Name: Observing and Quantifying Fibroblast-mediated Fibrin Gel Compaction (Video) | JoVE Journal
Uploaded: 2014-01-16T13:45:00.000+00:00
Duration: 10 min 37 s
Description: Learn time-lapse microscopy techniques to observe fibroblast-mediated fibrin gel compaction and fiber realignment over 48 hours. This method enables quantitative analysis of cell-driven matrix remodeling.

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5 minutes

We thank George Giudice and Steven Eliason for donating human dermal fibroblasts and Ramesh Raghupathy for help with the strain tracking algorithm.&#160;Support for this work was provided by a U.S. Department of Education Graduate Assistance in Areas of National Need Fellowship (GAANN P200A120071).

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&#160;fibrin gels are described in Sander et al.13&#160;A similar protocol by Ye et al.19 for making 3.3 mg/ml&#160;fibrin gels can also be viewed on the JoVE website.
<ol>
	<li>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&#160;of microbead stock solution and 0.149 ml&#160;of DMEM into a microcentrifuge tube.</li>
	<li>Sonicate this suspension for 10 min&#160;to disperse the beads and homogenize the solution.</li>
	<li>Fibrin Solution &#8212; 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.</li>
	<li>Thrombin Solution &#8212; 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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Seal up the bioreactor, insert the heating blocks, and connect the thermocouples to the temperature controller. Incubate the gel at 37 &#176;C for 15-30 min.</li>
</ol>
4. Cell Explant Preparation
<ol>
	<li>Remove medium from the T-75 flask containing the human dermal fibroblast cells.</li>
	<li>Carefully rinse the surface with approximately 5 ml&#160;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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Create explants by pipetting 0.3 &#181;l 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 &#181;l).</li>
	<li>Allow cells to settle and attach to the fibrin matrix for 1 hr&#160;at 37&#160;&#176;C.</li>
	<li>With the bioreactor still open, add approximately 5 ml&#160;of DMEM supplemented with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin, 0.1% amphotericin B, and 10 mg/ml&#160;aprotinin directly into the bioreactor chamber. DMEM is bicarbonate buffered and requires 5% CO2&#160;to maintain a neutral pH. Since the bioreactor is not supplied with CO2, condition the medium in an incubator with 5% CO2&#160;for 2-3 hr&#160;before use. Aprotinin is a serine protease inhibitor that is widely used to reduce the rate of fibrin degradation20.</li>
	<li>Reseal the bioreactor. Use a syringe to deliver an additional 5 ml&#160;of CO2&#160;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.</li>
	<li>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&#160;or 30 ml&#160;syringe filled with 5% CO2&#160;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&#160;1C).</li>
	<li>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&#160;beaker to collect waste.</li>
	<li>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&#160;1D for reference).</li>
	<li>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 &#176;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&#160;6 will not be accurate.</li>
</ol>
5. Time-lapse Imaging
<ol>
	<li>Once the sample has been prepared, reseal the bioreactor, reconnect the heating blocks and the thermocouples, and set the bioreactor temperature to 37 &#176;C.</li>
	<li>Set the bioreactor onto the microscope mounted motorized stage. Position the 20X&#160;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.</li>
	<li>Open the imaging software.</li>
	<li>Focus the objective on the area between the three explants.</li>
	<li>Save the coordinates (x, y, and z) of this location in the imaging software.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
6. Strain Tracking
For details and instructions on the strain tracking software (Figure&#160;2) used see Raghupathy et al.21&#160;The algorithm is a custom MATLAB code that can be downloaded from <a href="http://www.license.umn.edu/default.aspx">http://www.license.umn.edu/default.aspx</a>. 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.

<ol><li>Grinnell, F., Petroll WM, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cell+motility+and+mechanics+in+three-dimensional+collagen+matrices%5BTitle%5D)+AND+%22Annu.+Rev.+Cell.+Dev.+Biol%22%5BJournal%5D)">Cell motility and mechanics in three-dimensional collagen matrices.</a> Annu. Rev. Cell. Dev. Biol. 26, 335-361 (2001).</li>
<li>Sander, E. A., Barocas, V. H. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aSander%2C+EA+%22Biomimetic+collagen+tissues%3A+collagenous+tissue+engineering+and+other+applications.+In%3A+Collagen%3A+Structure+and+Mechanics%22">Biomimetic collagen tissues: collagenous tissue engineering and other applications. In: Collagen: Structure and Mechanics</a>. , Springer. New York. (2008).</li>
<li>Pedersen JA,, Swartz MA, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanobiology+in+the+third+dimension%5BTitle%5D)+AND+%22Ann.+Biomed.+Eng%22%5BJournal%5D)">Mechanobiology in the third dimension.</a> Ann. Biomed. Eng. 33 (11), 1469-1490 (2005).</li>
<li>Bell, E., Ivarsson, B., Merrill, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Production+of+a+tissue-like+structure+by+contraction+of+collagen+lattices+by+human+fibroblasts+of+different+proliferative+potential+in+vitro%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+U.S.A%22%5BJournal%5D)">Production of a tissue-like structure by contraction of collagen lattices by human fibroblasts of different proliferative potential in vitro.</a> Proc. Natl. Acad. Sci. U.S.A. 76 (3), 1274 (1979).</li>
<li>Guidry, C., Grinnell, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Studies+on+the+mechanism+of+hydrated+collagen+gel+reorganization+by+human+skin+fibroblasts%5BTitle%5D)+AND+%22J.+Cell.+Sci%22%5BJournal%5D)">Studies on the mechanism of hydrated collagen gel reorganization by human skin fibroblasts.</a> J. Cell. Sci. 79 (1), 67-81 (1985).</li>
<li>Fluck, J., Querfeld, C., Cremer, A., Niland, S., Krieg, T., Sollberg, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Normal+human+primary+fibroblasts+undergo+apoptosis+in+three-dimensional+contractile+collagen+gels%5BTitle%5D)+AND+%22J.+Invest.+Dermatol%22%5BJournal%5D)">Normal human primary fibroblasts undergo apoptosis in three-dimensional contractile collagen gels.</a> J. Invest. Dermatol. 110 (2), 153-157 (1998).</li>
<li>Rosenfeldt, H., Grinnell, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fibroblast+quiescence+and+the+disruption+of+ERK+signaling+in+mechanically+unloaded+collagen+matrices%5BTitle%5D)+AND+%22J.+Biol.+Chem%22%5BJournal%5D)">Fibroblast quiescence and the disruption of ERK signaling in mechanically unloaded collagen matrices.</a> J. Biol. Chem. 275 (5), 3088-3092 (2000).</li>
<li>Nakagawa, S., Pawelek, P., Grinnell, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Long-term+culture+of+fibroblasts+in+contracted+collagen+gels%3A+Effects+on+cell+growth+and+biosynthetic+activity%5BTitle%5D)+AND+%22J.+Invest.+Dermatol%22%5BJournal%5D)">Long-term culture of fibroblasts in contracted collagen gels: Effects on cell growth and biosynthetic activity.</a> J. Invest. Dermatol. 93 (6), 792-798 (1989).</li>
<li>Harris, A. K., Stopak, D., Wild, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fibroblast+traction+as+a+mechanism+for+collagen+morphogenesis%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Fibroblast traction as a mechanism for collagen morphogenesis.</a> Nature. 290, 249-251 (1981).</li>
<li>Stopak, D., Harris AK, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Connective+tissue+morphogenesis+by+fibroblast+traction%3A+I.+tissue+culture+observations%5BTitle%5D)+AND+%22Dev.+Biol%22%5BJournal%5D)">Connective tissue morphogenesis by fibroblast traction: I. tissue culture observations.</a> Dev. Biol. 90 (2), 383-398 (1982).</li>
<li>Sawhney, R. K., Howard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Slow+local+movements+of+collagen+fibers+by+fibroblasts+drive+the+rapid+global+self-organization+of+collagen+gels%5BTitle%5D)+AND+%22J.+Cell.+Biol%22%5BJournal%5D)">Slow local movements of collagen fibers by fibroblasts drive the rapid global self-organization of collagen gels.</a> J. Cell. Biol. 157 (6), 1083-1092 (2002).</li>
<li>Provenzano, P. P., Inman, D. R., Eliceiri, K. W., Trier, S. M., Keely, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Contact+guidance+mediated+three-dimensional+cell+migration+is+regulated+by+rho%2FROCK-dependent+matrix+reorganization%5BTitle%5D)+AND+%22Biophys.+J%22%5BJournal%5D)">Contact guidance mediated three-dimensional cell migration is regulated by rho/ROCK-dependent matrix reorganization.</a> Biophys. J. 95 (11), 5374 (2008).</li>
<li>Sander, E., Barocas, V., Tranquillo, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Initial+fiber+alignment+pattern+alters+extracellular+matrix+synthesis+in+fibroblast-populated+fibrin+gel+cruciforms+and+correlates+with+predicted+tension%5BTitle%5D)+AND+%22Ann.+Biomed.+Eng%22%5BJournal%5D)">Initial fiber alignment pattern alters extracellular matrix synthesis in fibroblast-populated fibrin gel cruciforms and correlates with predicted tension.</a> Ann. Biomed. Eng. 39 (2), 714-729 (2011).</li>
<li>Barocas, V. H., Tranquillo, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+anisotropic+biphasic+theory+of+tissue-equivalent+mechanics%3A+the+interplay+among+cell+traction%2C+fibrillar+network+deformation%2C+fibril+alignment%2C+and+cell+contact+guidance%5BTitle%5D)+AND+%22J.+Biomech.+Eng%22%5BJournal%5D)">An anisotropic biphasic theory of tissue-equivalent mechanics: the interplay among cell traction, fibrillar network deformation, fibril alignment, and cell contact guidance.</a> J. Biomech. Eng. 119, 137-146 (1997).</li>
<li>Neidert, M. R., Tranquillo, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tissue-engineered+valves+with+commissural+alignment%5BTitle%5D)+AND+%22Tissue+Eng%22%5BJournal%5D)">Tissue-engineered valves with commissural alignment.</a> Tissue Eng. 12 (4), 891-903 (2006).</li>
<li>Robinson PS,, Robinson PS, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Planar+biaxial+behavior+of+fibrin-based+tissue-engineered+heart+valve+leaflets%5BTitle%5D)+AND+%22Tissue+Eng.+Part+A%22%5BJournal%5D)">Planar biaxial behavior of fibrin-based tissue-engineered heart valve leaflets.</a> Tissue Eng. Part A. 15 (10), 2763-2772 (2009).</li>
<li>Grassl, E., Oegema, T., Tranquillo, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fibrin+as+an+alternative+biopolymer+to+type+I+collagen+for+the+fabrication+of+a+media+equivalent%5BTitle%5D)+AND+%22J.+Biomed.+Mater.+Res%22%5BJournal%5D)">Fibrin as an alternative biopolymer to type I collagen for the fabrication of a media equivalent.</a> J. Biomed. Mater. Res. 60 (4), 607-612 (2002).</li>
<li>Paten, J. A., Zareian, R., Saeidi, N., Melotti, S. A., Ruberti, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Design+and+performance+of+an+optically+accessible%2C+low-volume%2C+mechanobioreactor+for+long-term+study+of+living+constructs%5BTitle%5D)+AND+%22Tissue+Eng.+Part+C+Methods%22%5BJournal%5D)">Design and performance of an optically accessible, low-volume, mechanobioreactor for long-term study of living constructs.</a> Tissue Eng. Part C Methods. 17 (7), 775-788 (2001).</li>
<li>Ye KY,, Sulli van KE,, Black LD, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Encapsulation+of+cardiomyocytes+in+a+fibrin+hydrogel+for+cardiac+tissue+engineering%5BTitle%5D)+AND+%22J.+Vis.+Exp%22%5BJournal%5D)">Encapsulation of cardiomyocytes in a fibrin hydrogel for cardiac tissue engineering.</a> J. Vis. Exp. (55), (2011).</li>
<li>Ahmann, K. A., Weinbaum, J. S., Johnson, S. L., Tranquillo, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fibrin+degradation+enhances+vascular+smooth+muscle+cell+proliferation+and+matrix+deposition+in+fibrin-based+tissue+constructs+fabricated+in+vitro%5BTitle%5D)+AND+%22Tissue+Eng.+Part+A%22%5BJournal%5D)">Fibrin degradation enhances vascular smooth muscle cell proliferation and matrix deposition in fibrin-based tissue constructs fabricated in vitro.</a> Tissue Eng. Part A. 16 (10), 3261-3270 (2010).</li>
<li>Raghupathy, R., Witzenburg, C., Lake, S. P., Sander, E. A., Barocas, V. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identification+of+regional+mechanical+anisotropy+in+soft+tissue+analogs%5BTitle%5D)+AND+%22J.+Biomech.+Eng%22%5BJournal%5D)">Identification of regional mechanical anisotropy in soft tissue analogs.</a> J. Biomech. Eng. 133, (2011).</li>
<li>Robinson, P. S., Johnson, S. L., Evans, M. C., Barocas, V. H., Tranquillo, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Functional+tissue-engineered+valves+from+cell-remodeled+fibrin+with+commissural+alignment+of+cell-produced+collagen%5BTitle%5D)+AND+%22Tissue+Eng.+Part+A%22%5BJournal%5D)">Functional tissue-engineered valves from cell-remodeled fibrin with commissural alignment of cell-produced collagen.</a> Tissue Eng. Part A. 14 (1), 83-95 (2008).</li>
<li>Gabbiani, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+myofibroblast+in+wound+healing+and+fibrocontractive+diseases%5BTitle%5D)+AND+%22J.+Pathol%22%5BJournal%5D)">The myofibroblast in wound healing and fibrocontractive diseases.</a> J. Pathol. 200 (4), 500-503 (2003).</li>
<li>PaszeK, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tensional+homeostasis+and+the+malignant+phenotype%5BTitle%5D)+AND+%22Cancer+Cell%22%5BJournal%5D)">Tensional homeostasis and the malignant phenotype.</a> Cancer Cell. 8 (3), 241-254 (2005).</li>
<li>Grinnell, F., Rocha L, B., Iucu, C., Rhee, S., Jiang, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nested+collagen+matrices%3A+A+new+model+to+study+migration+of+human+fibroblast+populations+in+three+dimensions%5BTitle%5D)+AND+%22Exp.+Cell.+Res%22%5BJournal%5D)">Nested collagen matrices: A new model to study migration of human fibroblast populations in three dimensions.</a> Exp. Cell. Res. 312 (1), 86-94 (2006).</li>
<li>Miron-Mendoza, M., Seemann, J., Grinnell, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+differential+regulation+of+cell+motile+activity+through+matrix+stiffness+and+porosity+in+three+dimensional+collagen+matrices%5BTitle%5D)+AND+%22Biomaterials%22%5BJournal%5D)">The differential regulation of cell motile activity through matrix stiffness and porosity in three dimensional collagen matrices.</a> Biomaterials. 31 (25), 6425-6435 (2010).</li>
<li>Wakatsuki, T., Kolodney MS,, Zahalak GI,, Elson EL, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cell+mechanics+studied+by+a+reconstituted+model+tissue%5BTitle%5D)+AND+%22Biophys.+J%22%5BJournal%5D)">Cell mechanics studied by a reconstituted model tissue.</a> Biophys. J. 79 (5), 2353-2368 (2000).</li>
<li>De Jesus, A., Aghvami, M., Sander, E. Fibroblast-mediated fiber realignment in fibrin gels. ASME Summer Bioengineering Conference, 2013 Jun 26-29, Sunriver, OR, , (2013).</li>
<li>Aghvami, M., Barocas, V. H., Sander, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multiscale+mechanical+simulations+of+cell+compacted+collagen+gels%5BTitle%5D)+AND+%22J.+Biomech.+Eng%22%5BJournal%5D)">Multiscale mechanical simulations of cell compacted collagen gels.</a> J. Biomech. Eng. 135, (2013).</li>
</ol>

No conflicts of interest declared.

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 tissues2,22, reducing scar1,23, and understanding pathological tissue remodeling12,24. 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...

An important tool for studying cell-matrix interactions is the cell populated collagen gel1,2. 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 gel4,5. The contractile fibroblasts in free floating gels then transition into a quiescent state soon after the gel has fully reached compaction1,6,7. The fibroblasts in gels that are constrained at the boundaries remain in an active, synthetic state8&#160;and they generate fiber alignment in a manner dependent on gel geometry and external constraints5,9. 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.&#160;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 &#8212; which are dependent primarily on the interplay between cell traction forces, cell spatial distribution, gel geometry, and the mechanical constraints on the gel &#8212; 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 tissue14,15. 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.

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

observing and quantifying fibroblast-mediated fibrin gel compaction

Observing and Quantifying Fibroblast-mediated Fibrin Gel Compaction

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.

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...

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&#160;period.

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 ...

The overall goal of this procedure is to observe and analyze the effect of cell matrix interactions on fiber realignment and matrix remodeling activities in a fibrin gel system. This is accomplished by first polymerizing, a three dimensional fibrin gel containing microbeads onto cover glass contained within an environmentally controlled bioreactor. The second step is to prepare a highly concentrated cell suspension that is used to position clusters of fibroblasts or X explants into geometric patterns on the surface of the gel.Next, the sample is imaged every 15 minutes using differential interference contrast microscopy over the course of 48 hours. The final step is to measure the extent of fibrin reorganization and remodeling in the form of strain from different regions of the gel using digital image correlation. Ultimately, this explant system allows one to arrange the cells into simple geometric patterns, which makes it easier to visualize and probe the mechanisms underlying cell driven matrix remodeling and fiber realignment.This method can answer key questions in the field of mechanical biology, such as what are the effects of very spatial and temporal mechanical cues on cell behavior and the resulting changes these generate in the surrounding tissue matrix. Demonstrate in this procedure will be Arabic de Jesus, a graduate student from my lab Gently dissolve fibrinogen into prewarm saline at 37 degrees Celsius overnight to create a 33.3 milligram per milliliter stock solution. Prepare a stencil on perfil to lay out the location of each x explant following the desired geometry space.Each dot approximately one to two millimeters apart. This distance corresponds to an ideal spacing for generating fiber alignment between explants. Next, thoroughly clean all components of the bioreactor using 70%ethanol and place the cleaned components under the UV of a tissue culture hood for two to three hours prior to mentation.After the components of the bioreactor have been sterilized, attach the stencil underneath the area of the 60 millimeter diameter cover glass where the sample will be prepared using tape. Next, prepare the microbead suspension, followed by the fibrinogen and thrombin. Stock solutions combine 0.017 milliliters of the Microbead stock solution and 0.149 milliliters of DMEM into a micro centrifuge tube to achieve a concentration of 10 million beads per milliliter.Then sonicate the suspension for 10 minutes to disperse the beads and homogenize the solution. Add 0.22 milliliters of the fibrinogen stock solution to 0.44 milliliters of 20 millimolar heaps buffer, and 0.1667 milliliters of the microbead solution in a 15 milliliter centrifuge tube. Gently mix the components and then place the tube on ice in a separate 15 milliliter centrifuge tube.Mixed together. 32.8 microliters of thrombin dissolved in saline at 25 units per milliliter. 131 microliters of 20 millimolar heaps buffer, and 2.46 microliters of two molar calcium chloride.Next carefully mix the prepared thrombin and fibrinogen solutions by pipetting up and down five to 10 times until the solution is evenly distributed. Try to minimize introducing bubbles to the mixture by not fully discharging the pipette while mixing. Pipette the mixed solution into eight millimeters square PDMS molds on cover glass as soon as possible, allow the gel to polymerize at room temperature.The addition of thrombin will cost the solution to gel quickly in around 30 seconds following polymerization, seal the bioreactor and prepare the cell explants to begin harvest 70 to 90%confluent human dermal fibroblast cells from a T 75 flask using trypsin and spin them down at 200 times G for five minutes. Transfer the bioreactor to a biosafety cabinet and carefully remove the lid following aseptic conditions. Then aspirate off the media and resuspend the cell pellet to a final concentration of 20 million cells per milliliter in DMEM with 10%FBS.Next, create explants by loading cells into a micro pipette and injecting 0.3 microliters of the cell suspension into the polymerized fibrin gel. Following the pattern on the stencil, each explan should contain approximately 6, 000 cells. Once the explants have been positioned on the gel seal the bioreactor.Next, insert the heating blocks and connect the thermocouples to the temperature controller. Set the temperature controller to 37 degrees Celsius and incubate the gels for one hour to allow the cells to settle and attach to the fibrin matrix. Then at approximately five milliliters of DMEM that has been conditioned for two to three hours with 5%carbon dioxide and supplemented with 10%fetal bovine serum 1%penicillin streptomycin 0.1%amphotericin B and 10 micrograms per milliliter, aprotinin directly into the bioreactor chamber.Then reseal the bioreactor. Use a syringe to deliver the additional five milliliters of carbon dioxide conditioned medium via the barbed fitting on the inlet port to completely fill the bioreactor carefully remove any bubbles that form set up a syringe pump with a 10 milliliter or 30 milliliter syringe filled with additional 5%carbon dioxide conditioned medium. Connect the syringe directly to the inlet port on the bioreactor lid with lure lock fitted sterile tubing.Set the perfusion rate on the syringe pump to 0.01 milliliters per minute. Then connect modified pieces of tubing to both outlet ports and place the ends into a 100 milliliter beaker. To collect waste, 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 supply.Fresh 5%carbon dioxide conditioned medium to the bioreactor throughout the experiment. In order to maintain pH supply nutrients and remove waste products, a 10 milliliter syringe filled with medium will last about 16 hours. Position the 20 XDIC objective under the view port of the microscope and ensure that the polarizer analyzer and prism are all in place.Then open the imaging software. Focus the objective on the area between the three x explan and save the X, Y, and Z coordinates of this location in the imaging software. In order to image the entire area between and around the x explan, 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 large tiled image, set the light intensity and exposure time to the lowest values possible. This will help to avoid cell death caused by phototoxicity while still providing sufficient resolution to discriminate between cells, microbeads and fibrin fibers. Note that in this particular experiment, the fibrin fibers are not visible due to the high density of light scattering beads in the gel, reduce the bead density or gel thickness in order to observe fibrin fibers.Next, begin strain tracking. By first creating or downloading custom MATLAB code, then register images using microbeads throughout the gel as reference points. Registering the image will ensure that the images obtained are taken at the exact same location each time.Fiber realignment was observed between explan by capturing tiled images every 15 minutes. Over a 48 hour period, individual regions were extracted from the tiled images and analyzed with the strain tracking algorithm. In order to measure local strains from microbead displacement contour plots show the distribution of maximum principle strain in the area of fiber realignment corresponding to the strap.Once mastered, this technique can be completed in approximately three To four hours if performed properly. Following this procedure, other methods like fluorescent labeling of collagen and cytoskeletal proteins as well as quantification of cell migration can be performed in order to answer additional questions like what mechano biological pathways are involved in tissue remodeling, and how are these processes modulated by various biochemicals.

observing-and-quantifying-fibroblast-mediated-fibrin-gel-compaction

Research

JoVE Journal

Bioengineering

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Observing and Quantifying Fibroblast-mediated Fibrin Gel Compaction

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