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 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.
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.
1. Preparation of the Native Soluble Collagen Solution
2. Collagen Methylation
3. Collagen Succinylation
4. Verification of the Collagen Modifications
5. Fabrication of Microfluidic Devices and Cell Seeding
6. Layer-by-layer Collagen Deposition
7. Stabilization of Cell Phenotype and Function
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 ε-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 groups and increases the number of carboxyl groups, while methylation has no effect on the amino groups and reduces the number of carboxyl groups, compared to native collagen (Figure 1B). These modifications alter the charge characteristics of the collagen molecules. Succinylation removes positively charged groups and replaces them with negatively charged groups, and methylation removes negatively charged groups (Figure 1C).
Layer-by-layer deposition of the methylated and succinylated collagen solutions (COL LBL) creates an ultrathin collagen matrix assembly on top of charged surfaces, such as cells (Figure 2A). Primary hepatocytes seeded on fibronectin-coated glass within a microfluidic device (Figure 2B) can be coated with such a purely collagen matrix assembly (Figure 2C). Deposition of 10 bilayers on cells creates a collagen layer thickness of approximately 140 nm (Figure 2D).
The ultrathin collagen assembly layer deposited using the COL LBL technique stabilizes primary hepatocytes for over 14 days in microfluidic devices. Hepatocytes without a top matrix cover lose their differentiated phenotype, contract, and lift off the surface of microfluidic devices over time (Fig. 3A-C). In contrast, hepatocytes covered with an ultrathin collagen assembly maintain their differentiated morphology and polarization over 14 days (Figure 3D-F). The viability of the cells was maintained at greater than 90% (Figure 3G-I). In addition, the polarization of the hepatocytes treated with COL LBL increased over time, showing development of an apical biliary canalicular network (Figure 3J-L).
In addition to cell viability, morphology, and polarization, the COL LBL technique recovers and stabilizes the function of primary hepatocytes at levels similar to standard techniques for cells in plates. The secretion of albumin by hepatocytes is low immediately after plating, and remains low unless the cells are induced to polarize through a matrix covering. After COL LBL treatment, the albumin secretion by the hepatocytes increases over 8–10 days before stabilizing (Figure 4A). Urea production is high immediately after cell seeding and decreases over time in cells not covered with a matrix, urea production stabilizes after 8–10 days in hepatocytes stabilized with COL LBL (Figure 4B). Cytochrome P450 (CYP) enzyme expression in response to stimuli is one specialized function of hepatocytes. The induction of CYP1A1 in hepatocytes in response to 3-methylcholanthrene was recovered and stabilized up to 14 days by the COL LBL treatment (Figure 4C).
Figure 1. Rat tail collagen solutions were chemically modified to create net positively or net negatively charged polymer solutions. (A) Schematic representations of the succinylation and methylation reactions that modify carboxyl and ε-amino groups, respectively. (B) Hydrogen titration curve derivatives showing shifts in the relative numbers of H+ ions needed to alter solution pH after collagen modification. (C) The net charges (mean ± SD) of the modified collagen solutions calculated from the H+ shifts in (B) normalized to native collagen. MC: methylated collagen. SC: succinylated collagen. AA: amino acid residues. Re-print with permission from 9. Please click here to view a larger version of this figure.
Figure 2. Layer-by-layer deposition of modified collagen solutions created an ultrathin collagen matrix assembly on top of the hepatocytes. (A) Schematic representation of hepatocytes seeded on fibronectin on a microdevice and incubated with alternating methylated and succinylated collagen solutions. TEM cross-sections of representative (20 cells examined per group) control (B) and COL LBL-treated (C) hepatocytes after 2 days of culture. Arrows: LBL collagen layer. Scale bar: 2 µm. (D) COL LBL layer thickness (mean ± SD) per cell measured from TEM images (n=20 cells from 4 devices). Modified from 9. Please click here to view a larger version of this figure.
Figure 3. The COL LBL technique maintained hepatocyte morphology, viability, and polarization in microdevices over 14 days in culture. Hepatocytes with no top layer in microdevices (negative control) contract and lift off the surface over time (A–C). LBL deposition of modified collagens maintains hepatocyte morphology (D–F) and viability (G–I) in microdevices over 14 days. CMFDA is a generic cell marker that remains in the cytoplasm of non-polarized hepatocytes (J). Over time, the development of bile canaliculi can be seen by the excretion of the fluorophore into canalicular spaces (K) that develop around the cells over time (L). Image scale bar: 100 µm. Inset scale bar: 50 µm. Modified from 9. Please click here to view a larger version of this figure.
Figure 4. COL LBL stabilizes hepatocyte albumin and urea sections and CYP activity over 14 days in microfluidic devices. (A) Albumin secretion by hepatocytes in COL LBL started low and increased with time until reaching a plateau at day 10. (B) Urea secretion by COL LBL hepatocytes decreased with time until reaching a plateau at day 10. (C) The induction of CYP1A activity was low at day 2, but significantly increased at day 7, a level that was maintained through day 14. Mean ± SEM; COL LBL: hepatocytes cultured after LBL collagen deposition; NoTop: hepatocytes cultured with no top matrix layer; n=6–8 devices. Modified from9. Please click here to view a larger version of this figure.
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 stabilize the morphology, viability, and polarization of cells such as hepatocytes (Figure 3), after seeding in microfluidic devices, as well as their function (Figure 4).
For successful deposition of ultrathin collagen layers, the modification reactions creating the methylated and succinylated collagens are critical. Because the carboxyl group methylation reaction is not favorable, the collagen methylation reaction must be performed in acidified methanol to drive the reaction forward, based on Le Chatelier’s principle. Therefore, it is important to remove all excess water from the collagen after the pH precipitation before dissolving the precipitated collagen in the acidified methanol in order to increase the reaction efficiency. For the succinylation reaction, maintaining the pH of the solution above 9 while adding the succinic anhydride in acetone is critical. Continuous monitoring of the pH and slow addition of the succinic anhydride acetone solution are helpful for ensuring an efficient reaction. During the layer-by-layer deposition, it is critical to minimize the time that the cells go without media. The procedure for 10 bilayers takes approximately 20 min, which is about as long as most cells should be without media outside of the incubator. If more than 10 bilayers must be deposited, rest the cells in media in an incubator for 3–4 hr between collagen depositions to ensure the health of the cells. At the other extreme, too few layers will not be effective in maintaining cell morphology. In our testing, 3 layers did not maintain cell morphology, while 10 layers did.
Several modifications can be made to the procedures to troubleshoot problems. The time and temperature of the methylation reaction can be varied if problems arise. For example, decreasing the reaction temperature to 4 °C and increasing the time to 7 days may increase efficiency. Also, depending on the extent of polymerization during the pH extraction, the collagen will sometimes fully dissolve in the acidified methanol during the methylation reaction. If this occurs, add 10 M NaOH to bring the pH up to 9–10 after the reaction has completed in order to precipitate the collagen so that it can be pelleted during the next step. While applying the layer-by-layer deposition in microfluidic devices, small channels or ports can become clogged with collagen, indicated by an increase in the device fluidic resistance. Flushing the device with fresh media should remove any blockages, allowing deposition to continue.
The manual deposition of the layer-by-layer collagen as described here is a limitation in terms of the scaling and automation of microfluidic devices. However, this technique is compatible with standard microfluidic hardware, including valves and pumps that could be used to automate the technique and prepare arrays of devices at once. Another limitation of this technique for use in plate culture is that it requires a relatively large amount of native collagen. For microfluidic application, the volumes of methylated and succinylated collagens can be used to create ultrathin collagen layers in many devices. With standard tissue plate culture, on the other hand, the volumes required to coat wells become more prohibitive with increasing well size.
This layer-by-layer technique for the deposition of ultrathin collagen matrix layers could be further developed by using alternative or functional modifications, as well as by creating multiple layers of cells separated by the thin matrix assemblies. Although the relatively simple amino acid side chain modification reactions of methylation and succinylation were used here, alternative or additional reactions could be used instead in order to create functionalized collagens. The functionalized sidechains should still yield proteins with net overall positive and negative charges, but such functionalization may be useful for a variety of applications.
The authors have nothing to disclose.
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.
collagen type I, rat tail | Life Technologies | A1048301 | option for concentrated rat tail collagen |
collagen type I, rat tail | Sigma-Aldrich | C3867-1VL | option for concentrated rat tail collagen |
collagen type I, rat tail | EMD Millipore | 08-115 | option for concentrated rat tail collagen |
collagen type I, rat tail | R%D Systems | 3440-100-01 | option for concentrated rat tail collagen |
succinic anhydride | Sigma-Aldrich | 239690-50G | succinylation reagent |
anhydrous methanol | Sigma-Aldrich | 322415-100ML | methylation reagent |
sodium hydroxide | Sigma-Aldrich | S5881-500G | pH precipitation reagent |
hydrochloric acid | Sigma-Aldrich | 320331-500ML | pH precipitation reagent |
rat collagen type I ELISA | Chondrex | 6013 | option for detecting collagen content |
hydroxyproline assay kit | Sigma-Aldrich | MAK008-1KT | option for detecting collagen content |
hydroxyproline assay kit | Quickzyme Biosciences | QZBtotcol1 | option for detecting collagen content |