Scalable Matrigel-Free Suspension Culture for Generating High-Quality Human Liver Ductal Organoids

Abstract

Liver transplantation is currently the sole definitive treatment option for end-stage liver failure. However, a significant shortage of donors prevails due to high clinical demands. Recently, human liver organoids have shown significant potential in regenerative medicine for liver diseases. Nevertheless, current static cultures of organoids grown in well-plates heavily rely on extracellular matrix hydrogels (Matrigel), thereby limiting both the scalability and quantity of organoid culture. In this study, we present a groundbreaking culture mode that eliminates all reliance on extracellular matrix hydrogels, enabling the successful preparation of functional human liver ductal organoids (LDOs) based on the cell suspension culture mode in a mechanically stirred bioreactor. Initially, the developed suspension culture in a 6-well plate without matrigel was proven to support robust growth of liver ductal organoids with an average size 2.6 times larger than those obtained in static culture, and with a high organoid survival rate exceeding 90%. Also, the transcriptome profile reveals that suspension culture activates the phosphatidylinositol 3-kinase (PI3K) signalling pathway through mechanical signal transduction, thereby promoting hepatobiliary characteristics. Then, a controllable and scalable bioprocess for liver ductal organoid culture was developed and successfully scaled up to a 50 mL flask bioreactor with a working volume of 15 mL. Finally, animal experiments indicated that the transplantation of liver ductal organoids harvested from suspension culture can effectively alleviate liver injury and inflammation, demonstrating the feasibility of large-scale production of liver ductal organoids cultivated in suspension culture with an improved extracellular matrix environment.

Keywords

large-scale production / liver ductal organoids / liver transplantation / matrigel free suspension culture

References

[1] R. Pais, A. S. Barritt, Y. Calmus, et al., “NAFLD and Liver Transplantation: Current Burden and Expected Challenges,” Journal of Hepatology 65, no. 6 (2016): 1245-1257, https://doi.org/10.1016/j.jhep.2016.07.033.

[2] W. A. Dar, E. Sullivan, J. S. Bynon, H. Eltzschig, and C. Ju, “Ischaemia Reperfusion Injury in Liver Transplantation: Cellular and Molecular Mechanisms,” Liver International 39, no. 5 (2019): 788-801.

[3] F. Durand, J. Levitsky, F. Cauchy, H. Gilgenkrantz, O. Soubrane, and C. Francoz, “Age and Liver Transplantation,” Journal of Hepatology 70, no. 4 (2019): 745-758.

[4] H. Mergental, M. T. P. R. Perera, R. W. Laing, et al., “Transplantation of Declined Liver Allografts Following Normothermic Ex-Situ Evaluation,” American Journal of Transplantation 16, no. 11 (2016): 3235-3245, https://doi.org/10.1111/ajt.13875.

[5] A. Sanchez-Fueyo, G. Whitehouse, N. Grageda, et al., “Applicability, Safety, and Biological Activity of Regulatory T Cell Therapy in Liver Transplantation,” American Journal of Transplantation 20, no. 4 (2020): 1125-1136.

[6] G. Mazza, K. Rombouts, A. R. Hall, et al., “Decellularized Human Liver as a Natural 3D-Scaffold for Liver Bioengineering and Transplantation,” Scientific Reports 7, no. 5 (2015): 13079.

[7] O. B. van Leeuwen, S. B. Bodewes, V. A. Lantinga, et al., “Sequential Hypothermic and Normothermic Machine Perfusion Enables Safe Transplantation of High-Risk Donor Livers,” American Journal of Transplantation 22, no. 6 (2022): 1658-1670.

[8] L. Campana and J. P. Iredale, “Regression of Liver Fibrosis,” Seminars in Liver Disease 37, no. 1 (2017): 1-10.

[9] C. C. Bell, D. F. G. Hendriks, S. M. L. Moro, et al., “Characterization of Primary Human Hepatocyte Spheroids as a Model System for Drug-Induced Liver Injury, Liver Function and Disease,” Scientific Reports 4, no. 6 (2016): 25187.

[10] H. Hu, H. Gehart, B. Artegiani, et al., “Long-Term Expansion of Functional Mouse and Human Hepatocytes as 3D Organoids,” Cell 175, no. 6 (2018): 1591-1606.

[11] N. Prior, P. Inacio, and M. Huch, “Liver Organoids: From Basic Research to Therapeutic Applications,” Gut 68, no. 12 (2019): 2228-2237.

[12] G. Rossi, A. Manfrin, and M. P. Lutolf, “Progress and Potential in Organoid Research,” Nature Reviews. Genetics 19, no. 11 (2018): 671-687.

[13] R. Cruz-Acuna, M. Quiros, A. E. Farkas, et al., “Synthetic Hydrogels for Human Intestinal Organoid Generation and Colonic Wound Repair,” Nature Cell Biology 19, no. 11 (2017): 1326-1335.

[14] L. Chung, D. R. Maestas, F. Housseau, and J. H. Elisseeff, “Key Players in the Immune Response to Biomaterial Scaffolds for Regenerative Medicine,” Advanced Drug Delivery Reviews 114 (2017): 184-192.

[15] X.-L. Fan, Y. Zhang, X. Li, and Q.-L. Fu, “Mechanisms Underlying the Protective Effects of Mesenchymal Stem Cell-Based Therapy,” Cellular and Molecular Life Sciences 77, no. 14 (2020): 2771-2794.

[16] Z. Julier, A. J. Park, P. S. Briquez, and M. M. Martino, “Promoting Tissue Regeneration by Modulating the Immune System,” Acta Biomaterialia 53 (2017): 13-28.

[17] F. Cofano, M. Boido, M. Monticelli, et al., “Mesenchymal Stem Cells for Spinal Cord Injury: Current Options, Limitations, and Future of Cell Therapy,” International Journal of Molecular Sciences 20, no. 11 (2019): 2698, https://doi.org/10.3390/ijms20112698.

[18] J. Etulain, “Platelets in Wound Healing and Regenerative Medicine,” Platelets 29, no. 6 (2018): 556-568.

[19] G. Itakura, S. Kawabata, M. Ando, et al., “Fail-Safe System Against Potential Tumorigenicity After Transplantation of iPSC Derivatives,” Stem Cell Reports 8, no. 3 (2017): 673-684.

[20] Y. Nakamura, S. Miyagawa, S. Yoshida, et al., “Natural Killer Cells Impede the Engraftment of Cardiomyocytes Derived From Induced Pluripotent Stem Cells in Syngeneic Mouse Model,” Scientific Reports 9, no. 1 (2019): 10840.

[21] J. Shiota, L. C. Samuelson, and N. Razumilava, “Hepatobiliary Organoids and Their Applications for Studies of Liver Health and Disease: Are We There Yet?,” Hepatology 74, no. 4 (2021): 2251-2263.

[22] F. Sampaziotis, D. Muraro, and O. C. Tysoe, “Cholangiocyte Organoids Can Repair Bile Ducts After Transplantation in the Human Liver,” Science 371, no. 6531 (2021): 839-846.

[23] N. Glorevski, N. Sachs, A. Manfrin, et al., “Designer Matrices for Intestinal Stem Cell and Organoid Culture,” Nature 539, no. 7630 (2016): 560-564.

[24] M. T. Kozlowski, C. J. Crook, and H. T. Ku, “Towards Organoid Culture Without Matrigel,” Communications Biology 4, no. 1 (2021): 1387.

[25] M. A. G. Barbosa, C. P. R. Xavier, R. F. Pereira, V. Petrikaite, and M. H. Vasconcelos, “3D Cell Culture Models as Recapitulators of the Tumor Microenvironment for the Screening of Anti-Cancer Drugs,” Cancers 14, no. 1 (2022): 190.

[26] C. Jubelin, J. Munoz-Garcia, L. Griscom, et al., “Three-Dimensional In Vitro Culture Models in Oncology Research,” Cell and Bioscience 12, no. 1 (2022): 155.

[27] W. Kim, Y. Gwon, S. Park, H. Kim, and J. Kim, “Therapeutic Strategies of Three-Dimensional Stem Cell Spheroids and Organoids for Tissue Repair and Regeneration,” Bioactive Materials 19 (2023): 50-74.

[28] E. A. Aisenbrey and W. L. Murphy, “Synthetic Alternatives to Matrigel,” Nature Reviews Materials 5, no. 7 (2020): 539-551.

[29] N. Bock, F. Forouz, L. Hipwood, et al., “GelMA, Click-Chemistry Gelatin and Bioprinted Polyethylene Glycol-Based Hydrogels as 3D Ex Vivo Drug Testing Platforms for Patient-Derived Breast Cancer Organoids,” Pharmaceutics 15, no. 1 (2023): 261, https://doi.org/10.3390/pharmaceutics15010261.

[30] N. Carpentier, S. C. Ye, M. D. Delemarre, et al., “Gelatin-Based Hybrid Hydrogels as Matrices for Organoid Culture,” Biomacromolecules 25, no. 2 (2024): 590-604.

[31] S. Kim, S. Min, Y. S. Choi, et al., “Tissue Extracellular Matrix Hydrogels as Alternatives to Matrigel for Culturing Gastrointestinal Organoids,” Nature Communications 13, no. 1 (2022): 1692.

[32] T. Lokai, B. Albin, K. Qubbaj, A. P. Tiwari, P. Adhikari, and I. H. Yang, “A Review on Current Brain Organoid Technologies From a Biomedical Engineering Perspective,” Experimental Neurology 367 (2023): 114461.

[33] S. Price, S. Bhosle, E. Goncalves, et al., “A Suspension Technique for Efficient Large-Scale Cancer Organoid Culturing and Perturbation Screens,” Scientific Reports 12, no. 1 (2022): 5571.

[34] O. Chaudhuri, J. Cooper-White, P. A. Janmey, D. J. Mooney, and V. B. Shenoy, “Effects of Extracellular Matrix Viscoelasticity on Cellular Behaviour,” Nature 584, no. 7822 (2020): 535-546.

[35] F. Meng, C. Shen, L. Yang, et al., “Mechanical Stretching Boosts Expansion and Regeneration of Intestinal Organoids Through Fueling Stem Cell Self-Renewal,” Cell Regeneration 11, no. 1 (2022): 39, https://doi.org/10.1186/s13619-022-00137-4.

[36] M. Wang, X. Zhou, S. Zhou, et al., “Mechanical Force Drives the Initial Mesenchymal- Epithelial Interaction During Skin Organoid Development,” Theranostics 13, no. 9 (2023): 2930-2945.

[37] N. Kobayashi, S. Togo, T. Matsuzaki, et al., “Stiffness Distribution Analysis in Indentation Depth Direction Reveals Clear Mechanical Features of Cells and Organoids by Using AFM,” Applied Physics Express 13, no. 9 (2020): 7001-7004.

[38] T. Matsuzaki, Y. Shimokawa, H. Koike, et al., “Mechanical Guidance of Self-Condensation Patterns of Differentiating Progeny,” Iscience 25, no. 10 (2022): 105109.

[39] A. Elosegui-Artola, I. Andreu, A. E. M. Beedle, et al., “Force Triggers YAP Nuclear Entry by Regulating Transport Across Nuclear Pores,” Cell 171, no. 6 (2017): 1397-1410.

[40] T. Iskratsch, H. Wolfenson, and M. P. Sheetz, “Appreciating Force and Shape—The Rise of Mechanotransduction in Cell Biology,” Nature Reviews. Molecular Cell Biology 15, no. 12 (2014): 825-833.

[41] K. H. Vining and D. J. Mooney, “Mechanical Forces Direct Stem Cell Behaviour in Development and Regeneration,” Nature Reviews. Molecular Cell Biology 18, no. 12 (2017): 728-742.

[42] N. Aizarani, A. Saviano, N. Sagar, et al., “A Human Liver Cell Atlas Reveals Heterogeneity and Epithelial Progenitors,” Nature 572, no. 7768 (2019): 199-204.

[43] J. Chen, J. Zhang, L. Yang, and B. Zhao, “Facile Suspension Culture Protocol of the Liver Biliary Organoids,” Bio-Design and Manufacturing 6, no. 1 (2023): 74-81.

[44] J. C. Valdoz, N. A. Franks, C. G. Cribbs, et al., “Soluble ECM Promotes Organotypic Formation in Lung Alveolar Model,” Biomaterials 283 (2022): 121464.

[45] Y.-C. Lu, D.-J. Fu, D. An, et al., “Scalable Production and Cryostorage of Organoids Using Core-Shell Decoupled Hydrogel Capsules,” Advanced Biosystems 1, no. 12 (2017): 1700165, https://doi.org/10.1002/adbi.201700165.

[46] E. D. Wrenn, B. M. Moore, E. Greenwood, M. McBirney, and K. J. Cheung, “Optimal, Large-Scale Propagation of Mouse Mammary Tumor Organoids,” Journal of Mammary Gland Biology and Neoplasia 25, no. 4 (2020): 337-350.

[47] T. Zhou, B. Gao, Y. Fan, et al., “Piezo1/2 Mediate Mechanotransduction Essential for Bone Formation Through Concerted Activation of NFAT-YAP1-Ss-Satenin,” eLife 9 (2020): e52779.

[48] H.-Q. Xu, J.-C. Liu, Z.-Y. Zhang, and C.-X. Xu, “A Review on Cell Damage, Viability, and Functionality During 3D Bioprinting,” Military Medical Research 9, no. 1 (2022): 70.

[49] X. Di, X. Gao, L. Peng, et al., “Cellular Mechanotransduction in Health and Diseases: From Molecular Mechanism to Therapeutic Targets,” Signal Transduction and Targeted Therapy 8, no. 1 (2023): 282.

[50] B. C. Low, C. Q. Pan, G. V. Shivashankar, A. Bershadsky, M. Sudol, and M. Sheetz, “YAP/TAZ as Mechanosensors and Mechanotransducers in Regulating Organ Size and Tumor Growth,” FEBS Letters 588, no. 16 (2014): 2663-2670.

[51] L. Broutier, A. Andersson-Rolf, C. J. Hindley, et al., “Culture and Establishment of Self-Renewing Human and Mouse Adult Liver and Pancreas 3D Organoids and Their Genetic Manipulation,” Nature Protocols 11, no. 9 (2016): 1724-1743.

[52] Z. Zhao, X. Chen, A. M. Dowbaj, et al., “Organoids,” Nature Reviews Methods Primers 2, no. 1 (2022): 94, https://doi.org/10.1038/s43586-022-00174-y.

[53] H. Xu, X. Lyu, M. Yi, W. Zhao, Y. Song, and K. Wu, “Organoid Technology and Applications in Cancer Research,” Journal of Hematology and Oncology 11, no. 1 (2018): 116.

[54] S. Maharjan, C. Ma, B. Singh, et al., “Advanced 3D Imaging and Organoid Bioprinting for Biomedical Research and Therapeutic Applications,” Advanced Drug Delivery Reviews 208 (2024): 115237, https://doi.org/10.1016/j.addr.2024.115237.

[55] S. Gunti, A. T. K. Hoke, K. P. Vu, and N. R. London, “Organoid and Spheroid Tumor Models: Techniques and Applications,” Cancers 13, no. 4 (2021): 874.

[56] J. A. Brassard, M. Nikolaev, T. Huebscher, M. Hofer, and M. P. Lutolf, “Recapitulating Macro-Scale Tissue Self-Organization Through Organoid Bioprinting,” Nature Materials 20, no. 1 (2021): 22-29.

[57] X.-Y. Tang, S. Wu, D. Wang, et al., “Human Organoids in Basic Research and Clinical Applications,” Signal Transduction and Targeted Therapy 7, no. 1 (2022): 168.

[58] S. J. Mun, J.-S. Ryu, M.-O. Lee, et al., “Generation of Expandable Human Pluripotent Stem Cell-Derived Hepatocyte-Like Liver Organoids,” Journal of Hepatology 71, no. 5 (2019): 970-985.

[59] D. Garnier, R. Li, F. Delbos, et al., “Expansion of Human Primary Hepatocytes In Vitro Through Their Amplification as Liver Progenitors in a 3D Organoid System,” Scientific Reports 8, no. 1 (2018): 8222.

[60] J. Y. Co, M. Margalef-Català, D. M. Monack, and M. R. Amieva, “Controlling the Polarity of Human Gastrointestinal Organoids to Investigate Epithelial Biology and Infectious Diseases,” Nature Protocols 16, no. 11 (2021): 5171-5192.

[61] N. Brandenberg, S. Hoehnel, F. Kuttler, et al., “High-Throughput Automated Organoid Culture via Stem-Cell Aggregation in Microcavity Arrays,” Nature Biomedical Engineering 4, no. 9 (2020): 863-874.

[62] A. A. Mansour, J. T. Goncalves, C. W. Bloyd, et al., “An In Vivo Model of Functional and Vascularized Human Brain Organoids,” Nature Biotechnology 36, no. 5 (2018): 432-441.

[63] R. Morizane, A. Q. Lam, B. S. Freedman, S. Kishi, M. T. Valerius, and J. V. Bonventre, “Nephron Organoids Derived From Human Pluripotent Stem Cells Model Kidney Development and Injury,” Nature Biotechnology 33, no. 11 (2015): 1193-1200.

[64] Y.-W. Chen, S. X. Huang, A. L. de, de Rodrigues Toste Carvalho, et al., “A Three-Dimensional Model of Human Lung Development and Disease From Pluripotent Stem Cells,” Nature Cell Biology 19, no. 5 (2017): 542-549, https://doi.org/10.1038/ncb3510.

[65] J. Brugge, K.-C. Chang, F. Silvestri, et al., “Breast Organoid Suspension Cultures Maintain Long-Term Estrogen Receptor Expression and Responsiveness,” Research Square 3 (2024): 4463390.

[66] Y. Takahashi, S. Sato, Y. Kurashima, et al., “A Refined Culture System for Human Induced Pluripotent Stem Cell-Derived Intestinal Epithelial Organoids,” Stem Cell Reports 10, no. 1 (2018): 314-328.

[67] P. Kakni, R. Hueber, K. Knoops, et al., “Intestinal Organoid Culture in Polymer Film-Based Microwell Arrays,” Advanced Biosystems 4, no. 10 (2020): e2000126.

[68] V. Sander, A. Przepiorski, N. A. Hukriede, and A. J. Davidson, “ Large-Scale Production of Kidney Organoids From Human Pluripotent Stem Cells,” in Kidney Research: Experimental Protocols, vol. 2664 (Springer US, 2023), 69-83.

[69] J. P. P. Licata, K. H. H. Schwab, Y.-e. Har-el, J. A. A. Gerstenhaber, and P. I. I. Lelkes, “Bioreactor Technologies for Enhanced Organoid Culture,” International Journal of Molecular Sciences 24, no. 14 (2023): 11427.

[70] I. Dasgupta and D. McCollum, “Control of Cellular Responses to Mechanical Cues Through YAP/TAZ Regulation,” Journal of Biological Chemistry 294, no. 46 (2019): 17693-17706.

[71] H. Ryoo, H. Kimmel, E. Rondo, and G. H. Underhill, “Advances in High Throughput Cell Culture Technologies for Therapeutic Screening and Biological Discovery Applications,” Bioengineering and Translational Medicine 9, no. 3 (2024): e10627.

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