This work was funded by Lawrence Livermore National Lab LDRD 18-ERD-062 (to C.R.). Thanks to John Muschler for his kind gift of the DgKO and DgKI cells. Thank you to the staff at the University of California Berkeley Electron Microscope Laboratory for advice and assistance in electron microscopy sample preparation and data collection.

1. Thaw and aliquot laminin-111
NOTE: Purified laminin-111 is available commercially (typically purified from EHS matrix sold as Matrigel), but not all sources provide intact, nondegraded protein.
<ol>
	<li>Confirm that Ln1 is not degraded by sourcing Ln1 and running size exclusion chromatography26 or polyacrylamide gel electrophoresis26. Denatured Ln1 should have bands at 337 kDa, 197 kDa and 177 kDa, and native Ln1 should have a single band at 711 kDa.</li>
	<li>Thaw Ln1 on ice in a refrigerator overnight, and aliquot into low protein binding microcentrifuge tubes (typically aliquot at 100 &#181;g/tube for culture overlays, or 10 &#181;g for coatings) using low protein binding tips and sterile technique27. Freeze aliquots and store at -80 &#176;C.</li>
</ol>
2. Laminin polymerization using low pH buffers

<ol>
	<li>Make polyLM polymerization buffer: Weigh out and mix 1 mM CaCl2 and 20 mM sodium acetate in ultrapure water. Then adjust the pH to 4.0, using HCl or acetic acid. Sterile filter through 0.2 &#181;m filter, and store at 4 &#176;C.</li>
	<li>Make control polymerization buffer: Mix 1 mM CalCl2 and 20 mM Tris and adjust the pH to 7.0, using 10 N HCl or 12 N NaOH as necessary. Sterile filter through 0.2 &#181;m filter, and store at 4 &#176;C. 
	NOTE: Protocol can be paused here.</li>
	<li>Thaw the needed number of Ln-1 aliquots in a refrigerator overnight.</li>
	<li>Using sterile cell culture technique, add the appropriate buffer (either polyLM polymerization buffer from step 2.1 or control polymerization buffer from step 2.2) to laminin aliquots at a final concentration of 100 &#181;g/mL (i.e. add 0.5 mL to 50 &#181;g aliquots) and mix gently by pipetting.</li>
	<li>If using fluorescently tagged laminin for visualization, reconstitute and add at 1:50 wt/wt ratio (e.g. 2 &#181;g/100 &#181;g) and mix gently by pipetting. 
	NOTE: If using laminins as a substrate for cell attachment, follow steps 2.6-2.8:</li>
	<li>Immediately after dilution in appropriate buffer, transfer laminins onto culture plastic or glassware and incubate overnight in a cell culture incubator at 37 &#176;C. Note that when coating coverslips, add a sufficiently large volume as to produce an equilibrated drop (typically, use 50 &#181;L for a 13 mm round coverslip), and keep in a humidified chamber overnight at 37 &#176;C.</li>
	<li>Using sterile technique27, wash with 1x phosphate buffered saline (PBS).</li>
	<li>Remove the liquid carefully. Do not allow surface to be completely dry. 
	NOTE: If using laminins to cover cells for milk protein induction, follow steps 2.9-2.12:</li>
	<li>Culture mammary epithelial cells. Culture MepG dystroglycan knock in (DgKI) or MepG dystroglycan knockout cells (DgKO) cells in DMEM-F12 supplemented with 2% FBS, 0.01 mg/mL insulin, 0.005 &#181;g/mL EGF, 0.05 mg/mL gentamycin, and 0.05 g/mL normocin.</li>
	<li>Seed DgKO and DgKI cells for laminin stimulation at 10,500 cells/cm2 in well plates or coverslip bottom dishes</li>
	<li>Incubate diluted laminins in a cell culture incubator at 37 &#176;C for 30 min.</li>
	<li>Centrifuge polyLM aliquots at 2,000 x g for 20 minutes, but centrifuge LM in neutral pH at 20,000 x g for 20 minutes due to the smaller size of the protein entities and need for higher speeds to collect effectively.</li>
	<li>Using sterile technique27, remove the supernatant gently, and resuspend in an appropriate volume of induction medium of DMEM-F12 supplemented with 3 &#181;g/mL prolactin, 2.8 &#181;M hydrocortisone, and 5 &#181;g/mL insulin</li>
	<li>Transfer laminin in cell culture medium immediately to stimulate cells. We have successfully stimulated cells with 50-800 &#181;g/mL Ln1</li>
</ol>
3. Laminin polymerization using isolated glycoproteins
<ol>
	<li>Thaw recombinant nidogen-1 and Ln1 gently on ice in the refrigerator overnight.</li>
	<li>Mix Ln1 with nidogen-1 at a 1:1 wt/wt ratio in cell culture medium using low protein binding tips and tubes and using sterile technique. Use pure Ln1 in cell culture medium as a control.</li>
	<li>If using fluorescently tagged laminin for visualization, reconstitute and add at 1:50 wt/wt ratio (e.g., 2 &#181;g/100 &#181;g). Gently mix by pipetting.</li>
	<li>Incubate Ln1-nidogen or control Ln1 in a cell culture incubator at 37 &#176;C for 30 min.</li>
	<li>Centrifuge Ln1-nidogen copolymers at 2,000 x g for 30 min and the control Ln1 at 20,000 x g for 30 min.</li>
	<li>Gently remove the supernatant and resuspend in cell culture medium to a final concentration of 100-800 &#181;g total protein/mL.&#160; &#160;</li>
	<li>Use immediately to stimulate cells, or transfer to coverslips and allow to adhere overnight at 37 &#176;C in humidified cell culture incubators.</li>
	<li>Remove culture medium from coverslips, wash coverslips with 1x PBS, and then fix with 10% formalin (i.e., 4% formaldehyde in PBS) for 15 minutes at room temperature.</li>
</ol>
4. Laminin polymerization using dystroglycan expressing cells
<ol>
	<li>Culture mammary epithelial cells. Culture MepG dystroglycan knock in (DgKI) or MepG dystroglycan knockout cells (DgKO) cells in DMEM-F12 supplemented with 2% FBS, 0.01 mg/mL insulin, 0.005 &#181;g/mL EGF, 0.05 mg/mL gentamycin, and 0.05 g/mL normocin.</li>
	<li>Seed DgKI cells at 5,000 cells/cm2, and seed DgKO cells at 8,000 cells/cm2 due to differences in growth rates. Culture at 37 &#176;C in humidified incubators with media changes every 2-3 days and passages every 4-5 days using a rigorous sterile technique. Count cells carefully with a hemocytometer or automated cell counter27 to ensure appropriate growth rates.</li>
	<li>Seed cells for laminin stimulation at 10,500 cells/cm2 in well plates or coverslip bottom dishes and culture for 2 days, using DgKI cells to generate fractal Ln1 and DgKO cells as negative controls. Thaw Ln-1 gently overnight on ice in a refrigerator, and then mix with induction medium of DMEM-F12 supplemented with 3 &#181;g/mL prolactin, 2.8 &#181;M hydrocortisone, and 5 &#181;g/mL insulin. For a 35 mm dish, use 1 mL of induction media with 50-800 &#181;g of Ln1.</li>
	<li>If using fluorescent Ln1, mix in at 50:1 wt/wt ratio.</li>
	<li>Remove media from cells, and cover with laminin and induction media of DMEM-F12 supplemented with 3 &#181;g/mL prolactin, 2.8 &#181;M hydrocortisone, and 5 &#181;g/mL insulin &#160;</li>
	<li>Culture cells for 2 days, and then fix with 10% formalin.</li>
</ol>
5. Characterization of laminin microstructure via optical microscopy
<ol>
	<li>Collect fixed samples and wash 3 times with 1x PBS at room temperature for 5 min.</li>
	<li>Block and permeabilize samples in 1x PBS with 0.5% Triton X-100, 3% bovine serum albumin, 10% goat serum, and 40 &#181;g/mL anti-mouse blocking antibody fragment for 1 h at room temperature in a humidified chamber. 
	NOTE: Triton X-100 is hazardous. Handle with gloves and eye protection and dispose of appropriately.</li>
	<li>Mix anti-laminin antibody at a dilution of 1:100 with 1x PBS with 0.5% Triton X-100 and 3% BSA and add to samples for 2 h at room temperature or overnight at 4 &#176;C in a humidified chamber (typically use tip boxes with a wet laboratory wipe in the bottom).</li>
	<li>Remove primary antibody and wash 3 times with 1x PBS with 0.5% Triton X-100 and 3% BSA.</li>
	<li>Add an appropriate secondary antibody at a dilution of 1:500 to samples in 1x PBS with 0.5% Triton X-100 and 3% BSA and incubate at room temperature in the dark in a humidified chamber.</li>
	<li>Remove secondary antibody and wash 3 times with 1x PBS with 0.5% Triton X-100 and 3% BSA.</li>
	<li>For samples containing cells, add 6 &#181;M phalloidin for 45 min, followed by 3 washes with PBS with 0.5% Triton X-100 and 3% BSA, and followed by 3 washes with PBS. Then stain with 0.1 &#181;g/mL DAPI in PBS for 5 minutes.</li>
	<li>Mount samples if appropriate.</li>
	<li>Image ln-1 structure using high resolution confocal microscopy. Use a laser scanning microscope equipped with immersion objectives (water immersion Plan Apochromat 40X/1.1NA or oil immersion Plan Apochromat 63X/1.4NA).</li>
</ol>
6. Characterization of laminin constructs via box counting dimension
<ol>
	<li>Analyze for fractal properties using box counting dimension algorithms available in Matlab toolboxes or in ImageJ. These methods threshold images of laminin and plot on a log-log plot the number of boxes containing Ln1 compared to the size of the boxes. The slope of the relationship between these parameters is the box-counting dimension: non-fractal geometries will have a dimension of 0, 1, or 2, whereas fractal geometries have a non-integer dimension28. 
	NOTE: Acid-treated, glycoprotein-treated and DgKI cell-attached Ln1 all have a box-counting fractal dimension of ~1.7, whereas control Ln1 has a fractal dimension of 2.0.</li>
</ol>
7. Characterization of laminin constructs via electron microscopy
<ol>
	<li>Resuspend laminins in cell culture media and allow to adsorb to silica wafers for 90 min in a humid 37 &#176;C environment.</li>
	<li>Fix matrices in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer at pH 7.2. 
	NOTE: Glutaraldehyde is hazardous and should be handled in a fume hood with gloves and eye protection and disposed of as hazardous waste.</li>
	<li>Postfix matrices in 1% osmium tetroxide in 0.1 M sodium cacodylate buffer at pH 7.2. 
	NOTE: Osmium tetroxide is hazardous and should be handled in a fume hood with gloves and eye protection and disposed of appropriately. Cacodylate is hazardous and should be handled in a fume hood with gloves and eye protection and disposed of appropriately.</li>
	<li>Wash 3 times in sodium cacodylate buffer at pH 7.2.</li>
	<li>Dehydrate with increasing concentrations of ethanol.</li>
	<li>Sputter coat matrices with a thin layer of gold.</li>
	<li>Image using scanning electron microscopy.</li>
</ol>

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This work was performed under the auspices of the U.S. Department of Energy by Lawrence Livermore National Laboratory under Contract DE-AC52-07NA27344. IM release LLNL-JRNL-799443.

Laminins represent a key element of the ECM in epithelial, muscular, and neural organ systems, and have been shown in vitro to play essential roles in regulating the functional differentiation of cells. Growing evidence indicates that the structure of laminin displayed to cells regulates its signaling and resultant cell phenotype15,16, thus methods to control Ln1 microstructure should be of interest to basic biologists working in these fields, and to tissue engineers seeking to recapitulate normal behavior in vitro. More generally, the microstructure of a variety of extracellular matrix proteins is known to regulate biological response and understanding the mechanisms by which microstructure modulates cell function is an active area of research.
In this work, we describe three methods to polymerize laminin into networks with fractal microstructure characteristic of diffusion limited aggregation. These Ln1 polymers have been shown to signal differently to cells compared to aggregated Ln1, potentially due to altered adhesive domain display to cells. These three methods to control Ln1 microstructure may represent biological redundancy in assembly of Ln1, but this represents a challenge for experimental work. Controlling Ln1 microstructure using acidic buffer treatment will only show effects when added to dystroglycan knockout cells19,20. Thus, experimental design must consider the cells and the purity of Ln1 to be used. Commercially available Ln1 is commonly derived from EHS matrix, which contains a high percentage of nidogens and other glycoproteins; Ln1 in EHS matrix polymerizes correctly into fractal networks in neutral buffered systems. Recombinant Ln1 has recently become available commercially but is exceptionally costly.
Ln1 protein quality is crucial for this work as short arm fragments can block polymerization. Short arm fragments (i.e., the E4 fragment) interfere with network formation as each fragment induces a void in the network29. Previous data shows that inclusion of E4 fragments can block Ln1 induced phenotypes29,30. Likewise, we have found that network formation is disrupted by small quantities of laminin-332 (unpublished data), which like E4 fragments contains a single adhesive domain29,30. Thus, it is crucial to check that Ln1 is intact, and highly purified to remove other laminin species before starting work.
While these methods are relevant to researchers studying cell-matrix interactions, it should be noted that the laminin family is highly complex, involving 15 known members and additional splice variants31, and this family interacts with a wide range of receptors, glycoproteins, collagens and potentially growth factors4. Thus, polymerized Ln1 represents a reductionist state which can be expected to be modified rapidly by cell function. Furthermore, different organ systems sense, respond to, and remodel laminins differently: we have demonstrated that linkage of laminin to actin through dystroglycan is dispensable for the mammary gland19, whereas dystroglycan is absolutely necessary for myocyte function32.

Unlike growth factors or cytokines, extracellular matrix (ECM) proteins can assemble into structural networks. As ECM dysfunction is a key element of many disease conditions, the mechanisms by which cells sense and transduce signals from ECM composition, microstructure and biomechanics are attractive targets for therapeutic development. Growing evidence suggests that microstructure of these networks plays a major role in how these proteins signal to cells. To date, this linkage between ECM microstructure and cell function has been shown for collagen I1, fibronectin2, and fibrin3.
Laminin-111 (Ln1) is a trimeric, cross-shaped protein that is a key structural component of the extracellular matrix that contacts epithelial cells4, muscle cells5, and neural cells6. In the mammary gland, Ln1 is necessary for functional differentiation of mammary gland epithelial cells into milk producing cells 7,8,9, and Ln1 is crucial for induction of tumor reversion10,11. Ln1 and other laminins are necessary for development of cortical actin networks and sarcolemma organization in muscle12,13, and for neural cell migration and neurite outgrowth13 . Thus, understanding the mechanisms by which laminin signals to cells is an active area of research.
Ln1 contains multiple adhesive domains for a broad range of receptors including integrins, syndecans, dystroglycan, LBP-110 and LamR (Figure 1A)4,14. The E8 fragment, which contains the c-terminus globular domains of laminin &#945;1, is necessary for binding dystroglycan and integrins &#945;6&#946;1 and &#945;3&#946;115, and it is necessary for functional differentiation of epithelial cells15,16. In contrast, the short arms show different biological activity17, meaning that availability for recognition of these different Ln1 domain after network formation could alter cell behavior.
We have recently demonstrated that Ln1 arranged into a polymeric network exhibits fractal properties (i.e., a structure that is self-similar across multiple length scales)18. Fractal networks signal differently to epithelial cells compared to Ln1, which lacks this arrangement19. This fractal network indicates a particular assembly structure, where the long arm is preferentially exposed to cells18. As a result of this different long arm display, epithelial cells undergo functional differentiation into milk producing cells with well-organized tight junctions and suppression of actin stress fibers19.
We describe 3 methods to generate Ln1 networks from short arm-short arm binding interactions, which show a high display of the Ln1 long arm: 1) by cell-free assembly with acidic buffers, 2) by co-incubation with glycoproteins, or 3) by interaction with cell surface dystroglycan. These three methods generate similar laminin microstructures through three very different mechanisms, permitting flexibility in experimental design. Previous work suggests that these three methods could represent biological robustness: in mammary gland epithelia, either cell surface dystroglycan, glycoproteins, or artificially structured laminin can induce functional differentiation19. Thus, researchers can choose between these methods based on the study subject: dystroglycan expressing cells show no sensitivity to Ln-1 microstructure, as dystroglycan in the cell membrane induces correct polymerization into a fractal network19,20. Matrigel, a mix of laminins and glycoproteins21, represents the most economical source of Ln-1, and offers the necessary microstructure to induce dystroglycan knockout mammary cells to differentiate20, but contains a complex mixture of proteins with lot to lot variability. PolyLM represents the cleanest, most reductionist system that provides this function, but at increased cost and lower efficacy to induce mammary cell differentiation compared to Matrigel19.
These three methods have a fractal network structure characteristic of diffusion limited aggregation18,19, and show altered biological activity compared to aggregated laminin in multiple experimental systems22,23,24,25. Future studies using these methods could identify the receptors and signaling necessary for epithelial differentiation and determine to restore normal cell-matrix interactions in cells in abnormal microenvironments20, such as tumors.

<table><tbody><tr><td>10% formalin</td><td>Sigma Aldrich</td><td>HT501128</td><td></td></tr><tr><td>Anti-Laminin primary antibody</td><td>Sigma Aldrich</td><td>L9393</td><td></td></tr><tr><td>Anti-Rabbit Alexafluor 488 secondary antibody</td><td>Thermo</td><td>A32731</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma Aldrich</td><td>A9418</td><td></td></tr><tr><td>CaCl2</td><td>Sigma Aldrich</td><td>C4901 or similar</td><td></td></tr><tr><td>Cell Culture Incubator</td><td>Thermo</td><td>Heracell 150 or similar</td><td></td></tr><tr><td>Centrifuge for microcentrifuge tubes</td><td>Eppendorf</td><td>5418 or similar</td><td></td></tr><tr><td>Confocal Microscope</td><td>Zeiss</td><td>LSM 710 or equivalent</td><td></td></tr><tr><td>Coverslips</td><td>Thermo</td><td>25CIR-1 or similar</td><td></td></tr><tr><td>DMEM-F12, Liquid, High Glucose, +HEPES, L-glutamine, +Phenol red</td><td>Thermo</td><td>11330107</td><td></td></tr><tr><td>EGF (Epidermal Growth Factor)</td><td>Sigma Aldrich</td><td>11376454001</td><td></td></tr><tr><td>Fluorescently Labeled Laminin</td><td>Cytoskeleton Inc</td><td>LMN02</td><td></td></tr><tr><td>Gentamicin</td><td>VWR</td><td>VWRV0304-10G</td><td></td></tr><tr><td>Glutaraldehyde</td><td>Sigma Aldrich</td><td>G5882</td><td></td></tr><tr><td>HCl</td><td>Sigma Aldrich</td><td>320331 or similar</td><td></td></tr><tr><td>Hydrocortisone</td><td>Sigma-Aldrich</td><td>H0888-5G</td><td></td></tr><tr><td>Insulin</td><td>Sigma Aldrich</td><td>16634-50mg</td><td></td></tr><tr><td>MepG Dystroglycan Knockout Cells</td><td>LBNL</td><td>N/A</td><td></td></tr><tr><td>NaOH</td><td>Sigma Aldrich</td><td>S8045 or similar</td><td></td></tr><tr><td>Normocin</td><td>Invivogen</td><td>ant-nr-1</td><td></td></tr><tr><td>Osmium Tetroxide</td><td>Sigma Aldrich</td><td>75633</td><td></td></tr><tr><td>Ovine Prolactin</td><td>Los Angeles Biomedical Research Institute</td><td>Ovine Pituitary Prolactin</td><td></td></tr><tr><td>Phalloidin</td><td>Thermo</td><td>A22287</td><td></td></tr><tr><td>Phosphate Buffered Saline</td><td>Thermo</td><td>10010023 or similar</td><td></td></tr><tr><td>Purified Laminin-111</td><td>Sigma Aldrich</td><td>L2020-1MG</td><td></td></tr><tr><td>Recombinant human Nidogen-1, carrier free</td><td>R&amp;amp;D systems</td><td>2570-ND-050</td><td></td></tr><tr><td>Sodium Acetate</td><td>Sigma Aldrich</td><td>S2889 or similar</td><td></td></tr><tr><td>Sodium Cacodylate</td><td>Sigma Aldrich</td><td>C0250</td><td></td></tr><tr><td>Sterile filters</td><td>Millipore</td><td>SLGP033RS or similar</td><td></td></tr><tr><td>Tris</td><td>Sigma Aldrich</td><td>252859 or similar</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>X100</td><td></td></tr><tr><td>Ultra-low protein binding tubes</td><td>VWR</td><td>76322-516, 76322-522 or similar</td><td></td></tr><tr><td>Ultrapure water</td><td>Thermo</td><td>15230253 or similar</td><td></td></tr></tbody></table>

generating a fractal microstructure of laminin-111 to signal to cells

Laminin-111 (Ln1) is an essential part of the extracellular matrix in epithelia, muscle and neural systems. We have previously demonstrated that the microstructure of Ln1 alters the way that it signals to cells, possibly because Ln1 assembly into networks exposes different adhesive domains. In this protocol, we describe three methods to generate polymerized Ln1.

Laminin-111 is a trimeric protein with three self-assembling domains at the end of its short arms (Figure 1A). When treated suitably, the short arms can polymerize into a hexagonal lattice (Figure 1B,1C), which displays diffusion-limited aggregate fractal properties at larger spatial scales (Figure 2). Laminin incubated in calcium-containing neutral-pH buffers forms small aggregates visualized with an anti-Ln1 antibody (Figure 2A), which have a box counting fractal dimension of ~2, indicating no fractality. In contrast, Ln1 incubated in calcium-containing pH4 buffer (Figure 2B), or in neutral buffer with nidogen-1 (Figure 2C) form space filling lacy networks with a fractal dimension of ~1.7 (Figure 3). Ln1 cultured with dystroglycan expressing cells forms similar lacy networks (Figure 2D, inset shows cell nuclei).
When pH7Ln and polyLM are imaged with SEM with increasing resolution, the differences in microstructure become more apparent. At low resolution (Figure 2Ei and v), polyLM networks appear more spread compared to pH7-Ln, and at high resolution, pH7-Ln aggregates are apparent (Figure 2E iv) whereas a spread hexagonal lattice can be seen in polyLM at high resolution (Figure 2E viii).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61134/61134fig1.jpg" /> 
Figure 1: Background of Ln1 and polymerization method: A: Ln1 is a complex trimeric protein with multiple adhesive domains (receptor binding sites noted), which can bind a variety of integrin and non-integrin receptors (in black) and form polymeric networks by short arm-short arm binding (in tan). B: Ln1 can form a hexagonal lattice in the presence of calcium ions and either low pH, glycoproteins such as nidogen-1 or cell surface dystroglycan. C: example SEM image of polymerized Ln1 showing hexagonal lattice and proposed Ln1 assembly model. <a href="https://www.jove.com/files/ftp_upload/61134/61134fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61134/61134fig2.jpg" /> 
Figure 2: Representative data for Ln1 polymerization. A: Ln1 aggregates in neutral buffers with calcium. When visualized with an anti-Ln1 antibody, small aggregates can be seen. B: Ln1 in acidic calcium containing buffer polymerizes into polyLM, which shows fractal properties. C: Ln1- nidogen-1 mixture (1:1 wt/wt) forms similar polymers. D: Ln1 in neutral buffered cell culture media overlaid on dystroglycan expressing cells shows a similar lacy network. Inset: nuclear counterstaining showing cell morphology. E: Scanning electron microscopy of pH7-Ln and polyLM networks (spin down method) with increasing resolution. In pH7-Ln, low resolution scans (i &#38; ii) show relatively compact fibers, which resolve as aggregates in high resolution scans (iii). polyLM at the same concentration is much more spread (iv&#38;v), and high-resolution scans show a hexagonal, space filling lattice (vii). F: This microstructural change causes DgKO mammary gland epithelial cells to functionally differentiate into milk producing cells19. i: pH7-Ln stimulated cells show abundant actin stress fibers (red) and disorganized zo-1 containing tight junctions (green), whereas ii: polyLM stimulated cells contain less total filamentous actin, lateral localization of actin into the cell-cell boundary and well organized zo-1 containing tight junctions. <a href="https://www.jove.com/files/ftp_upload/61134/61134fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61134/61134fig3.jpg" /> 
Figure 3: Fractal dimension estimation with the box counting method. A&#38;B: Raw image of pH7-Ln and polyLM show differences in morphology. C&#38;D: these images are thresholded to give rise to a black and white image, E&#38;F: then the log of the ratio of the number of boxes containing the image to the size of the box is calculated. The average slope represents the box counting dimension Fd is calculated. pH7-Ln has a characteristic dimension of 2.0, indicating no fractal properties, whereas polyLM has a dimension of ~1.7, indicative of a diffusion limited aggregation process. <a href="https://www.jove.com/files/ftp_upload/61134/61134fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Generating a Fractal Microstructure of Laminin-111 to Signal to Cells at JoVE.com

Generating a Fractal Microstructure of Laminin-111 to Signal to Cells

We describe three methods to generate Ln1 polymers with fractal properties that signal to cells differently compared to unpolymerized Ln1.

Laminin-111 (Ln1) is an essential part of the extracellular matrix in epithelia, muscle and neural systems. We have previously demonstrated that the ...

generating-a-fractal-microstructure-of-laminin-111-to-signal-to-cells

Universidad Científica del Sur

Research

JoVE Journal

Bioengineering

985 Views.  Texas Heart Institute. We describe three methods to generate Ln1 polymers with fractal properties that signal to cells differently compared to unpolymerized Ln1.

Video: Generating a Fractal Microstructure of Laminin-111 to Signal to Cells

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

Preparation of Hydroxy-PAAm Hydrogels for Decoupling the Effects of Mechanotransduction Cues

It is now well established that many cellular functions are regulated by interactions of cells with physicochemical and mechanical cues of their extracellular matrix (ECM) environment. Eukaryotic cells constantly sense their local microenvironment through surface mechanosensors to transduce physical changes of ECM into biochemical signals, and integrate these signals to achieve specific changes in gene expression. Interestingly, physicochemical and mechanical parameters of the ECM can couple with each other to regulate cell fate. Therefore, a key to understanding mechanotransduction is to decouple the relative contribution of ECM cues on cellular functions.
Here we present a detailed experimental protocol to rapidly and easily generate biologically relevant hydrogels for the independent tuning of mechanotransduction cues in vitro. We chemically modified polyacrylamide hydrogels (PAAm) to surmount their intrinsically non-adhesive properties by incorporating hydroxyl-functionalized acrylamide monomers during the polymerization. We obtained a novel PAAm hydrogel, called hydroxy-PAAm, which permits immobilization of any desired nature of ECM proteins. The combination of hydroxy-PAAm hydrogels with microcontact printing allows to independently control the morphology of single-cells, the matrix stiffness, the nature and the density of ECM proteins. We provide a simple and rapid method that can be set up in every biology lab to study in vitro cell mechanotransduction processes. We validate this novel two-dimensional platform by conducting experiments on endothelial cells that demonstrate a mechanical coupling between ECM stiffness and the nucleus.

We present a new polyacrylamide hydrogel, called hydroxy-PAAm, that allows a direct binding of ECM proteins with minimal cost or expertise. The combination of hydroxy-PAAm hydrogels with microcontact printing facilitates independent control of many cues of the natural cell microenvironment for studying cellular mechanostransduction.

It is now well established that many cellular functions are regulated by interactions of cells with physicochemical and mechanical cues of their ...

ECM Protein Nanofibers and Nanostructures Engineered Using Surface-initiated Assembly

The extracellular matrix (ECM) in tissues is synthesized and assembled by cells to form a 3D fibrillar, protein network with tightly regulated fiber diameter, composition and organization. In addition to providing structural support, the physical and chemical properties of the ECM play an important role in multiple cellular processes including adhesion, differentiation, and apoptosis. In vivo, the ECM is assembled by exposing cryptic self-assembly (fibrillogenesis) sites within proteins. This process varies for different proteins, but fibronectin (FN) fibrillogenesis is well-characterized and serves as a model system for cell-mediated ECM assembly. Specifically, cells use integrin receptors on the cell membrane to bind FN dimers and actomyosin-generated contractile forces to unfold and expose binding sites for assembly into insoluble fibers. This receptor-mediated process enables cells to assemble and organize the ECM from the cellular to tissue scales. Here, we present a method termed surface-initiated assembly (SIA), which recapitulates cell-mediated matrix assembly using protein-surface interactions to unfold ECM proteins and assemble them into insoluble fibers. First, ECM proteins are adsorbed onto a hydrophobic polydimethylsiloxane (PDMS) surface where they partially denature (unfold) and expose cryptic binding domains. The unfolded proteins are then transferred in well-defined micro- and nanopatterns through microcontact printing onto a thermally responsive poly(N-isopropylacrylamide) (PIPAAm) surface. Thermally-triggered dissolution of the PIPAAm leads to final assembly and release of insoluble ECM protein nanofibers and nanostructures with well-defined geometries. Complex architectures are possible by engineering defined patterns on the PDMS stamps used for microcontact printing. In addition to FN, the SIA process can be used with laminin, fibrinogen and collagens type I and IV to create multi-component ECM nanostructures. Thus, SIA can be used to engineer ECM protein-based materials with precise control over the protein composition, fiber geometry and scaffold architecture in order to recapitulate the structure and composition of the ECM in vivo.

A method to obtain nanofibers and complex nanostructures from single or multiple extracellular matrix proteins is described. This method uses protein-surface interactions to create free-standing protein-based materials with tunable composition and architecture for use in a variety of tissue engineering and biotechnology applications.

The extracellular matrix (ECM) in tissues is synthesized and assembled by cells to form a 3D fibrillar, protein network with tightly regulated fiber ...

Revealing the Cytoskeletal Organization of Invasive Cancer Cells in 3D

Cell migration has traditionally been studied in 2D substrates. However, it has become increasingly evident that there is a need to study cell migration in more appropriate 3D environments, which better resemble the dimensionality of the physiological processes in question. Migratory cells can substantially differ in their morphology and mode of migration depending on whether they are moving on 2D or 3D substrates. Due to technical difficulties and incompatibilities with most standard protocols, structural and functional analysis of cells embedded within 3D matrices still remains uncommon. This article describes methods for preparation and imaging of 3D cancer cell cultures, either as single cells or spheroids. As an appropriate ECM substrate for cancer cell migration, we use nonpepsinized rat tail collagen I polymerized at room-temperature and fluorescently labeled to facilitate visualization using standard confocal microscopes. This work also includes a protocol for 3D immunofluorescent labeling of endogenous cell cytoskeleton. Using these protocols we hope to contribute to a better description of the molecular composition, localization, and functions of cellular structures in 3D.

This article presents a method to fluorescently label collagen that can be further used for both fix and live imaging of 3D cell cultures. We also provide an optimized protocol to visualize endogenous cytoskeletal proteins of cells cultured in 3D environments.

Cell migration has traditionally been studied in 2D substrates. However, it has become increasingly evident that there is a need to study cell migration ...

Medicine

Sandwich-like Microenvironments to Harness Cell/Material Interactions

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. However in vivo, most of the cells are completely surrounded by the extracellular matrix (ECM), resulting in a three-dimensional (3D) distribution of receptors. This may trigger differences in the outside-in signaling pathways and thus in cell behavior.
This article shows that stimulating the dorsal receptors of cells already adhered to a 2D substrate by overlaying a film of a new material (a sandwich-like culture) triggers important changes with respect to standard 2D cultures. Furthermore, the simultaneous excitation of ventral and dorsal receptors shifts cell behavior closer to that found in 3D environments. Additionally, due to the nature of the system, a sandwich-like culture is a versatile tool that allows the study of different parameters in cell/material interactions, e.g., topography, stiffness and different protein coatings at both the ventral and dorsal sides. Finally, since sandwich-like cultures are based on 2D substrates, several analysis procedures already developed for standard 2D cultures can be used normally, overcoming more complex procedures needed for 3D systems.

The following protocol describes the procedure to assemble sandwich-like cultures to be used as an intermediate stage between bi-dimensional (2D) and three-dimensional (3D) cellular environments. The engineered systems can have applications in microscopy, biomechanics, biochemistry and cell biology assays.

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. ...

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

This manuscript describes a soft lithography-based technique to engineer uniform arrays of three-dimensional (3D) epithelial tissues of defined geometry surrounded by extracellular matrix. This method is amenable to a wide variety of cell types and experimental contexts and allows for high-throughput screening of identical replicates.

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, ...

Ligand Nano-cluster Arrays in a Supported Lipid Bilayer

Currently there is considerable interest in creating ordered arrays of adhesive protein islands in a sea of passivated surface for cell biological studies. In the past years, it has become increasingly clear that living cells respond, not only to the biochemical nature of the molecules presented to them but also to the way these molecules are presented. Creating protein micro-patterns is therefore now standard in many biology laboratories; nano-patterns are also more accessible. However, in the context of cell-cell interactions, there is a need to pattern not only proteins but also lipid bilayers. Such dual proteo-lipidic patterning has so far not been easily accessible. We offer a facile technique to create protein nano-dots supported on glass and propose a method to backfill the inter-dot space with a supported lipid bilayer (SLB). From photo-bleaching of tracer fluorescent lipids included in the SLB, we demonstrate that the bilayer exhibits considerable in-plane fluidity. Functionalizing the protein dots with fluorescent groups allows us to image them and to show that they are ordered in a regular hexagonal lattice. The typical dot size is about 800 nm and the spacing demonstrated here is 2 microns. These substrates are expected to serve as useful platforms for cell adhesion, migration and mechano-sensing studies.

We present a protocol to functionalize glass with nanometric protein patches surrounded by a fluid lipid bilayer. These substrates are compatible with advanced optical microscopy and are expected to serve as platform for cell adhesion and migration studies.

Currently there is considerable interest in creating ordered arrays of adhesive protein islands in a sea of passivated surface for cell biological ...

Light-sheet Microscopy for Three-dimensional Visualization of Human Immune Cells

In vivo, activation, proliferation, and function of immune cells all occur in a three-dimensional (3D) environment, for instance in lymph nodes or tissues. Up to date, most in vitro systems rely on two-dimensional (2D) surfaces, such as cell-culture plates or coverslips. To optimally mimic physiological conditions in vitro, we utilize a simple 3D collagen matrix. Collagen is one of the major components of extracellular matrix (ECM) and has been widely used to constitute 3D matrices. For 3D imaging, the recently developed light-sheet microscopy technology (also referred to as single plane illumination microscopy) is featured with high acquisition speed, large penetration depth, low bleaching, and photocytotoxicity. Furthermore, light-sheet microscopy is particularly advantageous for long-term measurement. Here we describe an optimized protocol how to set up and handle human immune cells, e.g. primary human cytotoxic T lymphocytes (CTL) and natural killer (NK) cells in the 3D collagen matrix for usage with the light-sheet microscopy for live cell imaging and fixed samples. The procedure for image acquisition and analysis of cell migration are presented. A particular focus is given to highlight critical steps and factors for sample preparation and data analysis. This protocol can be employed for other types of suspension cells in a 3D collagen matrix and is not limited to immune cells.

Here, we present a protocol to visualize immune cells embedded in a three-dimensional (3D) collagen matrix using light-sheet microscopy. This protocol also elaborates how to track cell migration in 3D. This protocol can be employed for other types of suspension cells in the 3D matrix.

In vivo, activation, proliferation, and function of immune cells all occur in a three-dimensional (3D) environment, for instance in lymph nodes or ...

Immunology and Infection

Pattern Generation for Micropattern Traction Microscopy

Micropattern traction microscopy allows control of the shape of single cells and cell clusters. Furthermore, the ability to pattern at the micrometer length scale allows the use of these patterned contact zones for the measurement of traction forces, as each micropatterned dot allows for the formation of a single focal adhesion that then deforms the soft, underlying hydrogel. This approach has been used for a wide range of cell types, including endothelial cells, smooth muscle cells, fibroblasts, platelets, and epithelial cells. 
This review describes the evolution of techniques that allow the printing of extracellular matrix proteins onto polyacrylamide hydrogels in a regular array of dots of prespecified size and spacing. As micrometer-scale patterns are difficult to directly print onto soft substrates, patterns are first generated on rigid glass coverslips that are then used to transfer the pattern to the hydrogel during gelation. First, the original microcontact printing approach to generate arrays of small dots on the coverslip is described. A second step that removes most of the pattern to leave islands of small dots is required to control the shapes of cells and cell clusters on such arrays of patterned dots. 
Next, an evolution of this approach that allows for the generation of islands of dots using a single subtractive patterning step is described. This approach is greatly simplified for the user but has the disadvantage of a decreased lifetime for the master mold needed to make the patterns. Finally, the computational approaches that have been developed for the analysis of images of displaced dots and subsequent cell-generated traction fields are described, and updated versions of these analysis packages are provided.

We describe improvements to a standard method for measuring cellular traction forces, based on microcontact printing with a single subtractive patterning step of dot arrays of extracellular matrix proteins on soft hydrogels. This method allows for simpler and more consistent fabrication of island patterns, essential for controlling cell cluster shape.

Micropattern traction microscopy allows control of the shape of single cells and cell clusters. Furthermore, the ability to pattern at the micrometer ...

Light-Induced Molecular Adsorption of Proteins Using the PRIMO System for Micro-Patterning to Study Cell Responses to Extracellular Matrix Proteins

Cells sense a variety of extracellular cues, including the composition and geometry of the extracellular matrix, which is synthesized and remodeled by the cells themselves. Here, we present the method of Light-Induced Molecular Adsorption of Proteins (LIMAP) using the PRIMO system as a patterning technique to produce micro-patterned extracellular matrix (ECM) substrates using a single or combination of proteins. The method enables printing of ECM patterns in micron resolution with excellent reproducibility. We provide a step-by-step protocol and demonstrate how this can be applied to study the processes of neuronal pathfinding. LIMAP has significant advantages over existing micro-printing methods in terms of the ease of patterning more than one component and the ability to generate a pattern with any geometry or gradient. The protocol can easily be adapted to study the contribution of almost any chemical component towards cell fate and cell behavior. Finally, we discuss common issues that can arise and how these can be avoided.

Our overall aim is to understand how cells sense extracellular cues that lead to directed axonal growth. Here, we describe the methodology of Light-Induced Molecular Adsorption of Proteins, used to produce defined micro-patterns of extracellular matrix components in order to study specific events that govern axon outgrowth and pathfinding.

Cells sense a variety of extracellular cues, including the composition and geometry of the extracellular matrix, which is synthesized and remodeled by the ...