<i>Retun loss (RL)</i> and <i>f</i>_<i>min</i> of fluids exhibiting different polarities and osmolalities in comparison to purified water (aq. dest.) and phosphate buffered saline (PBS).

<p>(A) <i>f</i>_<i>min</i> decreased with lower polarity. Water; exhibiting the highest polarity showed the highest <i>f</i>_<i>min</i> compared to methanol (MeOH), ethanol (EtOH) and propanol (PrOH) with decreasing polarities. (B) <i>RL</i> decreased with lower polarity from aq. dest. to PrOH. (C) <i>f</i>_<i>min</i> decreased with higher osmolality from aq. dest. to sodium chloride (NaCl) solutions at concentrations ranging from 0.1 to 20%. (D) <i>RL</i> decreased with a higher osmolality. Bar diagrams show mean values and error bars depict standard deviation. Asterisks indicate statistical relevant differences (<i>p</i>-value ≤ 0.5). Each group comprised three individual samples that were measured in triplicate.</p

Measurement of cell-free dermal equivalents at different collagen concentrations.

<p>(A) A plastic compression system allowed generating dermal equivalents exhibiting collagen concentrations ranging from <i>c</i> = 6.67 mg/ml to <i>c</i> = 26.67 mg/ml. During processing, hydrogels were compressed to a defined volume of <i>V</i> = 3.6 cm² employing a linear motion engine. The squeezer (1) compressed the hydrogel (2) in the bioreactor (3). Pried water was aspirated via the port near the bottom of the chamber (4). (B) Comparison of dry to wet weight showed an increase in weight with a higher collagen concentration, whereas (C) <i>f</i>_<i>min</i> at the detected minimum declined with a higher collagen concentration, and (D) <i>RL</i> decreased at higher collagen concentration. Plots show mean values, error bars depict standard deviation. Each group comprised 3 individual samples that were measured in triplicate.</p

Microwave reflection signal of dermal equivalents at different osmolalities and cell concentrations.

<p>(A) Comparison of dry to wet weight ratio demonstrated that the weight of the dry component increased with a higher osmolality and cell concentration of the remaining substances. (B) <i>f</i>_<i>min</i> declined at higher cell concentrations (from 4.5x10⁵ to 9x10⁵ cells) and osmolalities, mimicking hypo-, iso- and hypernatraemia. (C) In analogy to <i>f</i>_<i>min</i>, <i>RL</i> dropped with a higher osmolality. Bar diagrams show mean values, error bars depict standard deviation. Asterisks indicate statistical relevant differences (<i>p</i>-value ≤ 0.5). Each group comprised 3 individual samples that were measured in triplicate.</p

Microwave signal of fluids and <i>in vitro</i> test models measurements at 8.2 GHz.

<p>(A) Microwave measurements with fluids. Compared to Control, <i>RL</i> showed increased values for polarity measurements (Polarity) at a constant frequency of 8.2 GHz with a lower polarity from aq. dest. to PrOH. Osmolality measurements displayed a decreasing <i>RL</i> with the highest osmolality at NaCl 20%. (B) Microwave measurements with <i>in vitro</i> models. <i>RL</i> displayed increasing values at a constant frequency of 8.2 GHz for measurements with cell-free collagen hydrogels (Collagen). <i>RL</i> dropped with higher osmolalities for both dermal (Dermis) and full-thickness 3D skin models (FT 3D skin). Bar diagrams depict mean values, error bars depict standard deviations. Asterisks indicate statistical relevant differences (<i>p</i>-value ≤ 0.5). Each group comprised 3 individual samples that were measured in triplicate.</p

Microwave reflection measurement.

<p>(A) A patch antenna, optimized at <i>f</i> = 7.9 GHz, was connected to a network analyzer (NWA) and microwaves were introduced to a substance under test (SUT). Following microwave emission, the reflected signal was detected. (B) Each SUT exhibited a specific return loss (<i>RL</i>) and frequency at the local minimum (<i>f</i>_<i>min</i>), exemplarily shown for purified water (aq. dest.) and air. Measurements were compared to a mathematical electromagnetic antenna model. The reflection minimum and frequency for air shifted in a range between 7000–9000 MHz for simulation and measurements.</p

Microwave signals of 3D skin models at different osmolalities.

<p>(A) H&E-staining of full-thickness 3D skin models (FT 3D skin) with intact and removed epidermal layer following treatment with PBS. (B) The dry to wet weight ratio increased with higher osmolality (from aq. dest. to NaCl), whereas (C) <i>f</i>_<i>min</i> decreased with a higher osmolality. (D) <i>RL</i> increased with a higher osmolality and was highest where no epidermal layer was present (PBS w/o rHE). All scale bars indicate 50 μm. Bar diagrams show mean values, error bars depict standard deviation. Asterisks indicate statistical relevant differences (<i>p</i>-value ≤ 0.5). Each group comprised 3 individual samples that were measured in triplicate.</p

Detection of tumor nodules with FDG-PET.

<p>(A) Gefitinib-treated and untreated A549 and HCC827 cells grown in 2D were incubated with ¹⁸F-FDG for 60 minutes and FDG uptake was quantified using a gamma-counter. Data were corrected for background and decay and related to the initially added activity (n = 4, p = 0.02, Man Whitney U test). (B-I) Cross sections of lungs recellularized with tumor cells and incubated with ¹⁸F-FDG were investigated by autoradiography (ARG). Regions of high radioactive intensity correlated with presence of tumor cells (arrows in B, C, D, E). Cells of both cell lines strongly expressed GLUT1 (F, G) while only a low amount of the tumor cells was proliferative as shown by Ki67 staining (H, I). Data are presented as arithmetic means ± SEM; *p<0.05, Man Whitney U test; scale bars: 50 μm. One representative experiment out of 3 is shown.</p

Comparison of native and decellularized rat lungs.

<p>(A) After decellularization using the H₂O-SDC protocol, no cellular remnants were observed while the alveolar architecture and extracellular matrix proteins are well-preserved as shown in histological and ultrastructural analyses (SEM, TEM). Conservation of the alveolar-capillary basement membrane was verified using SEM and TEM. (B) Quantification of the percentage of airspace revealed no difference between decellularized and native lungs. (C) DNA content was significantly reduced after decellularization (p = 0.04, Wilcoxon rank sum test). (D-E) Higher collagen concentration was found in the acellular scaffold, while elastin was maintained at similar levels to native lung tissue. Scale bars in histological images: 50 μm; scale bars in SEM images: 10 μm, scale bars in TEM images: 500 nm; data in B-E are presented as arithmetic means ± SEM; *p<0.05, Kruskal-Wallis test; n = 5.</p

Monitoring of treatment response to targeted therapy with FDG-PET in lung tumor models.

<p>Lungs recellularized with HCC827 (top panel, A) or A549 (bottom panel, D) were incubated with ¹⁸F-FDG-PET for 60 min and imaged using a μPET-scanner. Coronal views of exemplary lung scaffold cultures (n = 3) at baseline and following 24 h or 72 h treatment with gefitinib are shown. 3D regions of interest were defined at individual tumor nodules or scaffold tissue and mean tumor-to-background ratios (TBR) were calculated (B, E). Horizontal bars depict median values for the mean TBRs of individual nodules at baseline, 24 h and 72 h. (C, F) Relative change of tracer uptake in individual tumor clusters compared to ¹⁸F-FDG-PET-intensity at baseline is shown. Intensity of tracer uptake decreased over the course of 72 h treatment with gefitinib in HCC827-, while no dramatic effect was detected in A549-seeded lungs.</p

Tumor cell lines form nodules with characteristics of <i>in vivo</i> tumors on the lung scaffold.

<p>HCC827 and A549 cells were introduced through the trachea for recellularization of the airway structures. Both cell lines were able to repopulate the acellular lung scaffold. After 14 days of static culture, HCC827 (A, C) cells formed dense “tumor-like” clusters at the distal periphery of the lung. In contrast, A549 cells (B, D) displayed a scattered colonization, with more tumor-like appearance and higher tendency of cell accumulation at the periphery. Tumor cells of both cell lines were still proliferative after 14 days in static culture (E, F) and showed a high expression of Mucin-1 (G, H). Scale bars: 50 μm. (I, J) Proliferation of tumor cells was quantified by the number of Ki67-positive cells. HCC827 cell grown on the 3D scaffold exhibited a slightly reduced percentage of proliferative cells compared to 2D cell culture (75.25 ± 0.74% vs. 85.7 ± 0.06% p = 0.057, Wilcoxon rank sum test) (I), while only a low percentage of A549 cells was proliferative in the 3D scaffold (12.52 ± 0.91% vs. 81.78 ± 0.03, p = 0.057, Wilcoxon rank sum test) (J). Data are presented as arithmetic means ± SEM; n = 3.</p