Estructura porcentual del activo de las IFM

All the measurements were performed with the PT-2 flow channel and the measurement setup for 3D tissue models shown in chapter 4.3.1 Fig. 9, unless stated otherwise. Data evaluation was done using individual LabView-based software programs for MFT and RTC mode (written by J. Wegener).

4.3.5.1 Spheroid Size and Repositioning

The measurements described in this subchapter were all performed using a single flow channel directly connected to the impedance analyzer. The spheroids were all measured in culture medium.

Spheroids of the Same Seeding Density and Growth Duration

Spheroids with a seeding density of 3000 cells/well were prepared following the standard protocol (chapter 4.1.5). For impedance measurements seven or eight

day old spheroids were used. First, the empty, medium-filled flow channel was measured to record baseline impedance. Then, a spheroid was transferred from the 96-well plate into the flow channel (without pausing the measurement) and measured for ~ 40 min. Afterwards, the spheroid was removed and at least three baseline spectra were recorded before the next spheroid was inserted. This procedure was applied for a total of five spheroids.

Spheroids of Different Seeding Densities and the Same Growth Duration

MCF-7 spheroids with three seeding densities (1000, 3000 and 6000 cells/well) were prepared following the standard protocol (chapter 4.1.5). Eight days after seeding the impedance analysis was performed measuring two spheroids of each seeding density successively with at least three baseline spectra in between.

Spheroid Repositioning

This experiment was performed using spheroids with a seeding density of 3000 cells/well nine days after seeding. A spheroid was inserted in the flow channel and measured for ~ 40 min. Then, the spheroid was repositioned by carefully removing it from the central restriction and remaining it on the reservoir bottom for a few baseline spectra before it was positioned again at the restriction. This procedure was repeated in total three times per spheroid without stopping the measurement in between.

4.3.5.2 Cross-Linked Spheroids

The measurement was performed using 7 d old spheroids (3000 cells/well) that were pre-incubated for 1 h in different concentrations of PFA in PBS++ (0.01 / 0.1 / 0.5 %). In order to perform duplicate data sets per PFA concentration two flow channels were used simultaneously. The whole measurement was performed in culture medium. When the medium was equilibrated during baseline recording (without spheroid), the measurement mode was switched from MFT to RTC (5 min/channel, 200 Hz, 50 mV). Afterwards, the mode was switched back to MFT and two control spheroids (no PFA incubation) were inserted and measured for ~ 1 h before RTC time series were recorded. Thereafter, the spheroids were removed from the channels and a few baseline spectra were recorded. The spheroids that were pre-incubated in the three PFA concentrations were washed

with PBS++ and culture medium before they were measured successively in the same way than the control spheroids. The spheroids were also monitored with the digital microscope and photographs were taken in the beginning and in the end of each measurement.

4.3.5.3 Permeabilized Spheroids

Spheroids (7 / 8 d; 3000 cells/well) were pre-incubated for 4 h in different concentrations of saponin in EBSS++ (0 / 0.01 / 0.05 / 0.1 / 0.25 ‰). The measurement was performed in duplicate and baseline as well as spheroids were measured in EBSS++. The spheroids were washed with EBSS++, measured for 30 – 40 min in MFT mode and then RTC time series data was recorded. The digital microscope was used to monitor the spheroids at the central restriction.

4.3.5.4 Spheroids Under Osmotic Pressure

The iso-, hypo- and the two hyperosmotic buffers were prepared as outlined in chapter 4.1.9. Spheroids were generated (8 d; 3000 cells/well) and measured in duplicate without pre-incubation in isoosmotic buffer. The measurement procedure was the same for each buffer, starting with the baseline MFT in the respective buffer without spheroid. The measurement was switched to RTC as soon as the signal remained constant. Then, the measurement mode was switched back to MFT, spheroids were inserted into the channels and measured for ~ 1 h until the measurement in RTC mode. Afterwards, the spheroid and the buffer were removed from the flow channel. The channel was first washed and then filled with the next buffer. The spheroids were monitored during the measurement and photographs were taken using the digital microscope.

4.3.5.5 Spheroid Response to Cytochalasin D

In order to test the impact of actin disruption by cytochalasin D (cytD) on 7 d old spheroids (3000 cells/well) they were pre-incubated for 0 / 2 / 4 / 6 / 8 h in 5 µM cytochalasin D in culture medium at 37 °C. The baseline of the medium-filled channel without cytD and without spheroid was recorded in MFT and RTC mode. Then the control spheroids were analyzed in medium as described previously (MFT for ~ 1 h, RTC) followed by the spheroids, which were incubated in cytD.

Those were washed once with culture medium before they were inserted in the channel. The microscopic observation of the spheroids during the measurement was done using the digital microscope.

4.3.5.6 Spheroid Response to Hyperthermia

In order to investigate the spheroid response to hyperthermia, 7 d old spheroids (3000 cells/well) were used. They were exposed to 41 / 43 / 45 °C in culture medium filled 1.5 mL tubes for 1 h or 3 h using the Thermomixer compact. The control spheroids (37 °C) were taken directly from the 96-well plate in the cell culture incubator. The measurement was performed in culture medium in duplicate and the spheroids were monitored using the digital microscope. The time course of impedance spectra was followed for ~ 50 min and RTC time series were taken from the medium-filled channel with and without (baseline) spheroids. 4.3.5.7 Phototoxicity of CaAM on Monolayer Cells and Spheroids

The experiments with monolayer cells were performed by Romy Freund and Pierre Pütz using the self-made 8-well ITO arrays and the measurement setup shown in chapter 4.3.1, Fig. 11. The cells were prepared as explained in chapter 4.3.4. The confluent cell layers were washed with EBSS++ and incubated with 2 µM CaAM in EBSS++ for 90 min in a standard cell culture incubator. With the staining solution in the supernatant the cells were taken to the Nikon Diaphot for irradiation with three different excitation wavelengths (~ 355 / 483 / 553 nm) for 3 min using the 10× objective focused on the working electrodes. In the controll well(s) the cells were not irradiated. Then, the array was connected to the measurement setup and the time course of impedance was followed for 12 – 24 h by repeated frequency scans (MFT). When the impedance measurement was finished the cells were washed carefully with EBSS++ and a live/dead staining was performed following the protocol for monolayer cells in chapter 4.2.2. The measured impedance magnitude was corrected for the lead resistances and then normalized to the time point right after irradiation.

For the experiments with 3D tissue models spheroids were prepared (3000 cells/well) and measured on day seven or eight after seeding. For this purpose, spheroids were incubated in 2 µM CaAM in EBSS++ or in EBSS++ only

(control spheroids) for 3 h in the dark at RT and 75 rpm. Meanwhile, the baseline of the EBSS++ filled channel was measured and RTC data was recorded. Then, the CaAM-loaded and the control spheroids were washed and inserted in the flow channel. They were measured for ~ 30 min in MFT mode. The channels were disconnected and brought to the Nikon Diaphot microscope where the spheroids were photographed (phase contrast) and exposed to epi illumination at 420 – 490 nm for 10 min using the 10× objective. The channels were re-connected to the impedance analyzer and the MFT measurement continued. At the end of the measurement the spheroids in the channels were photographed again at the Nikon Diaphot.

4.3.5.8 Phototoxicity of Carbon Dots on Monolayer Cells and Spheroids

N-doped carbon dots (C-dots) from starch and L-tryptophan with an average diameter of (1.6 ± 0.8) nm were produced and characterized by Michael Lemberger. The particles show a good water dispersibility and the uptake into cells is in the time range of a few minutes. For low concentrated particle suspensions the excitation / emission maximum is ~ 370 / 450 nm. From cytotoxicity investigations on NRK (normal rat kidney) monolayer cells it is known that C-dot concentrations up to 1.1 mg/mL are uncritical in the dark. Furthermore, it is known that the particles are generating reactive oxygen species (ROS) upon photo-excitation and thus fulfill essential prerequisites of a potential photodynamic therapy agent.

The phototoxicity studies with monolayer cells were performed by Pierre Pütz using the self-made 8-well ITO array (R.F.) and the measurement setup for monolayer cells (chapter 4.3.1, Fig. 11). The array with the confluent cell monolayer (for preparation details see chapter 4.3.4) was measured in MFT mode. The initial baseline was recorded in medium. Then, the cells were washed twice with EBSS++ and in some wells the cells were incubated with 1 mg/mL C- dots in EBSS++ for 1 h while other wells served as controls. The C-dot solution was removed and the cells were washed twice with EBSS++. Afterwards, the measurement was interrupted to take the array to the Nikon Diaphot where the C- dot stained cells were exposed to epi illumination at 330 – 380 nm (UV-2A filter)

for 3 min using the 10× objective. The measurement was continued for ~ 20 h. Controls with or without C-dots were not irradiated.

For the phototoxicity studies with 3D tissue models, spheroids (3000 cells/well) were prepared and used on day seven after seeding. They were incubated in 1.5 mg/mL C-dots in EBSS++ for 2 h in a standard cell culture incubator. In the meantime, the baseline of the EBSS++ filled channels was measured in MFT mode. Then, the spheroids were washed, inserted in the channels and measured for ~ 30 min. Afterwards, both channels were brought to the Nikon Diaphot microscope to take phase contrast photographs and to expose one of the C-dot spheroids to epi illumination at 330 – 380 nm (UV-2A filter) for 15 min using the 10× objective. The other C-dot spheroid served as control and was not exposed to illumination. The channels were re-connected to the measurement setup and spheroids were followed in MFT mode. At the end of the measurement the spheroids in the channels were documented by phase contrast micrographs. 4.3.5.9 Testing of Carbon Dot Cytotoxicity on Spheroids

MCF-7 spheroids (3000 cells/well) were used for the cytotoxicity experiments 7 d after seeding. They were incubated in either 1.5 mg/mL C-dots in EBSS++ or in pure EBSS++ (control spheroid) for 2 h in a standard cell culture incubator. The baseline of the EBSS++ filled channel was measured before the spheroids were washed and inserted in the channels. The spheroids were monitored during the measurement using the digital microscope.