We previously reported the development of a three-dimensional cellulosic sponge capable of constraining hepatocytes within macropores to support the rapid formation of organoids with preserved hepatocyte functions for hepatotoxicity testing applications. Fabrication of this macroporous sponge involves conjugating allyl groups onto hydroxypropyl cellulose (HPC) to serve as crosslinking sites during gamma irradiation following thermally-induced phase separation. However, this method requires the use of moisture-sensitive reagents and unstable organic solvents which introduces batch-to-batch variability. To address this problem, we developed a cellulosic sponge system which replaces the use of allyl groups as cross-linkers with methacrylic groups to generate methacrylic-HPC (MA-HPC) under fully aqueous conditions. The resulting MA-HPC sponge contains macropores (94 ± 8 μm, 90% porosity) to constrain cells to form organoids, and has an average elastic modulus of 8.5 kPa that is close to the modulus of native rat and human livers. We demonstrate that similar to the allyl-based sponge, the MA-HPC sponge reliably supports human hepatocyte organoid culture and maintains high level cellular functions for at least 1 week in culture, thereby providing a reliable alternative to the existing allyl-based sponge for organoid culture.
This is a preview of subscription content, log in to check access.
We’re sorry, something doesn't seem to be working properly.
Please try refreshing the page. If that doesn't work, please contact support so we can address the problem.
Ananthanarayanan A, Nugraha B, Triyatni M et al (2014) Scalable spheroid model of human hepatocytes for hepatitis C infection and replication. Mol Pharm. https://doi.org/10.1021/mp500063y
Fang JM, Fowler PA, Sayers C, Williams PA (2004) The chemical modification of a range of starches under aqueous reaction conditions. Carbohydr Polym. https://doi.org/10.1016/j.carbpol.2003.10.003
Fong ELS, Harrington DA, Farach-Carson MC, Yu H (2016) Heralding a new paradigm in 3D tumor modeling. Biomaterials 108:197–213
Gao J, Haidar G, Lu X, Hu Z (2001) Self-association of hydroxypropylcellulose in water. Macromolecules. https://doi.org/10.1021/ma001631g
Haycock J (eds) (2011) 3D cell culture. Methods in molecular biology (methods and protocols), vol 695. Humana Press
Heinze T, Liebert T (2001) Unconventional methods in cellulose functionalization. Prog Polym Sci 26(9):1689–1762
Hollister SJ (2005) Porous scaffold design for tissue engineering. Nat Mater. https://doi.org/10.1038/nmat1421
Joabsson F, Rosén O (1998) Phase behavior of a “clouding” nonionic polymer in water Effects of hydrophobic modification and added surfactant on phase compositions. J Phys Chem B 102(16):2954–2959. https://doi.org/10.1021/jp980108p
Loh QL, Choong C (2013) Three-dimensional scaffolds for tissue engineering applications: role of porosity and pore size. Tissue Eng Part B Rev. https://doi.org/10.1089/ten.teb.2012.0437
Meyer U, Meyer T, Handschel J, Wiesmann HP (2009) Fundamentals of tissue engineering and regenerative medicine. Springer, Berlin
Mikos AG, Temenoff JS (2000) Formation of highly porous biodegradable scaffolds for tissue engineering. Electron J Biotechnol 3(2):23–24
Montalbetti CAGN, Falque V (2005) Amide bond formation and peptide coupling. Tetrahedron 61(46):10827–10852
Murphy SV, Atala A (2014) 3D bioprinting of tissues and organs. Nat Biotechnol 32(8):773
Novotna K, Havelka P, Sopuch T et al (2013) Cellulose-based materials as scaffolds for tissue engineering. Cellulose. https://doi.org/10.1007/s10570-013-0006-4
Nugraha B, Hong X, Mo X et al (2011) Galactosylated cellulosic sponge for multi-well drug safety testing. Biomaterials. https://doi.org/10.1016/j.biomaterials.2011.05.087
Pampaloni F, Reynaud EG, Stelzer EHK (2007) The third dimension bridges the gap between cell culture and live tissue. Nat Rev Mol Cell Biol 8(10):839
Roulot D, Czernichow S, Le Clésiau H et al (2008) Liver stiffness values in apparently healthy subjects: influence of gender and metabolic syndrome. J Hepatol. https://doi.org/10.1016/j.jhep.2007.11.020
Sachlos E, Czernuszka JT, Gogolewski S, Dalby M (2003) Making tissue engineering scaffolds work Review on the application of solid freeform fabrication technology to the production of tissue engineering scaffolds. Eur Cells Mater 5(29):39–40
Schafer ZT, Grassian AR, Song L et al (2009) Antioxidant and oncogene rescue of metabolic defects caused by loss of matrix attachment. Nature. https://doi.org/10.1038/nature08268
Souza GR, Molina JR, Raphael RM et al (2010) Three-dimensional tissue culture based on magnetic cell levitation. Nat Nanotechnol. https://doi.org/10.1038/nnano.2010.23
Tanaka H (2012) Viscoelastic phase separation in soft matter and foods. Faraday Discuss. https://doi.org/10.1039/c2fd20028g
Tasnim F, Toh YC, Qu Y et al (2016) Functionally enhanced human stem cell derived hepatocytes in galactosylated cellulosic sponges for hepatotoxicity testing. Mol Pharm. https://doi.org/10.1021/acs.molpharmaceut.6b00119
Tung YC, Hsiao AY, Allen SG et al (2011) High-throughput 3D spheroid culture and drug testing using a 384 hanging drop array. Analyst. https://doi.org/10.1039/c0an00609b
Turing A (1952) The chemical basis of morphogenesis. Philos Trans R Soc Lond B Biol Sci. https://doi.org/10.1098/rstb.1952.0012
Van Den Bulcke AI, Bogdanov B, De Rooze N et al (2000) Structural and rheological properties of methacrylamide modified gelatin hydrogels. Biomacromolecules. https://doi.org/10.1021/bm990017d
Yue Z, Wen F, Gao S et al (2010) Preparation of three-dimensional interconnected macroporous cellulosic hydrogels for soft tissue engineering. Biomaterials. https://doi.org/10.1016/j.biomaterials.2010.07.059
Material meso-properties characterisation facilities were provided by Department of Chemical Engineering, National University of Singapore with the assistance of Dr. Yuan Ze Liang. Imaging protocol and tools was kindly provided by Kapish Gupta from Mechanobiology Institute, National University of Singapore. Discussion with Dr. Bramasta Nugraha in University of Zurich contributed to the formation of this project. This work is supported in part by the Institute of Bioengineering and Nanotechnology, Biomedical Research Council, Agency for Science, Technology and Research (A*STAR), A*STAR (Project Number 1334i00051); NMRC (R-185-000-294-511); NUHS Innovation Seed Grant 2017 (R-185-000-343-733); MOE ARC (R-185-000-342-112); IAF (R-185-000-350-305); SMART BioSyM and Mechanobiology Institute of Singapore (R-714-006-008-271) funding to HYU.
Conflict of interest
Hanry Yu holds equity at InvitroCue, HistoIndex, Osteopore and Pishon Co. Ltd. There is no direct conflict of interest with the submitted work.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Performance of PHHs in sponges synthesized under different reaction conditions. (A) Gene expression of CYP1A2 and (B) CYP3A4, and (C) albumin and (D) urea production in PHHs at Day 4 and 7 of culture. Details on the conditions #1-11 are listed in Supplementary Table 1 (TIFF 5798 kb)
About this article
Cite this article
Liu, Z., Tasnim, F., Ong, S. et al. Cost-effective robust synthesis of methacrylic cellulosic sponge for organoid culture. Cellulose 27, 171–184 (2020). https://doi.org/10.1007/s10570-019-02768-4
- 3D culture
- Macroporous sponge