Force-induced fibronectin assembly and matrix remodeling in a 3D microtissue model of tissue morphogenesis.

Device fabrication and microtissue seeding Single layer and multilayer templates were created as described previously. 17 SU-8 photoresist (Microchem) was spin coated onto silicon wafers. Multilayer SU-8 masters were created using successive spin coat and exposure steps. To generate substrates for microtissues, SU-8 masters were cast with a prepolymer of PDMS (sylgard 184; Dow-Corning). Before cell seeding, the PDMS templates were sterilized in 70% ethanol followed by UV irradiation for 15 minutes and treated with 0.5% Pluronic F127 (BASF) to reduce cell adhesion. Liquid neutralized collagen I from rat tail (BD biosciences) was then added to the substrates on ice and templates were degassed under vacuum. Additional collagen and cells were then added to the mold and the entire assembly was centrifuged to drive the cells into the micropatterned templates. Excess solution was removed by dewetting the surface of the substrate prior to incubating at 37 °C to induce collagen polymerization. Fn containing media was then added to each substrate. All constructs in this study were prepared using a final concentration of 1.75 mg ml −1 collagen. Calculation of cantilever spring constant and microtissue force/stress Cantilever spring constants were calculated utilizing a capacitive MEMS force sensor mounted on a micromanipulator as described previously. 28 Images of the sensor tip and cantilever head were acquired during each test using an Olympus FV1000 confocal microscope with an air immersion 0.4 NA 10× objective. To account for local deformation of the PDMS material around the sensor, the spring constant of the MEMS sensor was calibrated against the side of the PDMS well which can be viewed as an elastic half space of the same material modulus as the PDMS cantilevers and was found to be 104 ± 1.9 nN µm −1 . This value was then used for the subsequent measurements of the force required for cantilever bending. For each measurement the sensor tip was placed 20 microns below the top of the post and the probe translated laterally against the outer edge of the cantilever using a custom written Lab View (National Instruments) script. The probe base was displaced approximately 150 microns for each measurement. The displacement of the probe tip (and thus of the cantilever head) was calculated from the spring constant measured above and the reported sensor force and was verified visually during the deformation. 5 cantilevers were measured across a substrate and measurements were repeated for three different substrates. Cantilevers were found to have linear responses up to approximately 40 microns of deformation. As the majority of cantilever deformations observed in this paper were below 40 microns, this section was fit using a linear fit with a spring constant of 148 ± 35 nN µm −1 which was then used for calculation of microtissue forces. Only microtissues that were uniformly anchored to the tips of the cantilevers were included in the analysis. Microtissue stress was calculated by imaging the entire volume of the microtissue within a 30 µm wide strip (measured along the long axis of the tissue) at the tissue midpoint. The cross-sectional area of this region was then measured from the confocal z -stack data using custom written Matlab (The Mathworks) scripts. Fibronectin isolation, fluorescent labeling and denaturation curves Fn was isolated from human plasma (Zurcher Blutspendedienst SRK) by affinity chromatography as described previously. 10 For cysteine (acceptor) labeling, unlabeled Fn was denatured in 4 M urea at a concentration of 1 g l −1 and incubated with 40-fold molar excess of Alexa Fluor 546-maleimide (Invitrogen) for 3 hours at room temperature. Cysteine labeled Fn was separated from free dye using by a size exclusion chromatography PD-10 column (Sephadex) equilibrated with PBS with 0.1 M NaHCO 3 [pH 8.5]. Cysteine labeled Fn was then incubated with 60-fold molar excess Alexa Fluor 488-succinimidyl ester for 3 hours at room temperature to label amines (donors) before separating Fn–donor/acceptor (Fn–DA) from free dye with a second PD-10 column equilibrated in PBS. Fn–DA was then diluted to 0.3 g l −1 in PBS, aliquoted and stored at −80 °C until needed. A labeling ratio of 4 acceptors and B7 donors per Fn dimer was determined by measuring the absorbances of Fn–DA at 280, 496 and 56 nm and using published extinction coefficients for dyes and Fn. Unlabeled Fn was isolated in a similar manner, dialyzed in PBS for 48 hours (Slide-a-lyzer dialysis cassette, 10 000 MW cutoff, Pierce) and diluted to 1 g l −1 in PBS before aliquoting and storing at −80 °C. Before use, Fn–DA or Fn was thawed on ice for 1 hour to avoid protein precipitation. After thawing, all samples were stored at 4 °C and used within 72 hours. All cell culture experiments utilized unlabeled Fn in 10-fold excess to Fn–DA in order to avoid signal from intermolecular FRET. Denaturation curves were obtained by diluting Fn–DA to a final concentration of 0.1 g l −1 in specified concentration of GdnHCl, and imaging with confocal microscopy under identical parameters to those used for cell culture.

Confocal microscopy

Z-stack images were acquired using an Olympus FV1000 confocal microscope with a water immersion 0.9 NA 40× objective. Four post microtissue images were acquired at 512 × 512 pixels per slice with 2 µm slice spacing for voxel dimensions of 0.621 × 0.621 × 2.0 µm. The mid-sections of 2 post microtissue images were acquired at 256 × 256 pixels with 1.5× zoom factor per slice with 2 µm slice spacing for voxel dimensions of 0.828 × 0.828 × 2.0 µm. All images were acquired with 3× Kalman line averaging. FRET images were acquired using a single photo multiplier tube (PMT) by sequentially acquiring images through a diffraction grating and slit. Donor and acceptor intensities were detected using 20 nm bandwidths centered at 510–530 nm and 560–580 nm, respectively. Serial imaging of the same volume confirmed that no photo bleaching was occurring. To ensure that no artifacts occurred due to the microscope setup, laser power, pixel dwell time and PMT voltage were kept constant between all conditions (all experiments and all time points) for imaging protein and DNA densities and Fn–DA FRET. Images of fluorescent beads within microtissues were acquired on a Zeiss LSM 710 laser scanning confocal microscope with a water immersion 1.1 NA 40× objective.

FRET calculation and colocalization analysis

FRET ratios are reported as the intensity of the acceptor ( I a ) divided by the intensity of the donor ( I d ). All images were 12-bit (4096 relative intensity units) and were processed with custom written Matlab (The Mathworks) scripts. First, background values were determined from tissue-free regions of each image and subtracted from the raw images. Then, images of each channel were binary thresholded using a cutoff value of 2× background (Fn donor and acceptor), 1.75× background (collagen), and 1.5× background (DAPI). These settings were determined empirically based on the intensity of each fluorescence signal and then kept constant for all experiments. Prior to calculation of the FRET ratio, donor and acceptor images were smoothed using a 2 × 2 pixel local averaging filter. Donor bleed through was calculated from images of microtissues labeled with Alexa-488 Fn and was determined to be a linear function of donor intensity with a slope of 0.224 ( i.e. approximately 20% of the donor intensity was present in the acceptor channel). Acceptor images were thus scaled appropriately prior to calculation of the FRET ratio. Any saturated pixels, or those below the threshold value were excluded from the FRET analysis. Median values were calculated for each tissue from all pixels above threshold with a single tissue considered as an independent observation. Colocalization analysis was performed on a voxel by voxel basis with Fn considered to be colocalized with collagen if each channel was above the aforementioned threshold values. These imaging conditions, combined with the thin profile of the microtissues, were optimized to avoid any confounding contributions due to aberration from the collagen based microtissues. However, we wanted to further validate that imaging into the depth of the microtissue would not cause spectral artifacts and thus confound our FRET data. To this end, we incorporated 0.5 µm fluorescent beads (Invitrogen) within the microtissues and imaged their intensities as a function of tissue depth. Imaging through the collagen rich micro-tissues had no appreciable effect on either the integrated brightness nor the peak intensities of beads with emission peaks at either 486 or 605 nm (similar to the FRET probes used in the study) ( Fig. S6, ESI† ).

Immunofluorescent staining and protein density mapping

Microtissues were cultured for the indicated periods of time before fixation with 4% formaldehyde in PBS for 1 h at 37 °C. Samples were subsequently rinsed for 30 minutes in PBS, before blocking overnight in 10% donkey serum. The exogenous collagen scaffold was labeled using primary antibodies directed against type-I rat collagen (Chondrex) and detected using fluorophore conjugated, isotype-specific, anti-IgG antibodies (Jackson ImmunoResearch). The use of primary antibodies specific for rat-tail collagen ensures that we only examine the localization of the original collagen scaffold and not collagen assembled de novo by the constituent NIH 3T3 cells (which are from mice). Cell nuclei were counter stained with DAPI. Density maps of protein and DAPI labeling were created by averaging the immunofluorescent (or FRET labeled Fn) data from individual microtissues. Cell-derived Fn was imaged utilizing an antibody specific to the EDA splice domain. 29 Plasma Fn density within microtissues was measured by computing the sum of the donor and acceptor fluorophores for Fn–DA excited at the donor wavelength. Masks were generated in Adobe Photoshop labeling the positions of each cantilever and used to align and crop the z-stacks of each microtissue. Optical slices from each tissue (10 tissues total) were then averaged to quantify protein and cell distributions for each condition. Because microtissues displayed small variations in thickness and in the vertical position at which they were tethered to the pillars, protein density maps were normalized by the thickness of each microtissue prior to averaging ( i.e. plotted from the uppermost to the lower most surface of the tissue at each x–y position). Volume renderings of microtissues were constructed from confocal image stacks using Fiji. Protein alignment was computed using principal component analysis of the image gradients from the confocal images as described previously. 17 , 30 The image gradient vector can be expressed as [ G x ( x , y ) G y ( x , y ) ] = sign ( ∂ I ( x , y ) ∂ x ) [ ∂ I ( x , y ) ∂ x , ∂ I ( x , y ) ∂ y ] T .

Principal component analysis

(PCA) computes a new orthogonal basis such that the variance of the projection on one axis is maximal while the variance of the projection on the orthogonal axis is minimal. This process is akin to finding the eigenvector decomposition of the autocovariance matrix C = [ G x x G x y G y x G y y ] . The principal eigenvector corresponds to the direction of fiber alignment. Alignment strength maps are computed from the eigen-values as str = λ 1 − λ 2 λ 1 + λ 2 . Highly aligned regions (str ~ 1) represent fibrillar regions with uniformly aligned fibers. Weakly aligned regions (str ~ 0) represent either fibrillar regions with heterogeneous orientations or non-fibrillar (diffuse) staining. x–y alignment and strength values were computed for each confocal slice separately. The alignment maps for each tissue were then averaged from all confocal slices. Final alignment maps represent the compiled average from 10 different microtissues. The integrated intensity and peak brightness of fluorescent beads within microtissues were computed utilizing code adapted from ref. 31 .

Statistical analysis

Statistical analysis was performed in Matlab utilizing the Kruskal–Wallis one-way analysis of variance and Tukey’s HSD (Honestly significant difference) tests to illustrate significance. Because the tests involve multiple comparisons, all statistical analysis are included in table format as Tables S1–S3 (ESI†) . Cell culture and reagents NIH 3T3 fibroblasts (American Type Culture Collection, ATCC) were cultured in 4.5 mg ml −1 glucose containing DMEM + l -glutamine (Gibco) supplemented with 10% bovine serum (Biowest). Cells were maintained at 37 °C and 5% CO 2 for all experiments. When indicated, Fn was added to the cell culture media at a total concentration of 50 µg ml −1 (5 µg ml −1 Fn–DA and 45 µg ml −1 unlabeled Fn). Blebbistatin (Sigma, 50 µM) treatment was performed for 2 hours at 37 °C and 5% CO 2 prior to fixation.

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