeded in Φ150-culture dishes (1 × 106 cells each). After 24 h of cell attachment in culture medium without N-NBs, half of culture dishes was replaced with the culture medium with N-NBs (experimental group), and the remaining half of culture dishes was replaced with the fresh culture medium without N-NBs (control group), followed by incubation for 48 h. Subsequently, the cells were detached from the dish using trypsin (Gibco®, Thermo Fisher Scientific, USA), and flow cytometry analysis was performed using FACS Canto™II (Becton, Dickinson and Company, USA).