Contractile forces regulate cell division in three-dimensional environments.

Cell culture Swiss 3T3 fibroblasts (passages 10–20) stably transfected either with GFP-actin or with mRuby-LifeAct (obtained as gifts from S. Fraser, University of Southern California, Los Angeles, CA) were cultured in DMEM medium supplemented with 10% fetal bovine serum and 1× nonessential amino acids in a 37°C humid incubator. Endothelial cells were included to promote fibroblast proliferation ( Hirschi et al., 1999 ).

Human umbilical vein endothelial cells

(HUVEC) stably transfected with RFP (Angio-Proteomie) were cultured in endothelial growth medium containing 5% serum, growth supplements, and 1× penicillin and streptomycin (passages 4–9; Angio-Proteomie) also in a 37°C humid incubator. 3D fibrin gel preparation 3T3 fibroblasts-GFP-actin (∼3,000 cells) and HUVEC-RFP (∼3,000 cells) were mixed with 20 µl of 5 mg/ml fibrinogen (Omrix Biopharmaceuticals). In a separate vial, red 0.5 µm carboxylated fluorescent particles (Ex 580/Em 605; Invitrogen) were mixed with 20 µl of a 20 U/ml thrombin solution (Omrix Biopharmaceuticals) and vortexed for 1 min to a final particle concentration of 0.05%. The thrombin suspension was placed on a No. 1.5 coverslip in a 35-mm dish (MatTek Corporation), and mixed gently with the fibrinogen suspension. The resulting fibrin gel was placed in the incubator for 15 min to fully polymerize, after which warm medium (50% fibroblast, 50% HUVEC medium) was added to cover the gel. For imaging of cell division without measurement of matrix displacements, fibrin gels were made in a similar way but without including fluorescent particles. 2D culture preparation 2D glass. 3T3 fibroblasts-GFP and HUVEC-RFP were mixed in a 1:1 ratio and plated on a coverslip bottom dish (No. 1.5; MatTek Corporation) uncoated or coated with fibrinogen solution (100 µg/ml, 2 h incubation at room temperature). The 3T3 cells were imaged after overnight incubation while maintaining low confluence. 2D fibrin. To prepare flat gels for studies of cell division on 2D fibrin substrates, 10 µl of 5 mg/ml fibrinogen was mixed with 10 µl of 20 U/ml thrombin, placed on a No. 1.5 coverslip in a 35-mm dish (MatTek Corporation), and incubated for 15 min. A sterile coverslip was placed on top of the gel to flatten it. After addition of phosphate-buffered saline to fill the well, the coverslip was removed and the cells were cultured on the gels. The thickness of the gel was measured by confocal imaging of gel-embedded fluorescent beads to be ∼200 µm. Fibrin gel labeling and fixation Alexa Fluor 546 carboxylic acid, succinimidyl ester (Invitrogen) was mixed with fibrinogen solution in a 7.5:1 molar ratio for 1 h at room temperature and then filtered through a HiTrap desalting column (GE Healthcare) packed with Sephadex G-25 resin to separate the unreacted dye. The labeled fibrinogen was then mixed with thrombin and cells to create labeled, cell-loaded fibrin gels. For the fixation protocols, labeled fibrin gels loaded with 3T3 GFP-actin cells for at least 6 h were immersed in 4% paraformaldehyde for 10 min and subsequently washed with PBS. The nuclear material was labeled with DAPI before imaging.

Fibrin gel characterization

The constitutive mechanical properties of fibrin gels without cells were measured using a stress-controlled AR1000 rheometer equipped with 8-mm-diameter aluminum parallel plates. Frequency sweep and creep tests were performed. The fibrin gel was prepared on the rheometer and placed between sheets of sandpaper to avoid slipping. The bulk material stiffness could not be used to compute traction forces owing to local matrix inhomogeneity at the scale of the cell.

Time-lapse microscopy

After overnight incubation, fibrin gels were imaged with a Swept Field confocal microscope mounted on a Ti stand (Nikon) outfitted with a 40× 1.15 NA Apochromat water immersion objective lens (Nikon) using the microscope’s 30-µm pinhole (for imaging the fluorescent particles), 45-µm pinhole (for imaging the fibrin gel), or 60-µm pinhole (for imaging the cells). Images were captured with a QuantEM:512SC camera (Photometrics) using NIS-Elements Ar software (Nikon). Imaging was performed in a 50/50% mix of fibroblast and HUVEC medium in a custom-built 37°C/5% CO 2 incubation chamber after allowing the system to equilibrate for ∼4 h. All experiments on cells embedded in the matrix were conducted on cells positioned at least 100 µm from the bottom surface of the gel. Confocal z-stacks capturing single fibroblasts were acquired every 15–30 min (for measurement of matrix displacements) or every 2–3 min (for visualizing dividing cells) for ∼6 h. Stacks were collected with a z step size of 0.4 µm (for displacement measurements) to 1 µm (for cell visualization). For experiments involving analysis of matrix displacements, blebbistatin (85 µM; Sigma-Aldrich) was added to the medium at the end of the experiment and stress-free stacks were acquired for an additional 4 h.

Measurement of matrix displacements

To compute matrix displacements, red 0.5 µm fluorescent particles (Invitrogen) were used to create a speckle pattern for a DVC algorithm ( Franck et al., 2007 ) implemented in MATLAB (MathWorks). Before running the DVC algorithm, 3D deconvolution was performed on the volume stacks of the particles in MATLAB using the Lucy-Richardson algorithm as described previously ( Franck et al., 2007 ). The DVC algorithm used a Fourier transform–based correlation to compute the 3D displacements at the center of a subset of 64 × 64 × 64 voxels on a grid of points within the image volume of 512 × 512 × 276 voxels. Real-time displacements were computed by using blebbistatin (final concentration of 85 µM) to inhibit the cell’s myosin II activity, thus allowing the gel to recover to an unstressed state. All stacks collected before injecting blebbistatin were correlated to the unstressed stack acquired after the addition of blebbistatin. To account for minor swelling or shrinking of the fibrin gels during displacement measurements, the mean normal matrix strains were computed. The displacement fields were then corrected by subtracting the displacements associated with mean normal matrix strains. To quantify the experimental error associated with the displacement measurements, control experiments were performed on fibrin gels without cells. In these experiments, confocal stacks were collected every 30 min for ∼6 h. Then, blebbistatin was injected into the medium to a concentration of 85 µM. Errors in matrix displacements computed with the DVC algorithm were found to be