This manuscript presents a detailed protocol for the fabrication of an emerging three-dimensional hepatocyte culture platform, the inverted colloidal crystal scaffold, and the concomitant techniques to assess hepatocyte behavior. The size-controllable pores, interconnectivity and ability to conjugate extracellular matrix proteins to the poly(ethylene glycol) (PEG) scaffold enhance Huh-7.5 cell performance.
The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics' cytotoxicity, studying virus infection and developing drugs targeted at the liver, requires a platform in which cells receive proper biochemical and mechanical cues. Recent liver tissue engineering systems have employed three-dimensional (3D) scaffolds composed of synthetic or natural hydrogels, given their high water retention and their ability to provide the mechanical stimuli needed by the cells. There has been growing interest in the inverted colloidal crystal (ICC) scaffold, a recent development, which allows high spatial organization, homotypic and heterotypic cell interaction, as well as cell-extracellular matrix (ECM) interaction. Herein, we describe a protocol to fabricate the ICC scaffold using poly (ethylene glycol) diacrylate (PEGDA) and the particle leaching method. Briefly, a lattice is made from microsphere particles, after which a pre-polymer solution is added, properly polymerized, and the particles are then removed, or leached, using an organic solvent (e.g., tetrahydrofuran). The dissolution of the lattice results in a highly porous scaffold with controlled pore sizes and interconnectivities that allow media to reach cells more easily. This unique structure allows high surface area for the cells to adhere to as well as easy communication between pores, and the ability to coat the PEGDA ICC scaffold with proteins also shows a marked effect on cell performance. We analyze the morphology of the scaffold as well as the hepatocarcinoma cell (Huh-7.5) behavior in terms of viability and function to explore the effect of ICC structure and ECM coatings. Overall, this paper provides a detailed protocol of an emerging scaffold that has wide applications in tissue engineering, especially liver tissue engineering.
The liver is a highly vascularized organ with a multitude of functions, including detoxification of the blood, metabolism of xenobiotics, and the production of serum proteins. Liver tissue has a complex three-dimensional (3D) microstructure, comprising of multiple cell types, bile canaliculi, sinusoids, and zones of different biomatrix composition and different oxygen concentrations. Given this elaborate structure, it has been difficult to create a proper liver model in vitro1. However, there is a rising demand for functional in vitro models hosting human hepatocytes as platforms for testing drug toxicity2 and studying diseases associated with the liver3.
Current liver tissue engineering platforms have simplified the complexity of the liver by isolating one, or focusing on a few, of the liver's parameters, namely co-culture of cells4, biochemical composition of the zonal microenvironments5, flow dynamics6,7 and the configuration of the biomatrix8. Configuration of the biomatrix can be broken into parameters such as scaffold materials, composition of extracellular matrix (ECM) proteins, matrix stiffness as well as the design and structure of the scaffold. There has been a rise in tissue engineering studies using synthetic hydrogels, especially poly(ethylene glycol) (PEG) hydrogels9, given the ability to tune the hydrogel's mechanical properties, bioactivity, and degradation rate. Regarding liver-related research, the biocompatible hydrogel was applied for virus infection study of liver disease3. As a hepatocyte platform design, numerous studies have utilized hepatocyte sandwich cultures10,11 and cell encapsulation within a hydrogel12,13 to provide the 3D environment and cell-ECM and cell-cell interaction which are essential to mimic in vivo microenvironment. However, these platforms do not possess a high degree of control and spatial organization, leading to non-uniform properties through the scaffold14.
The inverted crystal colloidal (ICC)14 scaffold is a highly organized 3D scaffold for cell culture that was first introduced in the early 2000s. The scaffold's unique structure can be attributed to the simple fabrication process using a colloidal crystal, an ordered lattice of colloidal particles of variable diameter. Briefly, to summarize the process, particles are neatly arranged and annealed using heat to form a lattice. The leaching of this lattice, by an organic solvent, in a polymerized hydrogel results in hexagonally packed spherical cavities15 with high surface area. This highly ordered scaffold has been previously made with both synthetic and natural materials, including but not limited to poly(acrylamide)16-21, poly(lactic-co-glycolic acid)15,22-30, poly(ethylene glycol)31,32, poly (2-hydroxyethyl methacrylate)21,33-35, and chitosan36-39. ICC scaffolds made of non-fouling materials tend to promote cellular spheroids within the cavities14,23,40. Multiple cell types have been shown to successfully proliferate, differentiate and function within this configuration, including chondrocytes41, bone marrow stromal cells42, and stem cells43,44. Regarding hepatocyte, studies have been conducted with ICC scaffolds made of Na2SiO3 and poly(acrylamide), but not PEG. With simple bioconjugation strategies (i.e., amine coupling through EDC/NHS), ECM proteins-conjugated PEG-based scaffolds can be fabricated, that can prove more cell binding sites to be a more in vivo like environment and enhance hepatic function.
In this manuscript and the associated video, we detail the fabrication of the ICC scaffold using poly(ethylene glycol) diacrylate (PEGDA) hydrogel and a polystyrene microsphere lattice, optimized for hepatocarcinoma (Huh-7.5) culture. We demonstrate the differences between the generally nonadhesive bare PEGDA ICC scaffolds and the collagen-coated PEGDA ICC scaffold in terms of scaffold topology and cell performance. Cell viability and function are measured qualitatively and quantitatively to assess Huh-7.5 cell behavior.
1. ICC Scaffold Fabrication (Figure 1)
Figure 1. Overview of ICC fabrication. PEG-based ICC scaffolds are fabricated using microfabrication techniques with and without ECM-functionalization. ECM-coated ICC scaffolds require PEG-NHS as well as PEGDA (as detailed in Figure 2). The PS lattice has a diameter of 6 mm and a height of 8-13 bead layers. PS, polystyrene; PEGDA, poly (ethylene glycol) diacrylate; UV, ultraviolet; THF, tetrahydrofuran; ECM, extracellular matrix. This figure has been modified and used with permission from Wiley47. Please click here to view a larger version of this figure.
2. ICC Structure Characterization
3. Huh-7.5 Cell Culture and Seeding
4. Cell Viability
5. Cell Function
The representative results for the structural characterization of the ICC scaffold and the comparison of each ICC scaffold condition's efficacy in culturing hepatocytes are shown and explained below. The ICC scaffold conditions used in these results are collagen coatings of 0 µg/ml (Bare), 20 µg/ml (Collagen 20), 200 µg/ml (Collagen 200), and 400 µg/ml (Collagen 400) and the initial Huh-7.5 cell seeding number is 1×106.
Characterization of ICC pore interconnections and scaffold topology.
SEM imaging, after fixation and freeze drying of the ICC scaffolds, was first used to determine whether proper interconnections between ICC pores were formed and to see the effect of the conjugation of varied collagen concentrations on the scaffold topology. Quantitative analysis of the two-dimensional bare ICC SEM images (Figure 3A) using ImageJ software, revealed an average pore diameter of 102.3 ± 9.3 µm and an average interconnection diameter of 38.6 ± 4.3 µm (Figure 3B, C). This interconnection between pores plays a significant role in proper diffusion of nutrients to cells as well as cell-cell interaction throughout the scaffold. The coating of collagen resulted in a fiber mesh network that was more defined at higher concentrations of collagen conjugation (Figure 4A). Confocal images reveal even layers of collagen on the cavity surfaces and higher surface area coverage with increases in the collagen concentration (Figure 4B).
Effect of ICC platform conditions on Huh-7.5 cell viability and function.
Huh-7.5 cells were subjected to the fluorescent Live/Dead staining assay (4 µM calcein AM and 8 µM EthD-1) and imaged using CLSM, as shown in Figure 5. Cells seeded in bare, non-adhesive ICC scaffolds tended to aggregate in the center of the pore and cell-cell interconnection between pores was seen in later times (Days 7 and 10). The presence of a collagen coating on the ICC scaffolds allowed cells to adhere to the scaffold surface as well a inter-pore cell-cell interaction as early as Day 1. Cell viability, indicated by the green stain, increased with time and was generally higher in collagen-coated scaffolds, as indicated by higher green fluorescence. Figure 6 shows the quantitative MSR results for further investigation of the effect of the ICC 3D structure and protein concentration on cell viability. A colorimetric cell viability assay was performed on cells cultured in the ICC scaffold conditions as well as on a 2D polystyrene culture plate and absorbance data (measured at 450 nm) was normalized to the control (Day 1). Cells cultured on 2D plates maintained cell viability but no increase in cell proliferation was observed. 3D bare ICC scaffold greatly enhanced cell proliferation compared to the 2D condition, indicating the importance of the 3D matrix. With the addition of the collagen coating, cell proliferation increased with time at all collagen concentrations and the maximum proliferation was found in the 200 µg/ml coated ICC scaffold on Day 14. This confirms qualitative observations in Figure 5.
Hepatocyte function was assessed by monitoring changes in albumin secretion as well the gene expression profile of three adhesion proteins in the different ICC scaffold conditions. Figure 7 illustrates the positive correlation between serum albumin secreted, as quantified by albumin ELISA, and the collagen concentration conjugated to the scaffold. On Day 14, Huh-7.5 cells cultured in the 400 µg/ml coated ICC scaffolds secreted more than three times the amount of albumin as those cultured in the bare scaffold. Results for the gene expression profiles of E-cadherin, N-cadherin, and integrin β1 proteins in the different ICC scaffold conditions appear in Figure 8. The rate of E-cadherin mRNA expression in 400 µg/ml coated ICC scaffolds increased by more than 4 folds as compared to the other coating conditions (Figure 8A). The fold change in N-cadherin gene expression was greater in the higher concentrations of collagen coating. However, the integrin β1 gene expression stayed relatively constant or decreased in all ICC scaffolds conditions except 200 µg/ml coated ICC scaffolds.
Figure 2. Conjugation of collagen to the PEGDA scaffold via NHS chemistry. Bioactive scaffolds use PEG-NHS that has an amine-reactive succinimidyl (NHS) ester that reacts to the amine group in collagen allowing conjugation to the scaffold. PEG, poly(ethylene glycol); NHS, N-hydroxysuccinimide; UV, ultraviolet. Please click here to view a larger version of this figure.
Figure 3. Analysis of the size of the ICC cavities and interconnections. (A) SEM micrographs of the ICC scaffold (scale bar = 100 µm) were analyzed using ImageJ software and quantitatively represented as histograms of (B) pore diameter and (C) interconnection diameter. This figure has been modified and used with permission from Wiley47. Please click here to view a larger version of this figure.
Figure 4. Influence of collagen coating on ICC scaffold topology. (A) SEM images and (B) confocal images were taken to qualitatively assess the surface topology of scaffolds coated with 20 µg/ml, 200 µg/ml, and 400 µg/ml of collagen. Red boxes surround the cavity shown in higher magnification images below. Scale bars are (A) 5 µm, (B) 200 µm and 100 µm (higher magnification). This figure has been modified and used with permission from Wiley47. Please click here to view a larger version of this figure.
Figure 5. Effect of ICC platform conditions on cell viability and morphology using qualitative Live/Dead assay. Confocal images of the Live/Dead stained Huh-7.5 cells seeded in ICC scaffolds with different collagen concentration coatings (0, 20, 200, and 400 µg/ml) were taken 1, 4, 7, and 10 days after seeding. Green stain (calcein) indicates live cells and red stain (Ethidium homodimer-1) indicates cell death. Scale bars indicate 200 µm. [This figure has been modified and used with permission from Wiley47] Please click here to view a larger version of this figure.
Figure 6. Effect of platform type and ICC platform conditions on cell viability using a quantitative colorimetric cell viability assay. Huh-7.5 cells seeded on a 2D polystyrene culture plate and in ICC scaffolds with different collagen concentration coatings (0, 20, 200, and 400 µg/ml) were subjected to the colorimetric MSR viability assay 1, 4, 7, 10, and 14 days after seeding. Absorbance was measured at 450 nm and the data were normalized to Day 1 absorbance values. (n = 3, mean ± SD; ***: P < 0.001 compared to MSR solution absorbance reading for Day 1 of each group.) [This figure has been modified and used with permission from Wiley47] Please click here to view a larger version of this figure.
Figure 7. Effect of ICC platform condition on Huh-7.5 function using secreted albumin concentration ELISA analysis. Albumin ELISA was used to quantify the serum albumin secretion in media collected from cell-laden ICC scaffolds with different collagen concentration coatings (0, 20, 200, and 400 µg/ml) on Days 1, 4, 7, 10, and 14 after seeding. Each data point represents an average of 3 samples and is normalized to the number of cells found using the colorimetric MSR cell viability assay and the created standard curve (n = 3, mean ± SD). [This figure has been modified and used with permission from Wiley47] Please click here to view a larger version of this figure.
Figure 8. Effect of ICC platform condition on Huh-7.5 function using gene expression analysis. Real-time quantitative PCR was used to profile gene expression of cells cultured in ICC scaffolds with different collagen concentration coatings (0, 20, 200, and 400 µg/ml) on Days 1, 4, and 7 after seeding. Three junction proteins were chosen, namely (A) E-cadherin, (B) N-cadherin, and (C) Integrin Beta 1. The mRNA expression levels were normalized by GAPDH. (n = 3, mean ± SD. *: P < 0.05. **: P < 0.01 compared to gene expression of Day 1 each group.) [This figure has been modified and used with permission from Wiley47] Please click here to view a larger version of this figure.
Tissue engineering scaffolds are rapidly evolving to provide all the physical and biochemical cues necessary to regenerate, maintain, or repair tissues for the application of organ replacement, studying disease, developing drugs, and many others57. In liver tissue engineering, primary human hepatocytes rapidly lose their metabolic functions once isolated from the body, creating a great need for engineering scaffolds and developing platforms to maintain the hepatic function. The current in vitro hepatocyte culture platforms have utilized different biomaterials. The research in this area has been centered on mimicking various features of the in vivo hepatic microenvironment, such as ECM protein configuration58,59, co-culture60,61, and micropatterning62. However, there has been a lack of scaffolds with controlled porosity and high spatial organization. In this regard, the described ICC cell culture platform, with its isotropic properties and high organization, addresses this void. We demonstrate the ICC PEGDA scaffold is a suitable platform for culturing hepatocarcinoma cells, a model cell type for hepatocytes, and that further collagen coating enhances cell behavior47.
In practice, the cavity size of the ICC scaffold can be tuned by the size of PS microspheres. In addition, the interconnected size of the scaffold can be controlled by the time and temperature of annealing process; higher annealing temperature or longer annealing time results in larger interconnection diameters. Therefore, these parameters should be selected carefully as larger interconnection diameters will compromise the mechanical stability of the scaffold. In this work, PS spheres of diameter 140 µm were chosen for hepatocyte cell culture to limit the size of the resulting hepatospheres in the bare ICC scaffolds in order to prevent necrosis of cell in the center of the hepatocyte spheroid.63 As shown in representative results, the 3D architecture of the ICC scaffold allows higher Huh-7.5 cell proliferation in comparison to 2D polystyrene plate cell culture. The results suggest that the interconnected uniform cavities of the ICC scaffold allow efficient cell-cell interaction and crosstalk between the cavities.
The concentration of the collagen type I coating affected cell morphology, viability and function. Collagen type I is an important ECM protein found in the Space of Disse, an area adjacent to the hepatocytes64. Nonadhesive, bare PEGDA ICC scaffolds promoted hepatocyte spheroid formation whereas, a collagen coating rendered the scaffold bioactive and Huh-7.5 cells adhered to the surface of the scaffold, utilizing the large surface area present. The ECM protein-coated ICC scaffolds improved both cell-cell interaction as well as cell-ECM interaction, as indicated by the upregulated E-cadherin, N-cadherin and Integrin β1 gene expressions, which play a role in regulating cell behavior. Collagen type I concentrations of 200 µg/ml and 400 µg/ml were suitable for Huh-7.5 culture, improving both cell proliferation and albumin secretion.
The main limitation posed by the ICC fabrication is control over the PS sphere alignment and number when making the lattices. The quick evaporation of ethanol while loading the spheres into the molds can cause different sphere densities, resulting in a different number of layers of spheres in lattices. However, the use of less volatile solution like water also has disadvantages. It lead to less ordered CCs in floating assembly system65, and there is a high possibility of a highly-curved scaffold surface due to meniscus formation. Another limitation is the proper alignment of spheres in the molds to ensure the highest efficiency of interconnections. The shaking step (step 1.1.5 and 1.1.6) is therefore critical to the ICC fabrication technique. If a good arrangement is not observed by microscopy, repeat step 1.1.6.
Overall, the ICC PEGDA scaffold, with its simple fabrication protocol, can be used conveniently as a platform for 3D cell culture applications. Bioactivation of the bare scaffold with proteins further enhances its functional characteristics14. In this manuscript, we tailored the fabrication protocol to and chose cell assessment assays for liver tissue engineering application. However, a range of cell types can be cultured within this unique scaffold and each cell type may require the change of certain parameters. Also layers of complexity in terms of multiple ECM protein types, co-culture, and dynamic culture using a bioreactor can all be added to enhance cell performance even further. This platform has the potential to aid in tissue regeneration and drug development, to study liver diseases, and to be used for transplantation.
The authors have nothing to disclose.
The authors wish to acknowledge support from a National Research Foundation Fellowship (NRF -NRFF2011-01) and Competitive Research Programme (NRF-CRP10-2012-07).
0.2 mL PCR tube | Axygen Scientific | PCR-02D-C | Boil-proof |
Gorilla Glue | Gorilla Glue, Inc. | Depends on vendor. This was purchased from a local store. | |
Glass slides | VWR | 631-1575 | Dimensions: 24×60 mm |
Polystyrene spheres | Fisher Scientific | TSS#4314A | Diameter = 140 um; 3×10^4 particles per milliliter and 1.4% size distribution |
Ethanol | Merck | 1.00983.1011 | absolute for analysis EMSURE; Dilute to 70% with Milli-Q water |
Ultrasonic Bath | Elma | S10H | Equiment |
Furnace | Nabertherm | N7/H | Equipment |
200 µL pipette tip | Axygen Scientific | T-210-Y-R-S | |
Rocking shaker | VWR | 444-0142 | |
Polyethylene Glycol (PEG) | Merck | 1.09727.0100 | Mw= 4kDa; acrylation of PEG monomers and purification of the resulting precipitate produces a PEGDA macromer with Mw = 4.6kDa |
Centrifuge | Beckman Coulter | 392932 | Equipment |
Acrylate-Poly (Ethylene Glycol) – Succinimidyl Valerate | Laysan Bio | ACRL-PEG-SVA-3400-1g | Mw = 3.4 kDa |
2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone | Sigma Aldrich | 410896 | |
Vortex | VWR | 58816-123 | Equipment |
Microcentrifuge | Eppendorf | 5404 000.413 | |
Paraffin Film | Parafilm M | #PM996 | Kept at 9" with allows intensity of 10.84 mW/cm^2 |
Bluewave 200 UV spotlight | Blaze Technology | 120008, 122300 | |
Tetrahydrofuran (THF) | Merck | 107025 | |
Orbital shaker | Heidolph | 543-123120-00-5 | From rat |
Collagen Type I | Sigma Aldrich | C3867-1VL | 1X, w/o CaCl & MgCl; Ph = 7.2 |
Phosphate Buffered Saline (PBS) | Gibco | 20012-027 | 16% W/V AQ. 10x10ml |
Paraformaldehyde | VWR | 43368.9M | Equipment |
Freezone 4.5 freeze drier | Labconco | 7750020 | Equipment |
Sputter coater | Jeol Ltd. | JFC-1600 | Equipment |
Scanning Electron Microscope | Jeol Ltd. | JSM 5310 | |
Anti-mouse primary antibodies against Collagen type I | Abcam | ab6308 | |
Anti-mouse secondary antibody conjugated with Alexa Fluor 488 | Life Technologies | A21121 | |
Plate, Tissue Culture 24 Well, Flat Bottom (Nunclon) | Bio-Rev PTE LTD | 3820-024 | |
Dulbecco's Modified Eagle's Medium(DMEM) 2.5 g/L Glucose w/ L-Gln |
Lonza | 12-604F | |
Fetal Bovine Serum (FBS) | Gibco | A15-151 | |
Penicillin-Streptomycin (P/S) | Life Tchnologies | 15140-122 E | |
APC49‐Huh ‐7.5 Cell Line | Apath | ||
100 mm Corning non-treated culture dishes | Sigma Aldrich | CLS430591 | |
0.25% Trypsin-EDTA | Gibco | 25200-056 | Equipment; 37°C, 5% Humidity |
Forma Steri-Cycle CO2 Incubators | Thermofisher Scientific | 371 | |
Hausser Bright-Line Phase Hemacytometer | Thermofisher Scientific | 02-671-6 | |
Live/Dead Viability/Cytotoxicity Kit 'for mammalian cells | Life Technologies | L3224 | |
CCK-8 Assay | Dojindo Laboratories | CK04-11 | Monosodium-salt reagent (MSR) |
Infinite 200 PRO microplate reader | Tecan | ||
Albumin Human ELISA kit | Abcam | ab108788 | |
Triton X-100 | Bio-Rad | #1610407 | |
Bovine Serum Albumin (BSA) | Sigma-Aldrich | A2153-50G | |
Anti-mouse primary antibodies (against CYP3A4, albumin) | Santa Cruz Biotechnology | sc-53850; sc-271605 | |
DAPI | Life Technologies | D3571 | |
Alexa Fluor 555 labelled Phalloidin | Life Technologies | A34055 | |
Trizol | Life Technologies | 15596-026 | |
Chloroform | VWR | 22706.326 | |
Isopropanol | Fisher Scientific | 67-63-0 | |
DPEC water | Thermofisher Scientific | AM9916 | |
Nanodrop 2000c Spectrophotometer | Thermofisher Scientific | ND-2000 | |
iScript Reverse Transcription Supermix | Bio-Rad Laboratories | 1708840 | |
SYBR select Master Mix for CFX | Life Technology | 4472937 | |
Primers (to be chosen) | |||
CFX96 Real-Time System, C-1000 Touch Thermal Cycler | Bio Rad Laboratories | SOFT-CFX-31-PATCH |