This work was supported by grants from the National Institutes of Health, including a microphysiological systems consortium grant from the National Center for Advancing Translational Sciences (UH2TR000503), a Ruth L. Kirschstein National Research Service Award Postdoctoral Fellowship (F32DK098905 for WJM) and pathway to independence award (DK095984 for AB) from the National Institute of Diabetes and Digestive and Kidney Diseases.

1. Preparation of the Native Soluble Collagen Solution
<ol>
	<li>Prepare or purchase 200 mg of acidified, soluble, type I collagen from rat tails at 1&#8211;3 mg/ml using standard isolation protocols, such as reported by Piez et al.15</li>
	<li>Scale the amount of starting material based on the desired end volume of modified collagen solutions. Approximately make 25&#8211;30 ml of methylated and 25&#8211;30 ml of succinylated collagen solutions, each at 3 mg/ml, from 200 mg of soluble native collagen.</li>
</ol>
2. Collagen Methylation
<ol>
	<li>Dilute 100 mg of the native, acidified (pH 2&#8211;3) collagen solution to a concentration of 0.5 mg/ml with ice cold sterile water and keep the solution on ice to prevent gelation.</li>
	<li>Adjust the pH of the collagen solution to 9&#8211;10 with a few drops of 1 N NaOH and stir at RT for 30 min. Observe the collagen precipitate, causing the solution to become cloudy.</li>
	<li>Spin down the precipitated collagen solution at 3,000 &#215; g for 25 min. A clear, gel-like precipitate should be visible in the bottom of the tubes. Aspirate and properly dispose of the supernatant fluid.</li>
	<li>Resuspend the precipitated collagen in 200 ml of methanol with 0.1 N HCl and allow the methylation reaction to occur with stirring at RT for 4 days. The collagen will not dissolve, but should break apart into very small visible pieces that will make the solution cloudy.</li>
	<li>After the methylation, centrifuge the solution at 3,000 &#215; g for 25 min to pellet the methylated collagen. Aspirate and dispose of the acidified methanol supernatant.</li>
	<li>Dissolve the methylated collagen in 25 ml of sterile PBS, giving a concentration of approximately 3 mg/ml, with repeated pipetting, and filter the solution through a 60 &#181;m cell strainer. Adjust the pH of the solution to 7.3&#8211;7.4 using 20 &#181;l increments of 1 N NaOH.</li>
	<li>Assess the concentration of the solution using a commercial rat collagen ELISA kit or hydroxyproline assay kit. Dilute the solution to 3 mg/ml with sterile PBS.</li>
	<li>Sterilize the methylated collagen solution by transferring it to a glass bottle with a screw cap, carefully layering 3 ml of chloroform at the bottom of the bottle, allow the bottle to set O/N&#160;at 4 &#176;C, and then aseptically remove and store the top layer, which is the methylated collagen.</li>
	<li>Store the solution at 4 &#176;C for use within 1 month.</li>
</ol>
3. Collagen Succinylation
<ol>
	<li>Dilute the other 100 mg of the native, acidified (pH 2&#8211;3) collagen solution to a concentration of 0.5 mg/ml with ice cold sterile water and keep the solution on ice to prevent gelation.</li>
	<li>Adjust the pH of the collagen solution to 9&#8211;10 with a few drops of 1 N NaOH and stir at RT for 30 min. The collagen should precipitate, causing the solution to become cloudy.</li>
	<li>Dissolve 40 mg of succinic anhydride (0.4 mg per mg of collagen) in 10 ml of acetone (1/20th the volume of the collagen solution). Slowly (in approximately 0.5 ml increments) add this mixture to the collagen solution with stirring while continuously monitoring the pH. Maintain the pH above 9.0 by adding 1 or 2 drops of 1 N NaOH as the pH approaches 9.0.</li>
	<li>Continue stirring at RT for 120 min after adding all of the succinic anhydride in acetone. Observe the solution to become clear as the succinylated collagen dissolves. Periodically check the pH to ensure it remains above 9.0.</li>
	<li>Adjust the pH of the solution to 4.0 with 20 &#181;l increments of 1 N HCl. Observe the solution cloudy again, as the succinylated collagen precipitates.</li>
	<li>After the succinylation, centrifuge the solution at 3,000 &#215; g for 25 min to pellet the succinylated collagen. Aspirate and dispose of the acidified supernatant with unreacted succinic anhydride.</li>
	<li>Dissolve the succinylated collagen in 25 ml of sterile PBS, giving a concentration of approximately 3 mg/ml, with repeated pipetting, and filter the solution through a 60 &#181;m cell strainer. Adjust the pH of the solution to 7.3&#8211;7.4 using 20 &#181;l increments of 1 N NaOH.</li>
	<li>Assess the concentration of the solution using a commercial collagen rat ELISA kit or hydroxyproline assay kit. Dilute the solution to 3 mg/ml with sterile PBS.</li>
	<li>Sterilize the succinylated collagen solution by transferring it to a glass bottle with a screw cap, carefully layering 3 ml of chloroform at the bottom of the bottle, allow the bottle to set O/N at 4 &#176;C, and then aseptically remove and store the top layer, which is the succinylated collagen.</li>
	<li>Store the solution at 4 &#176;C for use within 1 month.</li>
</ol>
4. Verification of the Collagen Modifications
<ol>
	<li>Prepare 1 ml samples from each of the native, methylated, and succinylated collagen solutions and dilute to a concentration of 0.1 mg/ml with sterile pure water for hydrogen ion titration. Further dilute the buffer at least 1,000-fold by dialysis against water through a 10 kDa cutoff membraneusing 3 solution changes for at least 4 hr&#160;each.</li>
	<li>Adjust the pH of the solutions to 7.3 with small amounts of NaOH and HCl. Using pH 7.3 as an arbitrary reference, perform hydrogen ion titration on each of the samples as described by Tanford16 to create titration curves for the native, methylated, and succinylated collagen solutions.</li>
	<li>Plot the change in pH per volume of acid added versus the number of bound H+ ions per molecule. The high pH &#8220;amino&#8221; range should show a shift in the succinylated collagen towards the neutral point (a loss of amine groups), and the low pH &#8220;carboxyl&#8221; range should show a leftward shift in the methylated collagen (a loss of carboxyl groups) and a rightward shift in the succinylated collagen (a gain in carboxyl groups), compared to the native collagen.</li>
	<li>Assess the efficacy of the succinylation reaction by determining the % of amino groups in the native collagen replaced by succinylation using the 2,4,6-trinitrobenzenesulfonic acid (TNBA) colorimetric method, following standard protocols17,18.</li>
</ol>
5. Fabrication of Microfluidic Devices and Cell Seeding
<ol>
	<li>Fabricate microfluidic devices using standard methods9. Use replica molding of PDMS from SU-8 masters on silicon defined using photolithography to create microfluidic cell culture chambers with 100 &#181;m tall, 0.4&#8211;1.5 mm wide, and 1&#8211;10 mm long channels for cell growth.</li>
	<li>Use a plasma cleaner to oxidize the surfaces of the device and a glass slide, and then press together to bond. After sterilizing the device by exposure to UV light for at least 30 min, fill the chamber with 50 &#181;g/ml fibronectin in sterile PBS and incubate at 37 &#176;C for 45 min.</li>
	<li>Seed the device with cells, such as primary rat or human hepatocytes, which require a collagen gel for stabilization of phenotype or differentiation state. We use 20 &#181;l of freshly isolated primary rat hepatocytes7,19 or commercially available cryopreserved primary human hepatocytes seeded at 14&#215;106 cells/ml per device.</li>
	<li>Allow the cells to attach for 4&#8211;6 hr, and then wash out unattached cells and replace the plating media with growth media. Incubate O/N to ensure full cell spreading and create a confluent monolayer of cells.</li>
</ol>
6. Layer-by-layer Collagen Deposition
<ol>
	<li>In a laminar flow tissue culture hood, prepare sufficient volumes of methylated and succinylated collagen solutions for 10 applications of each solution per device, as well as a few mls of media. Keep the solutions on ice. We use 20 &#181;l (10&#8211;15 times the total device volume) of collagen per layer per device.</li>
	<li>Beginning with the methylated (polycationic) solution, alternate flushing the devices with 20 &#181;l of methylated and then succinylated collagen solutions, waiting 1 min between each application. Flush the device a total of 10 times per solution, which should take approximately 20 min. Work quickly to minimize the amount of time the cells are without media.</li>
	<li>Observe collagen slowly accumulate at the inlet/outlet depending on its size. If the resistance to fluid flow increases, flush the device once or twice with media, and then continue layering.</li>
	<li>After applying all of the layers, rinse the device twice with fresh media and return to the incubator. Depending on the cell type, the extracellular matrix-induced morphological changes, such as enhanced polarization in hepatocytes, should be visible within a few hours.</li>
	<li>Prepare representative devices for transmission electron microscopy using standard methods9 to verify the presence of a collagen matrix assembly on top of the cultured cells.</li>
</ol>
7. Stabilization of Cell Phenotype and Function
<ol>
	<li>Image the cell morphology, viability, and polarity using standard methods for the cell type. For hepatocytes, collect images over 14 days using phase microscopy, LIVE/DEAD staining for viability, and CMFDA dye for apical polarization to demonstrate the effects of the collagen deposition.</li>
	<li>For cell morphology, flush the device through the inlet by pipette with 20 &#181;l of PBS to rinse, inject 20 &#181;l of PBS, and image the device using phase contrast microscopy.</li>
	<li>For LIVE/DEAD staining, flush the device through the inlet by pipette with 20 &#181;l of PBS to rinse, inject 20 &#181;l of LIVE/DEAD staining solution with DAPI prepared following the manufacturer&#39;s instructions, incubate for 30 min at 37 &#176;C, rinse the device again with 20 &#181;l of PBS, and image the device using fluorescence microscopy.</li>
	<li>For bile canaliculi staining, flush the device through the inlet by pipette with 20 &#181;l of PBS to rinse, inject 20 &#181;l of 2 &#181;M CMFDA staining solution with DAPI prepared following the manufacturer&#39;s instructions, incubate for 30 min at 37 &#176;C, rinse the device again with 20 &#181;l of PBS, and image the device using fluorescence microscopy.</li>
	<li>Collect the spent media and measure cellular metabolic products at appropriate time points over the culture duration to determine the cell function. For hepatocytes, measure the amount of albumin and urea in the spent media.</li>
	<li>Perform endpoint functional analyses on the cells themselves, such as assessing enzyme activity levels, antibody staining, or RNA analysis. For hepatocytes, induce and measure cytochrome P450 enzyme activities or phase II conjugation enzyme glutathione S-transferase.</li>
</ol>

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S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multilineage+differentiation+of+rhesus+monkey+embryonic+stem+cells+in+three&#8208;dimensional+culture+systems.">Multilineage differentiation of rhesus monkey embryonic stem cells in three&#8208;dimensional culture systems.</a> Stem Cells. 21 (3), 281-295 (2003).</li><li>Whelan, M. C., Senger, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagen+I+initiates+endothelial+cell+morphogenesis+by+inducing+actin+polymerization+through+suppression+of+cyclic+AMP+and+protein+kinase+A.">Collagen I initiates endothelial cell morphogenesis by inducing actin polymerization through suppression of cyclic AMP and protein kinase A.</a> J Biol Chem. 278 (1), 327-334 (2003).</li><li>Dunn, J. C., Tompkins, R. G., Yarmush, M. 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The authors have nothing to disclose.

Ultrathin pure collagen assemblies can be deposited on charged cells or material surfaces using layer-by-layer deposition of modified collagens. The results of this study demonstrate that methylation and succinylation of native collagen create polycationic and polyanionic collagen solutions (Figure 1) that can be used with the layer-by-layer technique to deposit ultrathin collagen matrix assemblies on cells (Figure 2) or other charged material surfaces. Such ultrathin matrix layers can s...

Although microfluidics allows for the exquisite control of the cellular microenvironment, culturing cells, especially primary cells, within microfluidic devices can be challenging. Many traditional cell culture techniques have been developed to sustain and stabilize cell function when cultured in tissue culture plates, but translating those techniques to microfluidic devices is often difficult. 
One such technique is the culture of cells on or sandwiched between collagen gels as a model of the physiological 3D cell environment.1 Type I collagen is one of the most frequently used proteins for biomaterials applications because of its ubiquity in extracellular matrix, natural abundance, robust cell attachment sites, and biocompatibility.2 Many cells benefit from 3D culture with collagen, including cancer cells3,45, microvascular endothelial cells6, and hepatocytes7, among others. While the use of collagen gels is easy in open formats, such as tissue culture plates, the limited channel dimensions and enclosed nature of microfluidic devices makes the use of liquids that gel impractical without blocking the entire channel.
To overcome this problem, we combined the layer-by-layer deposition technique8 with chemical modifications of native collagen solutions to create ultrathin collagen assemblies on top of cells cultured in microfluidic devices. These layers can stabilize cell morphology and function similar to collagen gels and can be deposited on cells in microfluidic devices without blocking the channels with polymerized matrix. The goal of this method is to modify native collagen to create polycationic and polyanionic collagen solutions and to stabilize cells in microfluidic culture by depositing thin collagen matrix assemblies onto the cells. This technique has been used to stabilize the morphology and function of primary hepatocytes in microfluidic devices.9
Although layer-by-layer deposition has previously been reported with natural and synthetic polyelectrolytes10 to cover hepatocytes in plate culture11,12 and as a seeding layer for hepatocytes in microfluidic devices13,14, this method describes the deposition of a pure collagen layer on top of hepatocytes, mimicking the 3D collagen culture techniques. In this protocol, we use hepatocytes as example cells that can be maintained using 3D collagen layers. The many other types of cells that benefit from 3D culture in collagen may similarly benefit from culture after layer-by-layer deposition of an ultrathin collagen matrix assembly.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>collagen type I, rat tail</td>
			<td>Life Technologies</td>
			<td>A1048301</td>
			<td>option for concentrated rat tail collagen</td>
		</tr>
		<tr>
			<td>collagen type I, rat tail</td>
			<td>Sigma-Aldrich</td>
			<td>C3867-1VL</td>
			<td>option for concentrated rat tail collagen</td>
		</tr>
		<tr>
			<td>collagen type I, rat tail</td>
			<td>EMD Millipore</td>
			<td>08-115</td>
			<td>option for concentrated rat tail collagen</td>
		</tr>
		<tr>
			<td>collagen type I, rat tail</td>
			<td>R%D Systems</td>
			<td>3440-100-01</td>
			<td>option for concentrated rat tail collagen</td>
		</tr>
		<tr>
			<td>succinic anhydride</td>
			<td>Sigma-Aldrich</td>
			<td>239690-50G</td>
			<td>succinylation reagent</td>
		</tr>
		<tr>
			<td>anhydrous methanol</td>
			<td>Sigma-Aldrich</td>
			<td>322415-100ML</td>
			<td>methylation reagent</td>
		</tr>
		<tr>
			<td>sodium hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td>S5881-500G</td>
			<td>pH precipitation reagent</td>
		</tr>
		<tr>
			<td>hydrochloric acid</td>
			<td>Sigma-Aldrich</td>
			<td>320331-500ML</td>
			<td>pH precipitation reagent</td>
		</tr>
		<tr>
			<td>rat collagen type I ELISA</td>
			<td>Chondrex</td>
			<td>6013</td>
			<td>option for detecting collagen content</td>
		</tr>
		<tr>
			<td>hydroxyproline assay kit</td>
			<td>Sigma-Aldrich</td>
			<td>MAK008-1KT</td>
			<td>option for detecting collagen content</td>
		</tr>
		<tr>
			<td>hydroxyproline assay kit</td>
			<td>Quickzyme Biosciences</td>
			<td>QZBtotcol1</td>
			<td>option for detecting collagen content</td>
		</tr>
	</tbody>
</table>

layer-by-layer collagen deposition in microfluidic devices for microtissue stabilization

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D culture of cells in collagen type I gels helps to stabilize cell morphology and function, which is necessary for creating microfluidic tissue models in microdevices. Translating traditional 3D culture techniques for tissue culture plates to microfluidic devices is often difficult because of the limited channel dimensions. In this method, we describe a technique for modifying native type I collagen to generate polycationic and polyanionic collagen solutions that can be used with layer-by-layer deposition to create ultrathin collagen assemblies on top of cells cultured in microfluidic devices. These thin collagen layers stabilize cell morphology and function, as shown using primary hepatocytes as an example cell, allowing for the long term culture of microtissues in microfluidic devices.

Native collagen can be modified using methylation and succinylation to create polycationic and polyanionic collagen solutions for use in layer-by-layer deposition. Succinylation modifies the &#949;-amino groups of native collagen with succinyl groups, and methylation modifies the carboxyl groups of native collagen with a methyl group (Figure 1A). These modifications to the collagen protein amino acid side chains alter the pH titration curves for the solutions. Succinylation reduces the number of amino gr...

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Layer-by-layer Collagen Deposition in Microfluidic Devices for Microtissue Stabilization

The creation of functional microtissues within microfluidic devices requires the stabilization of cell phenotypes by adapting traditional cell culture techniques to the limited spatial dimensions in microdevices. Modification of collagen allows the layer-by-layer deposition of ultrathin collagen assemblies that can stabilize primary cells, such as hepatocytes, as microfluidic tissue models.

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D ...

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9.3K Views.  Massachusetts General Hospital, Harvard Medical School. The creation of functional microtissues within microfluidic devices requires the stabilization of cell phenotypes by adapting traditional cell culture techniques to the limited spatial dimensions in microdevices. Modification of collagen allows the layer-by-layer deposition of ultrathin collagen assemblies that can stabilize primary cells, such as hepatocytes, as microfluidic tissue models.