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For the generation of cardiospheres from Cor.At® cells the hanging drop technique and the formation on a non-adhesive surface were pursued as described in chapters 4.1.4 and 4.1.5. The formation and beating of cardiospheres was observed and documented using phase contrast microscopy or the digital microscope.

Microscopic Characterization of Cor.At® Cardiospheres

Fig. 70: Typical micrographs documenting the development of two cardiospheres (A, B) over 4 d that were generated by the hanging drop technique with a seeding density of 6000 cells/drop (scale bars: 200 µm).

The hanging drop technique was chosen in the first set of experiments. Typical micrographs (Fig. 70) show the development of two cardiospheres (A, B) over four days. At the first day after seeding there are mainly small cell aggregates and single cells in a random distribution. The small aggregates are already contracting regularly, while the single cells show no beating. On the second day after seeding single cells and small cell aggregates accumulate. Until day four after seeding the aggregates adhere to each other and compact. They appear darker than the days before and beat very slowly. All cardiopheres that are generated by hanging drop culture have a very irregular shape.

Also shapes that are not appropriate for impedance measurements in the flow channel are obtained, like the cardiosphere in image A 4 d, which has a cell-free gap in the center.

Fig. 71: Typical micrographs documenting the development of a cardiosphere over 5 days generated by the non-adhesive surface technique with a seeding density of 6000 cells/well (scale bar: 200 µm).

In the second set of experiments higher numbers of Cor.At® cells were available and in addition to the hanging drop culture the formation of cardiospheres on a non-adhesive surface was tested. In Fig. 71 one typical cardiosphere prepared in the 96-well plate is followed for 5 d. The cells aggregated to each other much faster than compared to the hanging drop method. On the first day after seeding most cells are adhered to each other, forming a flat aggregate of regularly beating cells. Some single non-beating cells are observed in the periphery of the aggregate. On the second day the aggregate is smaller than on day one and more single cells are observed in close proximity of the aggregate. Three and four days after seeding the compaction of the aggregate to a cardiosphere can be observed. This is accompanied by a further size reduction and a darker appearance of the cardiosphere. The shape is reproducibly smoother and more spherical than for cardiospheres from hanging drop culture. On day five after seeding a decomposition of the spheroid is observed. The size is again reduced and several single cells that detached from the cardiosphere are lying around it. The aggregates on different days show all a regular beating behavior. Due to the more reproducible shape of cardiospheres prepared in the coated 96-well plate they were preferred over hanging drop spheroids and used for most impedance measurements.

Impedimetric Characterization of Cardiospheres in the PT-1 Flow Channel

All measurements with cardiospheres were performed with the PT-1 flow channel on ITO-glass. Due to the different channel dimensions and the new spheroid model frequency scans were performed to identify a suitable monitoring frequency.

Fig. 72: Typical impedance spectra conducted with (w) and without (wo) cardiosphere in the PT-1 flow channel on ITO-glass measured in Cor.At® culture medium (A) or EBSS++(B). From this data the respective relative impedance was calculated and plotted (C, D). T = 34 °C.

Fig. 72 shows the impedance spectra of a medium- (A) or buffer-filled (B) flow channel with or without a cardiosphere. Spectra from spheroid-free channels show a horizontal, frequency independent curve between ~ 101 and ~ 105 Hz at ~ 80 k for the medium-filled channel and ~ 60 k for the EBSS++-filled channel. These impedance values are much higher than compared to the PT-2 flow channel values with ~ 10 kdue to the different dimensions. The decline in the

frequency range above 105 Hz reflecting the parasitic stray capacitance shows similar values (Apara: 2 – 3·10-13

Fsn-1cm-2, npara = 0.99) than for PT-2 flow channels. The increase in impedance after the introduction of a cardiosphere into the flow channel is in this example 30 – 40 k (A, B). From the measured spectra the relative imedance IZIrel was calculated and plotted against the frequency (Fig. 72, C and D). The plateau phase is observed for the medium- and buffer- filled channel at ~ 50 – 60 % in the frequency range between 101

and 104 Hz. For MCF-7 spheroids measured in the PT-2 flow channel a similar frequency range is observed including the monitoring frequency of 200 Hz. For this purpose, 200 Hz was used also as monitoring frequency for the presentation of impedance time courses of Cor.At® cardiospheres measured in the PT-1 ITO-glass flow channel. Preliminary to the impedimetric results for cardiospheres measured in three different experimental scenarios a typical detrended RTC time course of impedance at 200 Hz for the medium-filled cardiosphere-free flow channel is shown in Fig. 73 A. The time course only exhibits small fluctuations that are related to electrical noise with a mean amplitude of 0.031 ± 0.009 k. In contrast, an exemplary time course of a cardiosphere in medium (B) shows distinct spikes with a mean amplitude of 1.3 ± 0.2 k

Fig. 73: Typical detrended RTC data of the medium-filled PT-1 on ITO-glass without cardiosphere (A) (also shown with a smaller y axis scale) and with cardiosphere (10000 cells/well, 4 d) (B). T = 34 °C.

A typical micrograph of a cardiosphere positioned in the central channel of the PT-1 flow channel is shown in Fig. 74. The beating of cardiospheres at the aperture was observed using the digital microscope.

Fig. 74: Micrograph of a cardiosphere prepared by the formation on a non-adhesive surface (6000 cells/well, 5 d) at the aperture of the PT-1 channel (ITO-glass substrate) (scale bar: 150 µm).

7.2 Impedimetric Analysis of Cardiospheres in Absence of