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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Bioengineering

Fabrication of Inverted Colloidal Crystal Poly(ethylene glycol) Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

Published: August 27th, 2016

DOI:

10.3791/54331

1School of Materials Science and Engineering, Nanyang Technological University, 2School of Chemical and Biomedical Engineering, Nanyang Technological University

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 ....

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1. ICC Scaffold Fabrication (Figure 1)

  1. Prepare the polystyrene (PS) lattices (diameter = 6 mm; 8-13 layers of beads).
    1. To prepare the mold, cut the tips off from 0.2 ml boil-proof microcentrifuge tubes at the 40 µl level. Adhere the top of the cut-tubes to 24 x 60 mm2 microscope cover glass slips with water-proof glue.
    2. Put the PS spheres (diameter = 140 µm) contained within a water suspension into a 20 ml vial, carefully pipette out the water suspension, and add 18 m.......

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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 1x106.

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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 hepato.......

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The authors wish to acknowledge support from a National Research Foundation Fellowship (NRF -NRFF2011-01) and Competitive Research Programme (NRF-CRP10-2012-07).

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Name Company Catalog Number Comments
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; 3x10^4 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 

  1. Yamada, M., et al. Controlled formation of heterotypic hepatic micro-organoids in anisotropic hydrogel microfibers for long-term preservation of liver-specific functions. Biomaterials. 33 (33), 8304-8315 (2012).
  2. Abboud, G., Kaplowitz, N. Drug-induced liver injury. Drug Safety. 30 (4), 277-294 (2007).
  3. Cho, N. J., et al. Viral infection of human progenitor and liver-derived cells encapsulated in three-dimensional PEG-based hydrogel. Biomed Mater. 4 (1), (2009).
  4. Revzin, A., et al. Designing a hepatocellular microenvironment with protein microarraying and poly (ethylene glycol) photolithography. Langmuir. 20 (8), 2999-3005 (2004).
  5. Sato, A., Kadokura, K., Uchida, H., Tsukada, K. An in vitro hepatic zonation model with a continuous oxygen gradient in a microdevice. Biochem Bioph Res Com. 453 (4), 767-771 (2014).
  6. Domansky, K., et al. Perfused multiwell plate for 3D liver tissue engineering. Lab Chip. 10 (1), 51-58 (2010).
  7. Hegde, M., et al. Dynamic interplay of flow and collagen stabilizes primary hepatocytes culture in a microfluidic platform. Lab Chip. 14 (12), 2033-2039 (2014).
  8. Flaim, C. J., Chien, S., Bhatia, S. N. An extracellular matrix microarray for probing cellular differentiation. Nat methods. 2 (2), 119-125 (2005).
  9. Underhill, G. H., Chen, A. A., Albrecht, D. R., Bhatia, S. N. Assessment of hepatocellular function within PEG hydrogels. Biomaterials. 28 (2), 256-270 (2007).
  10. Dunn, J., Tompkins, R. G., Yarmush, M. L. Hepatocytes in collagen sandwich: evidence for transcriptional and translational regulation. J cell biol. 116 (4), 1043-1053 (1992).
  11. Dunn, J. C., Tompkins, R. G., Yarmush, M. L. Long-term in vitro function of adult hepatocytes in a collagen sandwich configuration. Biotechnol progr. 7 (3), 237-245 (1991).
  12. Ling, Y., et al. A cell-laden microfluidic hydrogel. Lab Chip. 7 (6), 756-762 (2007).
  13. Kim, M., Lee, J. Y., Jones, C. N., Revzin, A., Tae, G. Heparin-based hydrogel as a matrix for encapsulation and cultivation of primary hepatocytes. Biomaterials. 31 (13), 3596-3603 (2010).
  14. Kotov, N. A., et al. Inverted Colloidal Crystals as Three-Dimensional Cell Scaffolds. Langmuir. 20 (19), 7887-7892 (2004).
  15. Shanbhag, S., Woo Lee, J., Kotov, N. Diffusion in three-dimensionally ordered scaffolds with inverted colloidal crystal geometry. Biomaterials. 26 (27), 5581-5585 (2005).
  16. Lee, Y. H., Huang, J. R., Wang, Y. K., Lin, K. H. Three-dimensional fibroblast morphology on compliant substrates of controlled negative curvature. Integr Biol. 5, 1447-1455 (2013).
  17. da Silva, J., Lautenschlager, F., Kuo, C. H. R., Guck, J., Sivaniah, E. 3D inverted colloidal crystals in realistic cell migration assays for drug screening applications. Integr Biol. 3, 1202-1206 (2011).
  18. da Silva, J., Lautenschlager, F., Sivaniah, E., Guck, J. R. The cavity-to-cavity migration of leukaemic cells through 3D honey-combed hydrogels with adjustable internal dimension and stiffness. Biomaterials. 31, 2201-2208 (2010).
  19. Lee, J., Lilly, G. D., Doty, R. C., Podsiadlo, P., Kotov, N. A. In vitro toxicity testing of nanoparticles in 3D cell culture. Small. 5, 1213-1221 (2009).
  20. Lee, J., Kotov, N. A. Notch ligand presenting acellular 3D microenvironments for ex vivo human hematopoietic stem-cell culture made by layer-by-layer assembly. Small. 5, 1008-1013 (2009).
  21. Liu, Y., et al. Rapid aqueous photo-polymerization route to polymer and polymer-composite hydrogel 3D inverted colloidal crystal scaffolds. J Biomed Mater Res. Part A. 83, 1-9 (2007).
  22. Ma, P. X., Choi, J. W. Biodegradable polymer scaffolds with well-defined interconnected spherical pore network. Tissue Eng. 7, 23-33 (2001).
  23. Cuddihy, M. J., Kotov, N. A. Poly (lactic-co-glycolic acid) bone scaffolds with inverted colloidal crystal geometry. Tissue Eng Part A. 14, 1639-1649 (2008).
  24. Choi, S. W., Zhang, Y., Xia, Y. Three-dimensional scaffolds for tissue engineering: the importance of uniformity in pore size and structure. Langmuir. 26, 19001-19006 (2010).
  25. Choi, S. W., Zhang, Y., Thomopoulos, S., Xia, Y. In vitro mineralization by preosteoblasts in poly(DL-lactide-co-glycolide) inverse opal scaffolds reinforced with hydroxyapatite nanoparticles. Langmuir. 26, 12126-12131 (2010).
  26. Choi, S. W., Zhang, Y., Macewan, M. R., Xia, Y. Neovascularization in biodegradable inverse opal scaffolds with uniform and precisely controlled pore sizes. Adv Healthc Mater. 2, 145-154 (2013).
  27. Zhang, Y., Choi, S. W., Xia, Y. Modifying the Pores of an Inverse Opal Scaffold With Chitosan Microstructures for Truly Three-Dimensional Cell Culture. Macromol Rapid Commun. 33, 296-301 (2012).
  28. Cai, X., et al. Investigation of neovascularization in three-dimensional porous scaffolds in vivo by a combination of multiscale photoacoustic microscopy and optical coherence tomography. Tissue Eng. Part C, Meth. 19, 196-204 (2013).
  29. Zhang, Y. S., Yao, J., Wang, L. V., Xia, Y. Fabrication of Cell Patches Using Biodegradable Scaffolds with a Hexagonal Array of Interconnected Pores (SHAIPs). Polymer. 55, 445-452 (2014).
  30. Zhang, Y. S., Regan, K. P., Xia, Y. Controlling the Pore Sizes and Related Properties of Inverse Opal Scaffolds for Tissue Engineering Applications. Macromol Rapid Commun. 34, 485-491 (2013).
  31. Stachowiak, A. N., Bershteyn, A., Tzatzalos, E., Irvine, D. J. Bioactive Hydrogels with an Ordered Cellular Structure Combine Interconnected Macroporosity and Robust Mechanical Properties. Adv Mater. 17, 399-403 (2005).
  32. Stachowiak, A. N., Irvine, D. J. Inverse opal hydrogel-collagen composite scaffolds as a supportive microenvironment for immune cell migration. J Biomed Mater Res. Part A. 85, 815-828 (2008).
  33. Liu, Y., Wang, S. 3D inverted opal hydrogel scaffolds with oxygen sensing capability. Colloids and surfaces. B, Biointerfaces. 58, 8-13 (2007).
  34. Bryant, S. J., Cuy, J. L., Hauch, K. D., Ratner, B. D. Photo-patterning of porous hydrogels for tissue engineering. Biomaterials. 28, 2978-2986 (2007).
  35. Bhrany, A. D., Irvin, C. A., Fujitani, K., Liu, Z., Ratner, B. D. Evaluation of a sphere-templated polymeric scaffold as a subcutaneous implant. JAMA facial plastic surgery. 15, 29-33 (2013).
  36. Kuo, Y. C., Chiu, K. H. Inverted colloidal crystal scaffolds with laminin-derived peptides for neuronal differentiation of bone marrow stromal cells. Biomaterials. 32 (3), 819-831 (2011).
  37. Yang, J. T., Kuo, Y. C., Chiu, K. H. Peptide-modified inverted colloidal crystal scaffolds with bone marrow stromal cells in the treatment for spinal cord injury. Colloids Surf. B, Biointerfaces. 84, 198-205 (2011).
  38. Kuo, Y. C., Tsai, Y. T. Inverted colloidal crystal scaffolds for uniform cartilage regeneration. Biomacromolecules. 11, 731-739 (2010).
  39. Choi, S. W., Xie, J., Xia, Y. Chitosan-Based Inverse Opals: Three-Dimensional Scaffolds with Uniform Pore Structures for Cell Culture. Adv Mater. 21, 2997-3001 (2009).
  40. Long, T. J., Sprenger, C. C., Plymate, S. R., Ratner, B. D. Prostate cancer xenografts engineered from 3D precision-porous poly(2-hydroxyethyl methacrylate) hydrogels as models for tumorigenesis and dormancy escape. Biomaterials. 35, 8164-8174 (2014).
  41. Kuo, Y. C., Tsai, Y. T. Inverted colloidal crystal scaffolds for uniform cartilage regeneration. Biomacromolecules. 11, 731-739 (2010).
  42. Kuo, Y. C., Chiu, K. H. Inverted colloidal crystal scaffolds with laminin-derived peptides for neuronal differentiation of bone marrow stromal cells. Biomaterials. 32, 819-831 (2011).
  43. Lee, J., Cuddihy, M. J., Cater, G. M., Kotov, N. A. Engineering liver tissue spheroids with inverted colloidal crystal scaffolds. Biomaterials. 30 (27), 4687-4694 (2009).
  44. Galperin, A., et al. Integrated bi-layered scaffold for osteochondral tissue engineering. Adv Healthc Mater. 2, 872-883 (2013).
  45. Waters, D. J., et al. Morphology of Photopolymerized End-linked Poly(ethylene glycol) Hydrogels by Small Angle X-ray Scattering. Macromolecules. 43 (16), 6861-6870 (2010).
  46. Elbert, D. L., Hubbell, J. A. Conjugate addition reactions combined with free-radical cross-linking for the design of materials for tissue engineering. Biomacromolecules. 2 (2), 430-441 (2001).
  47. Kim, M. H., et al. Biofunctionalized Hydrogel Microscaffolds Promote Three-Dimensional Hepatic Sheet Morphology. Macromol Biosci. , (2015).
  48. Ferreira, T., Rasband, W. . ImageJ User Guide. , (2012).
  49. JoVE Science Education Database. . General Laboratory Techniques. Introduction to Fluorescence Microscopy. , (2015).
  50. Tominaga, H., et al. A water-soluble tetrazolium salt useful for colorimetric cell viability assay. Anal Commun. 36 (2), 47-50 (1999).
  51. JoVE Science Education Database. . General Laboratory Techniques. Introduction to the Microplate Reader. , (2015).
  52. JoVE Science Education Database. . Basic Methods in Cellular and Molecular Biology. The ELISA Method. , (2015).
  53. Nolan, T., Hands, R. E., Bustin, S. A. Quantification of mRNA using real-time RT-PCR. Nat Protoc. 1, 1559-1582 (2006).
  54. JoVE Science Education Database. . Essentials of Environmental Microbiology. RNA Analysis of Environmental Samples Using RT-PCR. , (2016).
  55. JoVE Science Education. . Essentials of Environmental Microbiology. , (2015).
  56. Jeong, S., et al. The evolution of gene regulation underlies a morphological difference between two Drosophila sister species. Cell. 132 (5), 783-793 (2008).
  57. Griffith, L. G., Naughton, G. Tissue engineering--current challenges and expanding opportunities. Science. 295 (5557), 1009-1014 (2002).
  58. Hegde, M., et al. Dynamic Interplay of Flow and Collagen Stabilizes Primary Hepatocytes Culture in a Microfluidic Platform. Lab Chip. 14, 2033-2039 (2014).
  59. Kim, Y., Lasher, C. D., Milford, L. M., Murali, T., Rajagopalan, P. A comparative study of genome-wide transcriptional profiles of primary hepatocytes in collagen sandwich and monolayer cultures. Tissue Eng Pt C. 16 (6), 1449-1460 (2010).
  60. Baimakhanov, Z., et al. Efficacy of multi-layered hepatocyte sheet transplantation for radiation-induced liver damage and partial hepatectomy in a rat model. Cell Transplant. , (2015).
  61. Li, C. Y., et al. Micropatterned Cell-Cell Interactions Enable Functional Encapsulation of Primary Hepatocytes in Hydrogel Microtissues. Tissue Eng Pt A. 20 (15-16), 2200-2212 (2014).
  62. Shlomai, A., et al. Modeling host interactions with hepatitis B virus using primary and induced pluripotent stem cell-derived hepatocellular systems. P Natl A Sci USA. 111 (33), 12193-12198 (2014).
  63. Curcio, E., et al. Mass transfer and metabolic reactions in hepatocyte spheroids cultured in rotating wall gas-permeable membrane system. Biomaterials. 28, 5487-5497 (2007).
  64. Martinez-Hernandez, A., Amenta, P. The hepatic extracellular matrix. Vichows Archiv A Pathol Anat. 423, 1-11 (1993).
  65. Liu, Y., Wang, S., Lee, J. W., Kotov, N. A. A Floating Self-Assembly Route to Colloidal Crystal Templates for 3D Cell Scaffolds. Chem Mater. 17 (20), 4918-4924 (2005).

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