Rtca sp instrument

Real-time cell electronic sensing was used to follow cell damage and/or protection in living barrier forming cells. The RTCA-SP instrument (ACEA Biosciences, San Diego, CA, USA) registered the impedance of cell layers every 10 min and at each time point the cell index was defined as (Rn − Rb)/15, where Rn is the cell-electrode impedance of the well when it contains cells and Rb is the background impedance of the well with the medium alone. The 96-well E-plates with built-in gold electrodes were coated with collagen type I (50 μg/mL for both) and dried for 20 min under sterile air-flow. Culture medium (60 μL) was added to each well for background readings, then 50 µL of HCE-T suspension was dispensed at the density of 5 × 103 cells/well. When cells reached a steady growth phase, they were treated.
For cell viability measurements, the originally prepared liquid formulations such as NLC1, NLC2, NLC3, and NLC4 (Table 1) formulations and HPMC solution were prepared as 10×, 30× and 100× dilutions in cell culture media.

Kinetics of RBEC and hCMEC/D3 cells responses to SLNs treatment were monitored by impedance measurement (RTCA-SP instrument; ACEA Biosciences, San Diego, CA, USA). Impedance measurement is label-free, real time, non-invasive, and correlates linearly with adherence and growth of cells. After background measurements, cells were seeded at a density of 6 × 10³ cells/well in collagen coated 96-well plates with integrated gold electrodes (E-plate 96, ACEA Biosciences, San Diego, CA, USA). Cells were cultured for 5–7 days in CO₂ incubator at 37 °C. When the growth of cultures reached a plateau phase, cells were treated with SLNs (1 and 10 µg/mL) diluted in culture medium and monitored for 24 h. Cell index was defined as R_n-R_b at each time point of measurement, where R_n is the cell-electrode impedance of the well when it contains cells, and R_b is the background impedance of the well with the medium alone. Cell index values reflect cell number and viability.

The effect of nanocarriers on the viability of hBECs was monitored by the label-free, real-time, non-invasive measurement of cell layer impedance (RTCA-SP instrument, Agilent Technologies, Santa Clara, CA, USA). Changes in the impedance values correlate linearly with cellular viability [44 (link),52 (link)]. The hBECs (P6) were seeded at a density of 6 × 10³ cells/well in 96-well plates with integrated gold electrodes (E-plate 96, Agilent), coated with 0.2% gelatin. To differentiate hBECs, the cells received a culture medium mixed with a PC-conditioned medium (50–50%) for 48 h. The confluent layers of hBECs in the plateau phase of growth were treated with 3-PLG or 3-PLG-A-GSH nanocarriers (1, 10, 20 or 100 µg/mL), diluted in hBEC culture medium. The impedance was followed every 5 min for 24 h. The cell index was defined as R_n-R_b at each time point of measurement, where R_n is the cell-electrode impedance of the well when it contains cells, and R_b is the background impedance of the well with the medium alone. The cell index was normalized in each well to the value measured at the last time point before the treatment.

The kinetics of the epithelial and endothelial cell reaction to the different treatments were monitored by impedance measurement at 10 kHz (RTCA-SP instrument, Agilent, Santa Clara, CA, USA). The impedance measurement is label-free, non-invasive, and correlates linearly with the adherence, growth, number, and viability of cells in real-time [86 (link),87 (link)]. For background measurements, the 50 μL cell culture medium was added to the wells. Then, the cells were seeded at a density of 2 × 10⁴ RPMI 2650 cells/well and 6 × 10³ hCMEC/D3 cells/well in 96-well plates coated with integrated gold electrodes (E-plate 96, Agilent, Santa Clara, CA, USA). The cells were cultured for 5–7 days in a CO₂ incubator at 37 °C and monitored every 10 min until the end of experiments. In addition, the cells were treated at the beginning of the plateau phase of growth. The insulin, insulin NPs, and HCl solution were diluted in a cell culture medium and the effects were followed for 20 h. Triton X-100 detergent (1 mg/mL) was used as a reference compound to induce cell toxicity. The cell index was defined as Rn-Rb at each time point of measurement, where Rn is the cell-electrode impedance of the well when it contains cells and Rb is the background impedance of the well with the medium alone.