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Cost-effective robust synthesis of methacrylic cellulosic sponge for organoid culture

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Abstract

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.

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Acknowledgments

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.

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Correspondence to Eliza Li Shan Fong.

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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.

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Supplementary material 1 (DOCX 29 kb)

Supplementary Fig. 1

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)

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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

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Keywords

  • 3D culture
  • Hepatocyte
  • Organoids
  • Macroporous sponge