Application of Multichannel Collagen Gels in Construction of Epithelial

Article pubs.acs.org/journal/abseba

Application of Multichannel Collagen Gels in Construction of Epithelial Lumen-like Engineered Tissues Kazuya Furusawa,*,† Takeomi Mizutani,† Hiromi Machino,‡ Saki Yahata,‡ Akimasa Fukui,† and Naoki Sasaki† †

Faculty of Advanced Life Science, and ‡Division of Biological Sciences (Macromolecular Functions), School of Science, Hokkaido University, Kita-ku Kita 10 Nishi 8, Sapporo, Hokkaido Japan S Supporting Information *

ABSTRACT: Introduction of epithelial lumen-like structures such as blood and lymphatic vessels, as well as renal tubules, is a prerequisite for successful construction and function of artificially engineered giant tissues. Here, we demonstrate a methodology for construction of various epithelial lumen-like structures by using multichannel collagen gels (MCCGs). MCCGs were prepared and used as template scaffolds for constructing epithelial lumen structures in a controlled fashion. The effect of NaCl concentration on the multichannel structure of MCCGs was investigated by using confocal laser scanning microscopy along with fluorescent staining. The channel diameter increased with increasing NaCl concentrations in the collagen solution and the phosphate buffer solution. In contrast, the channel number decreased with increasing NaCl concentrations. Engineered tissues with various lumen-like structures were constructed by seeding and culturing Madin−Darby canine kidney cells on MCCGs. The diameter of the lumen and the number of lumens per unit area were controllable by regulating the multichannel structure of cylindrical MCCG. We believe that our methodology for the construction of engineered tissues possessing epithelial lumen-like structures will prove helpful in regeneration of giant tissues with various hierarchical structures. KEYWORDS: collagen gel, multichannel structure, phase separation dynamics, tissue engineering, epithelial lumen, Madin−Darby canine kidney cells

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INTRODUCTION Transport ducts, such as blood vessels, lymphatic vessels, and kidney tubules, contribute to efficient passage of oxygen, nutrients, cells, and waste products in the human body and their optimum functioning in tissues and organs is vital for maintaining health. Transport ducts in native tissues and organs vary in dimensions and number and form a highly organized structural network. Introduction of transport ducts and reconstruction of their structures are crucial for the construction of artificially engineered tissues with the dimensions larger than several centimeters and biological functions. In this context, hydrogels with multichannel structures can be used as template scaffolds to construct transport ducts. Several approaches for constructing transport ducts by using hydrogels with multichannel structures have already been reported.1−3 For example, blood vessels with large diameters have been constructed by using a combination of electrochemical detachment of cells with gelatin methacrylate hydrogel photopatterning.1,2 However, no studies constructing transport ducts that mimic the highly organized structural network in native tissues and organs have been reported. Hydrogels with multichannel structures prepared by dialysis of solutions of polysaccharides, such as alginate and carboxymthylcellulose (CMC), against solutions of multivalent metal © XXXX American Chemical Society

cations, such as copper chloride and calcium chloride, might be used as a scaffold for constructing highly organized structural networks.4,5 The polysaccharide hydrogels with multichannel structures (multichannel polysaccharide hydrogel: MCPG) have been used for constructing various engineered tissues. For example, MCPGs have been used for constructing blood vessels3 and as a scaffold to directionally guide the growth of axons.6 Furthermore, MCPG have been also prepared by dialysis of acidic chitosan solutions to alkaline solutions.7 However, because the alginate, CMC, and chitosan are not components in ECM in human body, the human cells cannot adhere on the as-prepared MCPGs. Therefore, modification of the polysaccharides with adhesion factors, such as a RGD peptide, fibronectin, and poly lysine, must be required for efficiently seeding cells on MCPGs.3,6,8 In addition, because biodegradation rates of alginate, CMC, and chitosan are very slow, the hydrogels with multichannel structures could be remained for long-term in human body.9,10 The low biodegradability of the polysaccharides possibly inhibits tissue remodeling due to various cell behaviors, such as cell invasion and cell migration. To overcome the low Received: January 5, 2015 Accepted: May 21, 2015

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DOI: 10.1021/acsbiomaterials.5b00003 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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ACS Biomaterials Science & Engineering

highly organized structural network by using MCCG as a template cell scaffold. For this purpose, MCCGs with various multichannel structures are useful for controlling the highly organized structural network. Therefore, we must establish the method to control the multichannel structure of MCCG. In the previous study, we suggested that the formation of multichannel structures within MCCGs is attributed to phase separation and gelation of the collagen solution.16 We studied the phase separation dynamics of collagen solution during dialysis and indicated that this separation is attributed to spinodal decomposition. Phase separation of two-component solutions generally results in two macroscopic phases in an equilibrium state.18,19 In contrast, the phase separation of collagen solutions that occurs during the dialysis process does not result in two macroscopic phases, because phase separation of collagen solutions is constrained by the formation of the collagen gel network. Thus, the final structure of MCCG is determined by the kinetics of phase separation and gelation of collagen solution. Therefore, our previous study suggested that the multichannel structure is controlled by regulating the kinetics of phase separation and gelation of collagen solutions. However, no studies concerning liquid−liquid phase separation of collagen solution have been reported. By contrast, the gelation of collagen solution has been well-investigated.20−23 Several reports have reported that fibrillogenesis of collagen limits the gelation rate of collagen solutions.20,21 Furthermore, the rate of fibrillogenesis is affected by various physicochemical properties, such as temperature, ionic strength and pH of collagen solution.24−28 Thus, the dynamics of collagen gelation are controlled by regulating the temperature, ionic strength, and pH during the dialysis of collagen solution. In fact, we showed that the multichannel structure of MCCG was controlled by regulating the gelation temperature.17,29 However, it is difficult to prepare MCCG at a constant gelation temperature under aseptic conditions. Therefore, alternative methodologies that can be used to prepare MCCG under aseptic conditions are needed. Control of salt concentrations in collagen solutions and phosphate buffer solutions under aseptic conditions is easy and does not require any special equipment. Salt concentration probably affects intermolecular collagen interactions and the interaction between collagen and water molecules in solution. Changes in the intermolecular collagen interactions and the interaction between collagen and water molecules could affect not only gelation kinetics but also the phase behavior of collagen solutions. Therefore, we focused on the concentration effect of sodium chloride (NaCl) on the structure of MCCGs. In this study, we developed a method to control the multichannel structure of MCCG by regulating the concentration of NaCl in PBS buffered collagen solution. Further, we constructed epithelial lumen-like structures by culturing MadinDarby Canine Kidney cells (MDCK cells) on MCCGs. We demonstrated that the epithelial luminal structure can be controlled using this method. Since the MCCG mainly consists of atelocollagen, cells can adhere on the MCCG without any modification with the adhesion factors. In addition, because many cells in the human body can degrade the type I collagen via secreting matrix metalloproteases, the MCCG does not inhibit the tissue remodeling due to various cell behaviors. Therefore, our methodology would allow to simplify the procedure for constructing the engineered tissues. Furthermore, since the antigenic sites were enzymatically removed from type I and type III collagen molecules during the manufactural processes of atelocollagen, allergic effects of atelocollagen on tissues and

biodegradability of the polysaccharides, various methods to improve and control the biodegradability of the polysaccharides have been developed. For example, biodegradability of alginate can be controlled by partially oxidation.11 However, the modifications to improve various properties of polysaccharides may complicate the procedure for constructing engineered tissues and rise a cost of construction of engineered tissues. Furthermore, it has been reported that hydrogels prepared by using multivalent metal cations release significant amount of cross-linking metal cations into medium.12,13 The released multivalent metal cations possibly induce unfavorable cellular behaviors in tissues and organs. For example, the released calcium ion (or increase in calcium ion concentration in the medium) induces up-regulation of expression levels of inflammatory cytokines and chemokines secreted by dendritic cells.13 Furthermore, the increase in concentration of extracellular calcium ion could lead to cell apoptosis.14 Therefore, hydrogels with multichannel structures prepared from ECM components and without multivalent metal cations are important for construction of more biomimetic engineered tissues with high biocompatibility. Previously, we reported the development of a collagen gel possessing anisotropic and multichannel structures; multichannel collagen gel, MCCG; using dialysis of atelocollagen into phosphate buffer solutions.15,16 By using MCCG as a cell scaffold, toroidal structures containing osteoblastic cells (MC3T3-E1 cells) were constructed.17 The toroidal structures shared morphological similarity to epithelial lumens, such as blood and lymphatic vessels, and renal tubules. Furthermore, as shown in Figure 1, the channel diameter and the channel number

Figure 1. Optical photograph of multichannel collagen gel.

increases and decreases with increasing the distance from the surface of gel, respectively. The gradient properties of multichannel structure of MCCG mimic the highly organized structural network in native tissues and organs. Because of this similarity, MCCGs can be used as template scaffolds for construction of various transport ducts. The purpose of this study is to develop the methodology for reconstructing the B


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prepared by boring an 8 mm-diameter hole. The lower silicone rubber sheet was placed on the 60 mm polystyrene dish, resulting in an 8 mm diameter well on the dish. The collagen solution was injected into the well. The well was sealed using a dialysis membrane (Spectra/Por Membrane MWCO: 3,500, Spectrum Laboratories, Inc., CA, USA) and the upper silicone rubber sheet was placed on the dialysis membrane. Ten milliliters of PBg was poured into the polystyrene dish to initiate dialysis for the preparation of MCCGs. The effects of varying salt concentration (CNaCl) on the multichannel structure of the MCCG were investigated by using different collagen and PBg solutions (containing identical CNaCl).

organs are little. Therefore, we would also provide the hydrogels with multichannel structure that is more biocompatible than MCPGs and that can be used for constructing biomimetic epithelial lumen-like structures.

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MATERIALS & METHODS

Materials. Atelocollagen solution was purchased from Koken Co., Ltd. (Tokyo, Japan). Disodium hydrogen phosphate (Na2HPO4), potassium dihydrogen phosphate (KH2PO4), and sodium chloride (NaCl) were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). A 50 mM NaCl solution was prepared in 1 mM hydrochloric acid (HCl). Collagen solutions containing various concentrations of NaCl (0, 1, and 2 mM) were obtained by mixing the collagen and NaCl solutions. The phosphate buffer solution used to induce formation of MCCG (PBg) was prepared in Milli-Q water and contained Na2HPO4 and KH2PO4 at 20 mM and 13 mM, respectively. PBg containing various concentrations of NaCl (CNaCl, 0, 1, and 2 mM) was prepared. pH of PBg was approximately 7.1, regardless of CNaCl. Dulbecco’s phosphate buffered saline (PBS(−)), containing 2.7 mM KCl, 1.5 mM KH2PO4, 8.1 mM Na2HPO4, and 137 mM NaCl, was used for washing samples and cells. Collagenase type I derived from clostridium histolyticum was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Earle’s balanced salt solution (EBSS) that contain calcium and magnesium cations was purchased from Life Technologies, Carlsbad, CA). The collagenase type I was dissolved in EBSS at 1 mg/mL, and then the solution was sterilized by filtration through a syringe filter “Minisart® Syringe Filter” (Sartorius Stedim Biotech GmbH, Gëttingen, Germany) with the pore size of 0.2 μm. MDCK cells were provided by RIKEN cell bank (Ibaraki, Japan). DMEM was purchased from Nacalai Tesque Co. Ltd. (Kyoto, Japan). Fetal bovine serum (FBS) was purchased from BioWest (Nuaillé, France) (Catalog Number: S1650−500, Lot Number: S05246S1650). DMEM supplemented with 10% FBS and 1% penicillin-streptomycin was used as a growth medium for culturing MDCK. Control of Multichannel Structure of MCCG. Preparation of MCCGs. A dialysis chamber, shown in Figure 2A, was constructed for

Characterization of the Multichannel Structure of MCCGs. MCCGs prepared as described above were fixed using 4% paraformaldehyde solution for 6 h at 4 °C. MCCGs were washed twice with PBS(−). Anticollagen alpha I (sc-25974, Santa Cruz Biotechnology, Inc., CA, USA) antibody (200 μg/mL) was used as a primary antibody solution after 200-fold dilution in PBS(−). Samples were incubated overnight in the primary antibody solution at 37 °C, following which they were incubated in the secondary antibody solution (1 μg/mL of antigoat IgG labeled with Alexa 633, Molecular Probes, Life Technologies Corporation, CA, USA) for 6 h. Multichannel structure of the MCCGs was observed using a confocal laser scanning microscope (Leica TCS-SP5). Dynamics of Multichannel Structure Formation. Anticollagen alpha I (SC25974) antibody (0.2 mg/mL) and 1 mg/mL of antigoat IgG labeled with Alexa 633 solution were diluted 200-fold and 1000-fold using 5 mg/mL atelocollagen solution, respectively. As shown in Figure 2(B), a chamber used for observing the dynamics of multichannel

structure formation, was set on the stage of the confocal laser scanning microscope (CLSM, Leica TCS-SP5). The lower silicone rubber sheet was equipped with a well for injection of collagen solution. The dimensions of the well were 5 mm × 5 mm × 1 mm. For measuring dynamics of structure formation, a glass ring with an inner diameter of 2 cm and height of 2 cm was used instead of the upper silicone rubber shown in Figure 2A. An imaging area, 100 μm apart from the dialysis membrane, was focused for observation of multichannel structure formation. Three mL of PBg was poured into the glass ring, following which the time course-dependent changes in diameter of diluted phase during dialysis was observed using CLSM. The time resolution was 1 image per second. Diameter of the diluted phase was measured by using an image manipulation program, ImageJ, available at the public domain NIH image program (http://rsb. info.nih.gov/nih-image).

the preparation of MCCGs. The dialysis chamber was composed of two silicone rubber sheets set on a 60 mm polystyrene dish. The thickness of the lower silicone rubber sheets, which also determined the thickness of the MCCGs, was 1 mm. The upper silicone rubber sheet was 3 mm thick. Both rubber sheets were

Construction of Engineered Tissues Displaying Various Epithelial Lumen-like Structures. Preparation of MCCG Scaffolds. MCCGs bearing varying multichannel structures were prepared at CNaCl between 0 mM to 2 mM in a 6-well cell culture plate. A homogeneous collagen gel membrane, which inhibited the invasion of epithelial cells into the inner part of MCCG, was observed at the MCCG surface irrespective of CNaCl, (Figure 3). To promote invasion of epithelial

cells into MCCG, the collagenous gel membrane was enzymatically removed by treatment with 200 μL of 1 mg/mL collagenase type I solution at 37 °C for 30 min in 5% CO2 incubator. The activity of type I collagenase, and therefore the resulting thickness of the gel membrane, was controlled by varying the time of incubation. After desired incubation with collagenase, its activity was inhibited by washing the MCCGs twice with a 5 mM EDTA solution. The MCCGs were further incubated overnight in a 5 mM EDTA solution. The resulting MCCGs were pliant, and shrunk after the above treatment by cell seeding. This indicated that the mechanical properties of MCCGs needed improvement in order to reduce gel shrinkage. For this purpose, we used genipin, a chemical cross-linker with low cell

Figure 2. Schematic illustration of the dialysis chamber used (A) to prepare MCCGs and (B) to observe the dynamics of MCCG multichannel structure formation. C


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following which they were seeded on MCCG scaffolds at a seeding density of 1.0 × 106 cells/well. Samples were cultured in the growth medium for 11 days. The growth medium was exchanged every 2 days. Confocal Scanning Laser Microscopy of MDCK Cells Seeded on MCCG Scaffolds. Morphological features of samples, collagen molecules, actin fibers, epithelial polarities (apical, lateral and basal domains), and nuclei were visualized by fluorescent staining. Collagen was visualized by immunofluorescence staining. Actin fibers were stained by Alexa Fluor 488- or 594- conjugated phalloidin (Molecular probes, Life technologies Corporation, CA, USA). Nuclei were stained by SYTOX Blue (Molecular probes). Engineered tissues were washed 4 times with PBS(−), and fixed by incubating in 4% paraformaldehyde solution for 6 h at 4 °C. Samples were washed 4 times with PBS(−) after paraformaldehyde treatment, and then incubated in 0.5% Triton X-100 in PBS(−) at 4 °C for 1 h. After detergent treatment, samples were washed twice with PBS(−), following which they were incubated with a blocking solution containing 1% bovine serum albumin in PBS(−) at 37 °C for 12 h. Following the blocking procedure, samples were washed twice with PBS(−), and later incubated with 1 μg/mL of anticollagen alpha I (sc-25974, Santa Cruz Biotechnology, Inc., CA, USA), antiezrin (apical marker; 610602, BD Transduction Laboratories, NJ, USA), anti-α-catenin (lateral marker; C2081, Sigma, MO, USA), and antilaminin-β1 (basal marker; 2E8, Developmental Studies Hybridoma Bank, IA, USA) at 37 °C for 12 h. Following a 2X wash with PBS(−), samples were incubated with 1 μg/ mL of Alexa633 conjugated antigoat IgG, Alexa488 conjugated antirabbit IgG or Alexa546 conjugated antimouse IgG solution (Molecular probes, Life Technologies Corporation, CA, USA), 5 U/ mL Alexa488-phalloidin solution, and 5 mM SYTOX Blue solution in PBS(−) at 37 °C for 6 h. Confocal scanning laser microscopy (Leica TCS-SP5 or Nikon C1) was employed to visualize morphologies of fluorescently labeled samples after a 2X PBS(−) wash. To characterize the epithelial lumen-like structure, we obtained Z-stack CLSM images by stacking the XY optical sections to the Z-direction with the thickness of 1 μm. The Z-stack CLSM images were obtained at three depth levels: Z = 0−30 μm, Z = 30−60 μm, and Z = 60−90 μm. Sample morphologies were characterized by measuring the average inner diameter of the lumen (DL) and the number of lumens per unit area (NL). Images were analyzed using ImageJ. To observe the epithelial polarities, Z-stack CLSM images for the sample labeled with the fluorescence antibodies for the polarity markers were recorded at the depth level of Z = 30−60 μm.

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RESULTS AND DISCUSSION Effect of NaCl Concentration on Multichannel Structure of MCCG. Figure 4A shows the CLSM images for the MCCGs prepared at various NaCl concentrations. The average channel diameter (Dc) and channel number (Nc) were measured as a function of the distance from the top of the gel (Z). Zdependences of Dc are shown in Figure 4B. The Dc values as a function of Z, for samples prepared at zero salt concentration, i.e., CNaCl of 0 mM, was similar to that at CNaCl of 1 mM. In contrast, the increase in Dc was rapid near top of the gel at CNaCl of 2 mM. Furthermore, in the Z-range of 40 to 200 μm, Dc significantly increased with increasing CNaCl (p < 0.050). Nc values as a function of Z are shown in Figure 4C. Nc was observed to decrease with increasing Z. Furthermore, Z-dependence of Nc decreased with increasing CNaCl. In the Z-range of 20 to 300 μm, the Nc values decreased significantly with increasing CNaCl at the same Z-position (p < 0.050). These results indicate that the multichannel structure of MCCGs is regulable by varying CNaCl. The total area of the channel region (Sc) in CSLM images can be approximated by the following equation: Sc = Ncπ(Dc/2)2 (1). Sc values as a function of Z are shown in Figure 4D. Although Sc was observed to increase with increasing Z near the top of gel, it was independent of Z in the deeper parts irrespective of CNaCl. In the

Figure 3. CLSM image for the MCCG prepared at 0 mM CNaCl. The magnification is 252× (including digital zoom factor of 4.0 × ). Red arrowhead indicates the Z-coordinate where YZ and ZX optical crosssections were obtained. Blue and green arrow heads indicate the Y and X coordinates where YZ and ZX optical cross-sections were obtained, respectively. White arrow indicates the homogeneous gel membrane formed near top of the MCCG. (A) XY optical cross-section was obtained at the homogeneous gel membrane. (B) XY optical crosssection was obtained at a deeper part of MCCG.

toxicity,30,31 to improve mechanical properties of MCCGs. Chemical cross-linking was allowed to occur by overnight incubation with 1 mg/mL genipin. Following this, MCCGs were washed twice with PBS(−), and further incubated overnight in PBS(−) to remove free genipin and EDTA molecules. This treatment resulted in MCCG scaffolds bearing open multichannel structures. Use of MCCG Scaffolds for Construction of Engineered Tissues. MDCK cells (number of passages, 17) were seeded on a 100 mm polystyrene dish at a seeding density of 1.0 × 106 cells/dish and were cultured for 2 days before use in subsequent experiments. Cells attached to the polystyrene dish were detached by trypsin-EDTA treatment, D


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Figure 4. (A) CLSM images of the cross-section perpendicular to the Z-axis of the MCCGs prepared at various NaCl concentrations. (B) Plot of Dc as a function of Z for MCCGs prepared under various CNaCl conditions. (C) Plot of Nc as a function of Z for MCCGs prepared under various CNaCl conditions. (D) Plot of Sc as a function of Z for the MCCGs prepared under various CNaCl conditions. Error bar indicates standard error of the mean (n = 9).

with fluctuating polymer concentration, which results in randomly mixed regions with high and low polymer concentrations in the solution. The late stage is characterized by coarsening of the regions where the polymer concentrations are approximately equal to those at the equilibrium state. The coarsening of the regions results in an increase in the size of a particular region and thus a decrease in the total number of regions. In the formation of MCCG, phase separation occurs after neutralization of pH, but does not result in a complete separation into two macroscopic phases due to hindrance offered by the collagen gel network. The changes in the dependence of Z on Sc with increasing CNaCl might be attributed to the difference in the stage at which phase separation of collagen solution is constrained by the collagen gel network. As observed from the CLSM images shown in Figure 4A, the fluorescence intensity contrast between the channel regions and the gel matrix region was weak near the top of cylindrical MCCG, indicating that the difference in collagen concentrations between channel regions and gel matrix regions was small. Therefore, this finding was

Z-range of 20 to 300 μm, the Sc values significantly decreased with increasing CNaCl at the same Z-position (p < 0.050). As shown in Figure 4C, Nc for the MCCG prepared at CNaCl = 0 mM was rapidly decreased with increasing Z. By contrast, Nc for the MCCG prepared at CNaCl = 2 mM was almost independent of Z. The difference in Z-dependence of Nc is important in controlling the architecture of epithelial lumens constructed in MCCG. For example, the MCCG prepared at CNaCl = 0 mM could be useful to reproduce the vascular networks where the number of vessels rapidly increases with increasing the distance from the center of tissue. By contrast, the vascular networks in which the number of vessels is almost independent of the distance from the center of tissue might be reconstructed by using the MCCG prepared at CNaCl = 2 mM. We previously demonstrated that the formation of multichannel structures resulted from phase separation and gelation of the collagen solution.16 The phase separation of a polymer solution generally consists of two stages: an initial stage and a late stage.32,33 The initial stage is characterized by a growth process E


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suggestive that the phase separation of collagen solution near the top of the cylindrical MCCG (at small Z) was constrained at the initial stage of phase separation. In contrast, a high fluorescence intensity contrast was observed between the channel regions and the gel matrix region within the deeper parts of the MCCG, indicating a large difference in collagen concentrations among these regions. Therefore, this finding suggested that phase separation of collagen solution within the deeper parts was constrained at the late stage of phase separation. At the initial stages of phase separation, the total volume of channel regions increases with progression of phase separation, because the fluctuation of collagen concentration increases with progression of phase separation. In contrast, the total volume of channel regions is not dependent on the progress of phase separation at the late stage, because collagen concentrations in the diluted and concentrated phases at the late stage of phase separation could be approximately equal to those in macroscopic diluted and concentrated phases at the completely phase separated state,19,34 respectively. CLSM images suggested that phase separation progressed with increasing Z. Therefore, Sc was dependent on Z near the gel surface, whereas it was independent of Z within deeper parts of the gel. A significant decrease in Sc with increasing CNaCl could be attributed to the change in the shape of the phase diagram of collagen solution. According to the Lever rule (the condition of volume conservation), the decrease in the total volume of channel regions resulted in the increase and decrease in the volume fraction of collagen in the channel region and gel matrix region, respectively.19 However, it is difficult to obtain the phase diagram of collagen solution, because we cannot induce the liquid−liquid phase separation of collagen solution without gelation. Therefore, the mechanism of liquid−liquid phase separation of collagen solution should be investigated in future works. In this study, characterization of the multichannel structure was limited near the surface region of MCCG, because the

Figure 5. (A) Time-course changes in the average diameter of low collagen concentration regions. Solid line represents the power law function with the exponent of 1/3. (B) Effect of CNaCl on tp. Error bar indicates standard error of the mean (n = 9). * indicates a significance of p < 0.05.

Figure 6. CLSM image for the epithelial lumen-like structure constructed in MCCG prepared at CNaCl of 0 mM. F


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Figure 7. Cellular polarity of MDCK cells in the epithelial lumen-like structure constructed in MCCG prepared at CNaCl of 0 mM. (A) Upper panels show the localized expression of ezrin (red), α-catenin (green), and F-actin (blue). (B) Lower panels show the localized expression of laminin (red), αcatenin (green), and F-actin (blue). The arrow heads indicate the laminin domains localized at the basal membrane of MDCK cells.

structure of the MCCG. As shown in Figure S2, however, we found that the multichannel structure of MCCG could be scaled by a gel size. The result suggests that the range where the significant difference in multichannel structure exists proportionally increases with increasing the gel size. Therefore, the difference in multichannel structure could not be ignored, even if we prepare the large MCCGs. Effect of NaCl Concentration on Dynamics of Multichannel Structure Formation of MCCGs. As mentioned above, salt concentration affected the phase behavior of collagen solution. Since the composition of collagen solution at the equilibrium state is dependent on its phase behavior, the concentrations of collagen in the diluted phases, which later transform into channel regions, and the concentrated phase, which transforms into the gel matrix region, could change with NaCl concentration. Several studies have shown that collagen concentration affects the kinetics of collagen gelation.21,22 The rate of collagen gelation increases with increasing concentration. As shown in the previous study, formation of a collagen gel network was shown to restrain phase separation.16 The changes in collagen concentration of concentrated phase affected the time from onset of phase separation to accomplishment of collagen gel network, which was represented as the time of pinning (tp). tp was determined by studying the process of collagen phase separation using time-lapse CLSM. Phase separation of collagen solution during its dialysis in PBg was observed at Z = 100 μm and under various CNaCl conditions (CNaCl = 0, 1, and 2 mM). Supporting Movies S1, S2, and S3 show the time course of structural changes in collagen solution during dialysis process. Several pores and a macroscopic network-like structure appeared rapidly during the process of dialysis. The pores represent regions of low collagen concentration (diluted

samples were too thick to observe the multichannel structures at deeper parts by using CLSM. By contrast, we can characterize the multichannel structure near the bottom of MCCG by observing from the bottom side of MCCG. Dc, Nc, and Sc were measured as a function of the distance from the bottom of gel (Z′), respectively. Z′-dependences of Dc, Nc, and Sc were shown in Figure S1. Near the bottom of MCCG, Dc did not depend on Z′, irrespective of CNaCl. In addition, no significant effect of CNaCl on Dc was observed. By contrast, Nc increased with increasing Z′, irrespective of CNaCl. Furthermore, in the Z′-range of 40 to 200 μm, Nc significantly decreased with increasing CNaCl (p < 0.050). Sc gradually increased with increasing Z′, irrespective of CNaCl. In the Z′-range of 100 to 280 μm, Sc for the sample prepared at CNaCl = 0 mM significantly larger than that for the samples prepared at CNaCl = 1 mM and CNaCl = 2 mM (p < 0.050). These results suggest that the multichannel structure near the bottom of MCCGs is also regulable by varying CNaCl. As mentioned above, Sc might be determined by the thermodynamic state of collagen solution. Therefore, the Z′-dependence of Sc near the bottom of MCCG might be attributed to the change in thermodynamic state of collagen solution during the formation process of MCCG. At small Z′, the collagen solution contacts with the surface of polystyrene dish, where the collagen molecules interact with the surface of polystyrene dish. Therefore, we speculate that the change in thermodynamic state of collagen solution is attributable to the interaction between the collagen molecules and the surface of polystyrene dish. In this study, the thickness of MCCGs prepared in this study was only 1 mm. Therefore, one may think that the difference in multichannel structure is ignored for the MCCGs which is much larger than gels prepared in this study and one may question the effectiveness of our method for controlling the multichannel G


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Figure 9. (A) Effect of NaCl concentration on the average inner diameter of the lumen-like structures (DL). (B) Effect of NaCl concentration on the number of epithelial lumen-like structures per unit area (NL) in the multichannel structure of MCCG. Error bar indicates standard error of the mean (n = 4).* indicates a significance of p < 0.05.

diluted phases to grow to larger diameters. An increase in the diameter of diluted phase can be attributed to the coarsening of this phase, which in turn is a result of diffusion of water molecules from the concentrated phase into the diluted phases. The diluted phases possessing smaller diameters were absorbed into the adjacent diluted phases with larger diameters following the phenomenon of “Ostwald ripening” (Movie S1 and S2). Coarsening of the diluted phases results not only in an increase in Dc but also in a decrease in Nc. Construction of Epithelial Lumen-Like Structures using MCCG. MDCK cells were cultured on MCCGs prepared under varying NaCl conditions to construct engineered tissues exhibiting complex hierarchical structures. The CLSM image for the epithelial lumen-like structures constructed in the MCCG prepared at CNaCl = 0 mM is shown in Figure 6, and it showed that the continuous epithelial lumen-like structure was constructed in the MCCG. Although the measurable length of epithelial lumen-like structure was approximately at least 160 μm, the actual length of the structure may be longer than the measured length. We investigated the cellular polarity of MDCK cells in the epithelial lumen-like structure. Figure 7 shows the localized expression of cellular polarity markers of MDCK cells in the epithelial lumen-like structure. Ezrin that is the apical marker was expressed at the inner surface of the epithelial lumen-like structure, whereas laminin that is the basal marker was localized at the basal part of MDCK cells. The results showed that the epithelial lumen-like structure has the apical-basal axis that is similar to the kidney tubules. Furthermore, the CLSM images for the α-catenin localization indicate that the cell−cell adhesion was

Figure 8. CLSM images of tissues constructed by culturing MDCK cells using MCCGs prepared under varying CNaCl concentrations. F-actin and nuclei are colored in green and blue, respectively.

phases), whereas the macroscopic network-like structure represents the region of high collagen concentration (concentrated phases). A boundary between the two phases, which was unclear initially, became sharper with increasing time. The diameter of diluted phases (Dpore) also simultaneously increased with time. However, this increase was slower at later stages of phase separation and may be attributed to formation of a collagen gel network. Ultimately, the collagen solution did not separate into two macroscopic phases, because a phase-separated structure formed during the phase separation was pinned by the formation of the network. Thus, the final collagen structure is determined by the time of pinning. The time course of changes in pore diameter during phase separation is shown in Figure 5A, and the changes are roughly expressed as a power law function with an exponent of 1/3. Our results were in good agreement with the results of a previous study, which indicated that phase separation of collagen solution results from spinodal decomposition.16,32,33,35 The effect of CNaCl on tp is shown in Figure 5B. tp was observed to increase significantly with increasing CNaCl. As mentioned above, increase in CNaCl results in the decrease in the volume fraction of collagen at equilibrium in the concentrated phase. Because the rate of collagen network formation decreases with increasing CNaCl, tp increases with increasing CNaCl. An increase in tp allows the H


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to PBg by using micropipette results in collagen gel fibers with multichannel structures. The multichannel structure of MCCGs can be controlled by the methodology established in this study. Therefore, the present study can provide a method for preparing MCCGs with various shapes and multichannel structures. In the previous study, we proposed a strategy for constructing giant engineered tissues by using the MCCG bead.29 The strategy is based on a bottom-up assembly of mini-engineered tissues constructed by using MCCGs. First, we construct spherical mini-engineered tissues by culturing cells in MCCG beads. The spherical mini-engineered tissue is used as a building block for constructing giant engineered tissues. Then, we assemble the spherical mini-engineered tissues into a sheetlike engineered tissue. The macroscopic structure of the sheet-like engineered tissues can be controlled by arranging the position or alignment of the mini-engineered tissues. Finally, we laminate the sheetlike engineered tissues for constructing the giant engineered tissue. In this strategy, the methodology developed in the present study can allow us to control the structure of miniengineered tissues, and to regulate hierarchical structures of giant engineered tissues from micrometers to centimeters. Therefore, we will investigate the effect of the hierarchical structure on biological functions of giant engineered tissues in future works.

established at the lateral part of MDCK cells. Therefore, MCCG can be used as a template scaffold for constructing the epithelial lumen-like structures with native cellular polarities. The morphological features for the samples prepared at various CNaCl concentrations are shown in Figure 8. As seen in Figure 8, the average inner diameter of lumen (DL) increased with increasing Z, whereas the lumen number per unit area (NL) decreased with increasing Z. The dependence of Z on DL and NL agreed with the dependence of Z on Dc and Nc, as shown in Figure 4B, C. This finding suggests that the multichannel structure of MCCG plays an important role as a template in the case of the epithelial lumen structures. These epithelial lumenlike structures can be constructed using MCCGs without genipin cross-linking, as seen in Figure S3. To evaluate the effect of MCCG multichannel structures on the morphological features of epithelial lumen-like structures, we compared DL and NL at Z = 30−60 μm for both samples. DL and NL for samples constructed using MCCGs prepared at 0 mM NaCl were significantly lower and higher than those at 2 mM NaCl, respectively (Figure 9A, B). These results clearly indicated that the morphologies of epithelial lumen-like structures are controlled by regulating the multichannel structure of MCCGs. Three-dimensional culture of MDCK cells in collagen gels prepared using conventional method lead to “cyst” formation.36 The cyst consists of a spherical monolayer sheet that encloses a central lumen. The hepatocyte growth factor (HGF) induces a morphological change in the epithelial architecture of the cyst into a tubule-like structure37,38 and the HGF-stimulated cyst has been used as a model system for studying the tubulogenesis of epithelial lumens. Interestingly, the epithelial lumen-like structure is constructed by using MCCG independent of HGF stimulation. This suggests that our methodology for construction of epithelial lumen-like structures using MCCG provides an alternative in vitro model system for investigations on tubulogenesis of epithelial lumens. This novel system will prove useful in elucidating the mechanism of tubulogenesis independent of soluble factors, such as HGF and vascular endothelial growth factors. Furthermore, we believe that the complex hierarchical structures of the transport ducts in native tissues and organs can be constructed by using combinations of our methodology with soluble factors.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsbiomaterials.5b00003. Characterization of multichannel structures near the bottom of MCCG (Figure S1), effect of gel size on the multichannel structure of MCCG (Figure S2), and photographs for the epithelial lumen-like structure constructed in MCCG without genipin cross-liking (Figure S3) (PDF) Time-lapse Movie S1 for the formation processes of multichannel structures of MCCGs (AVI) Time-lapse Movie S2 for the formation processes of multichannel structures of MCCGs (AVI) Time-lapse Movie S3 for the formation processes of multichannel structures of MCCGs (AVI)

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CONCLUSION In conclusion, we developed a method to control the multichannel structure of MCCGs. NaCl concentration exerted an effect on the time required to accomplish the formation of collagen gel network, which further restrained phase separation during dialysis. Thus, the final MCCG structure was amenable to change by regulation of NaCl concentration contained in both the collagen and phosphate buffer solution. The proposed methodology is easy to perform, and can be operated at low costs. Further, MCCGs were used as template cell scaffolds for construction of engineered tissues with epithelial lumen-like structures, which could be controlled by using the MCCGs possessing multichannel structures. This methodology allows introduction of transport ducts for oxygen, and nutrients into tissues, as well as removal of waste products from these engineered tissues. MCCGs with various shapes, such as bead, disk, fiber, and cylinder, can be prepared by choosing appropriate preparation methods. For example, we showed that MCCG bead with the diameters of 0.5−2.0 mm can be prepared by adding dropwise the collagen solution to PBg16. Injection of the collagen solution

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Fax: +81-11-706-4493. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS K.F. acknowledges support from the Grant-in-Aid for Scientific Research on Innovative Areas, “Hyper Bio Assembler for 3D Cellular Innovation” (26106703), from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT).

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Application of Multichannel Collagen Gels in Construction of Epithelial

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