Observing and Quantifying Fibroblast-mediated Fibrin Gel Compaction

Summary

Time-lapse microscopy and image processing techniques were used to observe and analyze fibroblast-mediated gel compaction and fibrin fiber realignment in an environmentally controlled bioreactor over a 48 hr period.

Abstract

Cells embedded in collagen and fibrin gels attach and exert traction forces on the fibers of the gel. These forces can lead to local and global reorganization and realignment of the gel microstructure. This process proceeds in a complex manner that is dependent in part on the interplay between the location of the cells, the geometry of the gel, and the mechanical constraints on the gel. To better understand how these variables produce global fiber alignment patterns, we use time-lapse differential interference contrast (DIC) microscopy coupled with an environmentally controlled bioreactor to observe the compaction process between geometrically spaced explants (clusters of fibroblasts). The images are then analyzed with a custom image processing algorithm to obtain maps of the strain. The information obtained from this technique can be used to probe the mechanobiology of various cell-matrix interactions, which has important implications for understanding processes in wound healing, disease development, and tissue engineering applications.

Introduction

An important tool for studying cell-matrix interactions is the cell populated collagen gel^1,2. The gel provides a 3D environment that is closer to the in vivo character of the tissue and better suited for understanding cell behavior than is offered by traditional 2D cultures³. Early studies in which fibroblasts were homogenously distributed within a collagen gel found that the cells rapidly consolidate the collagen fibers and compact the gel^4,5. The contractile fibroblasts in free floating gels then transition into a quiescent state soon after the gel has fully reached compaction^1,6,7. The fibroblasts in gels that are constrained at the boundaries remain in an active, synthetic state⁸ and they generate fiber alignment in a manner dependent on gel geometry and external constraints^5,9. Differences in cell activity appear to be a result of the internal tension (or lack thereof) that develops as the cells exert traction forces via integrins on the collagen fibers in the gel.

A variant of this technique involves placing fibroblast explants (i.e. clumps of cells) a distance apart within a collagen gel and observing cell-matrix interactions and the gradual development of fiber alignment between the explants (sometimes called ligament-like straps)^10-12. The primary advantage of the explant system is that it allows one to arrange the cells into simple geometric patterns, which makes it easier to visualize and probe the mechanisms underlying cell-driven fiber realignment. These alignment patterns — which are dependent primarily on the interplay between cell traction forces, cell spatial distribution, gel geometry, and the mechanical constraints on the gel — are important to understand because they play a central role in global tissue organization, mechanical function, and local mechanical environment¹³.

In the field of tissue engineering, one strategy for producing mechanically functional, engineered-tissues involves controlling the fiber alignment pattern that develops from cell compaction so that the engineered tissue possesses fiber alignment that mimics that of the native tissue^14,15. Such alignment is believed necessary for engineered tissues to replicate the complex mechanical behavior of native tissues. A modification of this strategy is to replace the collagen gel with a fibrin gel¹⁶. The fibrin gel develops a similar alignment pattern as a collagen gel during compaction. Over time the fibrin is degraded and replaced with cell-synthesized ECM that follows the initial fibrin fiber alignment pattern. The resulting engineered construct has significantly improved mechanical properties compared to collagen gel derived constructs¹⁷.

The alignment process and subsequent remodeling events in fibrin gels proceed in a complicated and poorly understood manner. To better characterize these interactions and their effect on cell behavior and ECM remodeling, we have developed a procedure that is based on the explant method. In this method, fibroblast explants are positioned on a fibrin gel in different geometric patterns. The gels are maintained in an environmentally-controlled, microscope-mounted bioreactor¹⁸, and the process of compaction and fiber realignment is monitored with time-lapse differential interference contrast (DIC) microscopy. Displacement fields are quantified with custom algorithms. The data obtained from these experiments have wide ranging implications for a number of processes, including optimizing tissue engineering strategies, improving wound healing, and treating pathological tissue remodeling.

Protocol

1. Stencil Preparation

Prepare a stencil on Parafilm to layout the location of each explant, following the desired geometry (Figures 1A and 1B). Space each explant approximately 1-2 mm apart. This distance corresponds to an ideal spacing for generating fiber alignment between explants. Attach the stencil underneath the area of the coverglass where the sample will be prepared with tape.

2. Sterilization

Thoroughly clean all components of the bioreactor using 70% ethanol and sterilize for 2-3 hr under UV light prior to experimentation. If an alternative vessel is being used in lieu of a bioreactor then proper sterilization techniques should be used. See step 4.12 for comments on using a glass bottom Petri dish.

3. Fibrin Gel Preparation

After the components of the bioreactor have been sterilized, cast a thin layer of fibrin gel on the surface of the coverglass. Details for preparing fibrinogen and thrombin stock solutions for making 6.6 mg/ml fibrin gels are described in Sander et al.¹³ A similar protocol by Ye et al.¹⁹ for making 3.3 mg/ml fibrin gels can also be viewed on the JoVE website.

1. Prepare a solution of fluorescent microbeads in DMEM at a concentration of 10 million beads/ml. The beads will be used to help track gel displacements. To achieve this concentration, combine 0.017 ml of microbead stock solution and 0.149 ml of DMEM into a microcentrifuge tube.
2. Sonicate this suspension for 10 min to disperse the beads and homogenize the solution.
3. Fibrin Solution — In a 15 ml c-tube, mix 0.22 ml of fibrinogen stock solution with 0.44 ml of 20 mM HEPES buffer. Add the 0.1667 ml of DMEM with microbeads created in step 3.1.
4. Thrombin Solution — In a separate 15 ml c-tube, mix together 0.0328 ml of thrombin stock solution, 0.131 ml of 20 mM HEPES buffer, and 0.00246 ml of 2 M CaCl₂.
5. Carefully mix the thrombin solution (step 3.4) with the fibrinogen solution (step 3.3) by pipetting up and down 5-10x until the solution is evenly distributed. Avoid introducing bubbles as much as possible. To reduce the amount of bubbles produced, be careful not to fully discharge the pipette while mixing.
6. The addition of thrombin will cause the solution to gel quickly (~30 sec). Pipette the mixed solution onto the coverglass as soon as possible. Allow the gel to polymerize at RT.
7. Seal up the bioreactor, insert the heating blocks, and connect the thermocouples to the temperature controller. Incubate the gel at 37 °C for 15-30 min.

4. Cell Explant Preparation

1. Remove medium from the T-75 flask containing the human dermal fibroblast cells.
2. Carefully rinse the surface with approximately 5 ml of phosphate buffered saline (PBS) to remove serum proteins. Add 1 ml of trypsin-EDTA and incubate for 3 min, or until cells have lifted.
3. After the cells have been lifted, spin the suspension down in a centrifuge at 200 x g for 5 min. Remove the supernatant and resuspend the pellet in a volume of DMEM that will allow a final concentration of 20 million cells/ml.
4. While the cells are spinning down in the centrifuge disconnect the bioreactor from the heating blocks and the thermocouples. Transfer the bioreactor to a biosafety cabinet and carefully remove the lid following asceptic techniques.
5. Create explants by pipetting 0.3 µl of the cell suspension onto the polymerized fibrin gel, following the pattern on the stencil. Each explant should contain approximately 6,000 cells. Make sure that low volume micropipette tips are used (0.1-10 µl).
6. Allow cells to settle and attach to the fibrin matrix for 1 hr at 37 °C.
7. With the bioreactor still open, add approximately 5 ml of DMEM supplemented with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin, 0.1% amphotericin B, and 10 mg/ml aprotinin directly into the bioreactor chamber. DMEM is bicarbonate buffered and requires 5% CO₂ to maintain a neutral pH. Since the bioreactor is not supplied with CO₂, condition the medium in an incubator with 5% CO₂ for 2-3 hr before use. Aprotinin is a serine protease inhibitor that is widely used to reduce the rate of fibrin degradation²⁰.
8. Reseal the bioreactor. Use a syringe to deliver an additional 5 ml of CO₂ conditioned medium via the barbed fitting on the inlet port. Dispense the medium slowly and make sure the entire volume of the bioreactor chamber is filled. Carefully remove bubbles that form in the bioreactor.
9. Supply fresh, 5% CO₂ conditioned medium to the bioreactor throughout the experiment in order to maintain pH, supply nutrients, and remove waste products. Setup a syringe pump with a 10 ml or 30 ml syringe filled with 5% CO₂ conditioned medium. Connect the syringe directly to the inlet port on the bioreactor lid with Luer-lock fitted sterile tubing. Remove the male fitting and attach the tubing to the barbed fitting on the bioreactor (see Figure 1C).
10. Set the perfusion rate to 0.01 ml/min. Connect modified pieces of tubing to both outlet ports and place the ends into a 100 ml beaker to collect waste.
11. Use a lab jack to set the heights of the inlet and outlet feeds so that a pressure differential does not develop in the bioreactor (consult Figure 1D for reference).
12. If a bioreactor is not available, prepare samples in 35 mm glass bottom Petri dishes with glass tops. Use one with a coverslip size that is optimized for the specific set of objectives to be used. Samples that are prepared in Petri dishes should be maintained in an incubator at 37 °C and 5% CO₂.
    Note: Polystyrene depolarizes light and will interfere with DIC imaging, so glass tops should be used if DIC imaging will be conducted. Phase contrast is a suitable alternative imaging modality. Transferring dishes between the incubator and microscope will make image registration difficult. If images are not properly registered then the strain calculated in Section 6 will not be accurate.

5. Time-lapse Imaging

1. Once the sample has been prepared, reseal the bioreactor, reconnect the heating blocks and the thermocouples, and set the bioreactor temperature to 37 °C.
2. Set the bioreactor onto the microscope mounted motorized stage. Position the 20X DIC objective under the view-port. A lower magnification objective is acceptable, particularly if a precision motorized stage is not available. Ensure that the polarizer, analyzer, and prism are all in place. Alternatively, samples can also be imaged using phase contrast.
3. Open the imaging software.
4. Focus the objective on the area between the three explants.
5. Save the coordinates (x, y, and z) of this location in the imaging software.
6. In order to image the entire area between and around the explants select the option to acquire a large image and specify the size of the area. This will allow the acquisition of multiple images around the specified area and create a tiled image that represents a larger region of the sample.
7. Set the exposure time and light intensity to the lowest values possible to avoid cell death caused by phototoxicity, while still providing sufficient resolution to discriminate between cells, microbeads, and fibrin fibers.

6. Strain Tracking

For details and instructions on the strain tracking software (Figure 2) used see Raghupathy et al.²¹ The algorithm is a custom MATLAB code that can be downloaded from http://www.license.umn.edu/default.aspx. Note that DIC images often have enough texture for strain tracking. The microbeads are included to serve as a check on the calculated strain fields. If strain tracking will be done it is critical that the images obtained are taken at the exact same location so that the images are registered. Unregistered images will produce spurious strains.

Results

Tissue remodeling is a complex process that is driven in part by reciprocal physical interactions between cells and the surrounding matrix. The cells reorganize the surrounding fibers and generate tension in the fiber network. The alignment of fibers and mechanical environment in turn controls cell behavior, so that both cells and matrix globally reorganize to produce remodeled tissue. In this experiment, the cells of the explants, initially round in morphology, began to extend into the gel and adhere to the fibrin fiber...

Discussion

This protocol was developed for the purpose of observing and quantifying the mechanics involved in cell-mediated ECM remodeling. Such processes underlie a number of biological phenomena and have important implications for engineering tissues^2,22, reducing scar^1,23, and understanding pathological tissue remodeling^12,24. The use of time-lapse DIC microscopy allows one to resolve and quantify the displacement and alignment of fibrin fibers that occurs as a result of cell traction forces. The...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Sigma-Aldrich	F8630
Sigma-Aldrich	T4648
Gibco	11965-092
Gibco	15140-122
Sigma-Aldrich	A2942
Sigma-Aldrich	H0887
Sigma-Aldrich	223506
Gibco	25200-056
Invitrogen	3000
Lonza	DE14-701F
Molecular Probes	F8858
GIBCO	A10483-01
GIBCO	11430-030
Fisher-Scientific	SS264-1
Sigma-Aldrich	A3428-25MG
Biotense Bioreactor	ADMET
Ti-Eclipe Microscope	Nikon
# 0 35 mm Glass Bottom Petri Dish	MatTek	P35G-0-20-C
# 0 35 mm Glass Top Petri Dish	MatTek	P35GTOP-0-20-C
Plastic Luer fittings, PVC tubing with Luer ends	Cole-Parmer	30600-65