«Der Naturwissenschaftlichen Fakultät der Friedrich-Alexander-Universität Erlangen-Nürnberg zur Erlangung des Doktorgrades Dr. rer. nat. vorgelegt ...»
For random biopolymer networks, the probability distribution of the radii more closely resembles a normal distribution, where the maximum of the distribution is approximately the average pore size of the gel.
2.8 Evaluation of fiber orientation
We determined the direction vector of short ﬁber segments by treating their brightness distribution as a mass distribution, computing and diagonalizing the moment of inertia tensor, and ﬁnding the ”easy” axis of minimal inertia. This easy axis is pointing in the direction of the locally straight line segment. 105 spherical volume segments with a radius of 3 pixels, with each sphere containing at least 5 solid-phase voxels, were chosen randomly from the binarized stack. The voxels were treated as mass points, located at the voxel centers, with constant mass m=1 for all solid-phase voxels and m=0 for all liquid-phase voxels. After determining the easy axis of the inertia tensor, the corresponding unit direction vector in spherical coordinates was computed. This vector does not depend on the exact position of the sphere‟s center as long as the same solid voxels are enclosed. Finally, a histogram was generated for the polar angle θ of the direction vectors from all fiber segments.
2.9 Magnetic tweezer
Microrheologic stiffness measurements were performed with a magnetic tweezer setup (Fig 2.2, A) as described in . For stiffness measurements with collagen gels, fibronectin-coated 5 µm superparamagnetic beads were bound on top of collagen gels for 1h. For cell stiffness measurements, fibronectin and RGD coated beads were added to the cells. In order to prevent internalization of the beads, they were added only 30 minutes prior to the measurement. In all cases a staircase-like force step protocol was applied with 20 consecutive steps of 0.5 nN and 1 s duration for collagen stiffness measurements and 10 steps from 0.5 to 10 nN for cell stiffness measurements. The maximum force was in all cases 10 nN (Fig. 2.2, B). Bead displacements in response to force steps were measured with a microscope equipped with a CCD-camera (Orca ER, Hamamatsu) and a 20 x 0.4 NA objective under bright field illumination. Bead
12 II MATERIALS AND METHODSdisplacements followed a weak power law at all force steps (Fig. 2.2, C). The creep compliance was fitted to the equation [65, 66] () () ( )( ) where 1/J0 represents the stiffness. The exponent β is a measure for the fluidity of the material. An exponent of β = 0 represents a completely elastic material, whereas β = 1 represents a completely plastic material. Cells are normally viscoelastic, which means their behavior is a mixture between both and their β is therefore between 0 and 1.
Figure 2.2: Magnetic tweezer setup.
(A) Actual image of a magnetic tweezer. A steel bar, shaped like a needle on one end (1, tip radius below 10 µm) is surrounded by a copper coil (2, ~250 turns) which generates the magnetic field. (B) The force protocol which was used consists of 20 consecutive steps, each kept for 1s. Forces ranged from
0.5 to 10 nN. (C) From the registered bead displacement, the stiffness and the fluidity of the cell or the material can be derived by fitting a power law to each step.
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2.10 Extensional rheometer
For measuring the stress-strain relationship under uniaxial stretch, a cylinder of collagen was cast between two parallel plates with holes arranged in a checkerboard pattern (Fig 2.3). The plates were pretreated with Pluronic F-127 (Sigma-Aldrich, Germany) to prevent adhesion of the gel. The lower plate was connected to a precision scale (AND GR-200), and the upper plate was mounted to a motorized micromanipulator (Eppendorf Injectman). The gel was vertically stretched at a rate of 10 µm/s, and the weight change was continuously recorded. In this setup, the stretched gel has two different cross-sections, between the plates and in the holes. This geometry corresponds to a serial connection of two mechanical elements with different cross-sections (A) and length (l0). The Young‟s modulus E was calculated from the weight change F and the change in total extension l as ( ) The factor of 2 enters because there is an upper and a lower plate, both with the same hole geometry. The spring constant ( ) of the gel was corrected for the mechanical compliance of the device.
Figure 2.3: Extensional Rheometer Setup.
(A) Schematic setup of the Rheometer. A scale under the lower plate measured the change in weight when the upper plate was lifted with a motorized micromanipulator. (B) Image of the Rheometer plate setup. The gel was polymerized in the inner circle. The surrounding basin was filled with water to stabilize the setup.
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2.11 Cross-linking of collagen gels
To stiffen collagen fibers, collagen gels were cross-linked using 0.2% glutaraldehyde (25% stock solution, Merck, Darmstadt) in PBS (Invitrogen, Darmstadt, Germany). The cross-linker connects the collagen fibrils via the covalent binding of aldehyde groups from glutaraldehyde and amino groups from the collagen (Fig. 2.4).
Figure 2.4: The cross-linking of collagen.
(A) The cross-linker glutaraldehyde (red) connects the collagen fibrils, which then form the collagen fiber (Figure modified from  and ).
(B) The highly reactive aldehyde groups of glutaraldehyde bind to the amino groups of collagen.
An amount of 2 ml glutaraldehyde was kept for 1h on the gels. After removing glutaraldehyde, gels were washed with 2 ml of 20 mM TRIS buffer (Roth, Karlsruhe) for at least 24h to eliminate free, unbound aldehyde molecules, which would be toxic to cells. During that time the buffer was renewed every 2 hours. Before adding cells, TRIS Buffer was replaced with cell culture medium and renewed twice before cell seeding.
2.12 Cell culture
2.13 Invasion assay preparation and analysis 50,000 cells were seeded on top of the collagen gels and allowed to invade for 3 days at cell culture conditions (Fig. 2.5, A).
Afterwards, cells were fixed with 2 ml of 2.5 % glutaraldehyde (in PBS), and cell nuclei were stained with 1 µg/ml Hoechst 33342 (Sigma, Schnelldorf, Germany, Fig.
2.5, B) for 20 minutes. To analyze the invasion profile, 3D image stacks with a zdistance of 2 µm were obtained with a motorized Leica 6000 inverse fluorescence microscope. The z-position of cell nuclei as a function of the invasion depth (Fig. 2.5,
C) was determined in 36 fields of view using a custom-written Matlab script. The invasion profile was then computed as the cumulative probability to find a cell at or below a given depth. We defined a characteristic invasion depth as the invasion depth that was reached or exceeded by 5 % of the cells.
Figure 2.5: Principle of an invasion assay.
(A) Experimental setup of an invasion assay. Cells were seeded on top of a collagen gel. After an invasion time of 3 days in the incubator, the assay was stopped. (B) Overlay of a brightfield z-slice from an example stack and the fluorescence image of the same z-slice. Nuclei were stained with Hoechst (blue). (C) Evaluation of the invasion depth of the cell nuclei.
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2.14 MMP inhibitor treatment The broad spectra matrix metalloproteinase inhibitor GM6001 (Merck Millipore, Darmstadt, Germany) was used to block the activity of MMPs. 25mM GM6001 were added directly after seeding the cells on top of the collagen gels.
2.15 Transfection Transient transfection is used to introduce nucleic acids into cells. 200,000 per 35mm dish were seeded 24h prior to transfection. We performed transfection with the use of cationic liposomes. As a transfection reagent, Lipofectamine 2000 (Invitrogen, Carlsbad) was used (7 µl per dish). For each transfection, 1.5 µg of DNA was used. To increase transfection efficiency, we used Opti-MEM media (250 µl per dish), which additionally increases the permeability of the cell membrane. The solution was gently pipetted on 200,000 cells and incubated for 4h. Afterwards the solution was taken off and warm (37°C) cell culture DMEM medium was added.
Moreover we used a plus reagent provided by the distributor of lipofectamine (3 µl per dish), which helped form more stable DNA complexes for transfection. The very low amount of foreign DNA and the short incubation time of only 4 hours helped raising the survival rate of transfected cells to over 50%. From these 50 % about 70% were successfully transfected.
2.16 Live cell imaging - 2D tracking
30,000 cells were seeded in 35 mm culture dishes 24 h prior to the measurement. For the measurement of transfected cells, a 4-compartment culture dish (Greiner) was used, in order to image simultaneously cells transfected with different constructs from the same cell passage. For imaging an inverted microscope (DMI6000B, Leica) equipped with a 20x, 0.4 NA objective was used and cells were kept at 37 C° and 5% CO2 in an incubation chamber during measurements. Phase contrast images of the cells are obtained and analyzed using a customized Matlab program. Images are taken every 300 s over a time period of 24 h. Cell movements are tracked with a custom image
II MATERIALS AND METHODS 17processing software written in Matlab. From the cell trajectories, the mean squared displacement (MSD) is calculated  ( ) 〈( ⃗( ) ⃗( )) 〉 ( ) and described with a power-law relationship, where the slope of the MSD is a measure for the persistence of a movement. A slope of 1 denotes randomly migrating cells while a slope of 2 indicates completely persistent, ballistically migrating cells (Fig. 2.6).
Figure 2.6: The MSD as a measure of cell migration speed and directionality.
From the tracking algorithm the centroid of the cell is known and the x- and y-position can be plotted as a cell trajectory (blue crosses and blue dotted line). To derive the MSD, different lag times can be used. (Inset) Illustration of a MSD curves plotted over the lag time. A linear equation can fitted to the MSD while the slope indicates a totally directed ballistic (slope =2), a diffusive (slope = 1), a superdiffusive (1 slope2) or a superdiffusive (slope 1) motion.
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2.17 Cell shape analysis
For cell shape analysis of non-transfected cells in 3-D collagen gels, cells were fixed with 4% paraformaldehyde (ThermoFisherScientific, Germany) in PBS for 20 min, stained with phalloidin TRITC (0.2 µg/ml, Sigma, Germany) for 1h and then washed twice with PBS. Fluorescence image stacks (z-distance = 839.8 nm) of invaded cells were obtained with a confocal microscope (SP5X upright microscope equipped with a 20x dip in water immersion objective with NA=1.0, Leica Mannheim, Germany). After a maximum intensity projection, the cell outline was determined with an edge detection algorithm implemented in Matlab and fitted to an ellipse. Cell eccentricity was computed as the ratio of the distance between the foci of the ellipse and its major axis.
Eccentricities close to 0 indicate circular shapes, while values close to 1 indicate highly elongated shapes.
In the case of transfected cells, 15,000 transfected and non-transfected cells were seeded in 35 mm cell culture dishes and incubated for 24h. 2D-images were obtained with the Leica SP5X confocal microscope and a 20x water immersion dip-in lens. Cell spreading area was determined from brightfield images using an edge detection algorithm implemented in a custom Matlab script.
2.18 Extraction of primary breast cells and cultivation
PS, EJ and KB cells are primary breast cells which were provided from the laboratory for Molecular Medicine in Erlangen, where they were derived from patients. The fractionation protocol was extensively modified from stem cell technology. In brief, 20ml tissue was placed in a Falcon tube and incubated with PBS and PenicillinStreptomycin for 1 h. Afterwards, 20 ml of DMEM and 0.5x final collagenase/hyaluronidase were added. Falcons were then incubated for 16 h at 37°C with orbital shaking at 75 rpm. Subsequently, the solution was centrifuged at 88g for 30 s and the fat layer was pipetted off completely. The remaining cell pellet was resuspended in 5ml PBS and 1ml trypsin and incubated for 30 min in order to digest the mesenchymal cells from the epithelial like glands (organoids). Again the solution was centrifuged. The pellet now contained epithelial cells while the supernatant contained mesenchymal cells for further cultivation. Primary breast cells were cultivated in
II MATERIALS AND METHODS 19EMF5% Media (with 1% PenStrep and 10% FCS) and seeded in T75 flaks coated with Collagen I. (For detailed protocol, see appendix)
2.19 Amnion preparation The amnion is a membrane that protects an embryo in mammals and is attached via the chorion to the placenta (Fig.2.7). Amnion extraction from human placentas was performed at the Frauenklinik Erlangen (all patients sign a letter of agreement to donate the placenta before they are given to research groups and provided for the study in this work). Only amnions from C-sections were taken, because they are sterile. The Amnion was separated from the chorion and the placenta, and then cut into pieces that fit into 10 cm dishes. The pieces were washed with PBS 6 times, with each washing step about 2-3 minutes.
Figure 2.7: The amnion in cell culture I: Human placenta after birth with (A) the maternal side and (B) the side which is facing the fetus with attached umbilical cord.
The white/transparent layer in (B) is the amnion attached to the chorion and the placenta. Pictures were taken in the Hôptial Sainte-Justine, Montreal, Canada.
After the washing steps, the Amnion pieces were cut again, so that they fit into 35mm culture dishes, which were coated before with fibronectin. The membranes were placed with the epithelial layer face-up and the connective tissue face-down into fibronectin coated the dishes. To be able to flatten and fix the Amnion on cell culture dishes, we