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Bioengineering

Fabrication of Decellularized Spleen Matrix Derived from Rats

Published: February 9th, 2024

DOI:

10.3791/66520

1National Local Joint Engineering Research Center for Precision Surgery and Regenerative Medicine, The First Affiliated Hospital of Xi'an Jiaotong University, 2Department of Hepatobiliary Surgery, The First Affiliated Hospital of Xi'an Jiaotong University, 3Center for Regenerative and Reconstructive Medicine, Med-X Institute, The First Affiliated Hospital of Xi'an Jiaotong University, 4Department of Graduate School, Xi'an Medical University

The decellularized spleen matrix (DSM) holds promising applications in the field of liver tissue engineering. This protocol outlines the procedure for preparing rat DSM, which includes harvesting rat spleens, decellularizing them through perfusion, and evaluating the resulting DSM to confirm its characteristics.

Liver transplantation is the primary treatment for end-stage liver disease. However, the shortage and inadequate quality of donor organs necessitate the development of alternative therapies. Bioartificial livers (BALs) utilizing decellularized liver matrix (DLM) have emerged as promising solutions. However, sourcing suitable DLMs remains challenging. The use of a decellularized spleen matrix (DSM) has been explored as a foundation for BALs, offering a readily available alternative. In this study, rat spleens were harvested and decellularized using a combination of freeze-thaw cycles and perfusion with decellularization reagents. The protocol preserved the microstructures and components of the extracellular matrix (ECM) within the DSM. The complete decellularization process took approximately 11 h, resulting in an intact ECM within the DSM. Histological analysis confirmed the removal of cellular components while retaining the ECM's structure and composition. The presented protocol provides a comprehensive method for obtaining DSM, offering potential applications in liver tissue engineering and cell therapy. These findings contribute to the development of alternative approaches for the treatment of end-stage liver disease.

Liver transplantation remains the only definitive treatment for end-stage liver disease1,2,3. However, the critical shortage and declining quality of donor organs have heightened the need for alternative treatments4. In the realm of regenerative medicine, bioartificial livers (BALs) utilizing decellularized liver matrix (DLM) have emerged as promising solutions5,6,7. The DLM preserves the original liver structure, including its intricate microvascular network ....

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This study was approved by the Committee on the Ethics of Animal Experiments of Xi'an Jiaotong University and carried out in accordance with the guidelines for the Care and Use of Laboratory Animals.

1. Spleen harvesting

  1. Use male Sprague Dawley rats weighing 250-280 g. House the rats in rooms with controlled temperature and humidity, and provide them with food and water ad libitum, except for fasting before surgery.
  2. Subcutaneously inject bupr.......

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This protocol utilized a combination of repeated freeze-thaw cycles and perfusion with decellularization reagents for the decellularization of rat spleen. The complete decellularization of the spleen was achieved in approximately 11 h (Figure 2A). Throughout the decellularization process, the spleen's color gradually transitioned from deep red to a mottled, light red, and eventually, a white translucent appearance (Figure 2B). The overall morphology remained.......

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The BALs represent an effective approach for the treatment of end-stage liver disease, particularly in cases where liver transplantation is hindered by the current shortage of donor organs6. A promising option for creating BALs is the utilization of DLM, which preserves the native liver's natural ECM and vascular structure. However, the scarcity of human DLM and the potential risks of infection and immunogenicity associated with animal DLM pose significant limitations. To address this challeng.......

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This work was supported by the National Natural Science Foundation of China (82000624), Natural Science Basic Research Program of Shaanxi (2022JQ-899 & 2021JM-268), Shaanxi Province Innovation Capability Support Program (2023KJXX-030), Shaanxi Province Key R&D Plan University Joint Project-Key Project (2021GXLH-Z-047), Institutional Foundation of The First Affiliated Hospital of Xi'an Jiaotong University (2021HL-42 & 2021HL-21).

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NameCompanyCatalog NumberComments
Anesthesia MachineHarvard Apparatustabletopanimal anesthesia
bubble trapShandong Weigao Group Medical Polymer Co., Ltd.pore diameter: 5 μmprevent air bubbles
BuprenorphineTIPR Pharmaceutical Responsible Co.,Ltdan analgesic
Hemostatic ForcepsShanghai Medical Instruments  Co., LtdJ31020surgical tool
Heparinized SalineSPH No.1 Biochemical & Pharmaceutical Co., LTD prevent the formation of thrombosis 
IsofluraneRWD life Science Co.anesthetic:for the induction and maintenanceof anesthesia
Penicillin-Streptomycin Beyotime Biotechnology Co., Ltd.C0222antibiotics in vitro to prevent microbial contamination
Peristaltic PumpBaoding Longer Precision Pump Co., Ltd.BT100-1L
Phosphate-Buffered SalineShanghai Titan Scientific Co., Ltd.4481228phosphoric acid buffer salt solution
Silicone TubeBaoding Longer Precision Pump Co., Ltd.2.4×0.8mm
Silk SutureYangzhou Jinhuan Medical Instrument Factory6-0 and 3-0ligate blood vessels
Sodium Dodecyl SulfateShanghai Titan Scientific Co., Ltd.151-21-3ionic detergent, dissolves both cell and nuclear membranes
Syringe PumpShenzhen Mindray Bio-Medical Electronics Co., LtdBeneFusion SP5intravenous infusion
Triton X-100Shanghai Titan Scientific Co., Ltd.9002-93-1non-ionic detergent, disrupts lipid-lipid, lipid-protein, and DNA-protein interactions
Venous CatheterB. Braun Company24Ginserting the spleen artery

  1. Xu, X. State of the art and perspectives in liver transplantation. Hepatobiliary Pancreat Dis Int. 22 (1), 1-3 (2023).
  2. Hautz, T., et al. Immune cell dynamics deconvoluted by single-cell RNA sequencing in normothermic machine perfusion of the liver. Nat Commun. 14 (1), 2285 (2023).
  3. Cardini, B., et al. Live confocal imaging as a novel tool to assess liver quality: insights from a murine model. Transplantation. 104 (12), 2528-2537 (2020).
  4. Ding, Y., et al. Mesenchymal stem cell-derived exosomes: a promising therapeutic agent for the treatment of liver diseases. Int J Mol Sci. 23 (18), 10972 (2022).
  5. Yaghoubi, A., et al. Prednisolone and mesenchymal stem cell preloading protect liver cell migration and mitigate extracellular matrix modification in transplanted decellularized rat liver. Stem Cell Res Ther. 13 (1), 36 (2022).
  6. Uygun, B. E., et al. Organ reengineering through development of a transplantable recellularized liver graft using decellularized liver matrix. Nat Med. 16 (7), 814-820 (2010).
  7. Xiang, J., et al. The effect of riboflavin/UVA cross-linking on anti-degeneration and promoting angiogenic capability of decellularized liver matrix. J Biomed Mater Res A. 105 (10), 2662-2669 (2017).
  8. Liu, P., et al. Implantation strategy of tissue-engineered liver based on decellularized spleen matrix in rats. J South Med Univ. 38 (6), 698-703 (2018).
  9. Xiang, J., et al. Decellularized spleen matrix for reengineering functional hepatic-like tissue based on bone marrow mesenchymal stem cells. Organogenesis. 12 (3), 128-142 (2016).
  10. Gao, R., et al. Hepatocyte culture in autologous decellularized spleen matrix. Organogenesis. 11 (1), 16-29 (2015).
  11. Liu, P., et al. Hemocompatibility improvement of decellularized spleen matrix for constructing transplantable bioartificial liver. Biomed Mater. 14 (2), 25003 (2019).
  12. Somuncu, &. #. 2. 1. 4. ;. Decellularization concept in regenerative medicine. Adv Exp Med Biol. 1212, 71-85 (2020).
  13. Neishabouri, A., Soltani, K. A., Daghigh, F., Kajbafzadeh, A. M., Majidi, Z. M. Decellularization in tissue engineering and regenerative medicine: evaluation, modification, and application methods. Front Bioeng Biotech. 10, 805299 (2022).
  14. Brown, M., Li, J., Moraes, C., Tabrizian, M., Li-Jessen, N. Decellularized extracellular matrix: New promising and challenging biomaterials for regenerative medicine. Biomaterials. 289, 121786 (2022).
  15. Gui, L., Muto, A., Chan, S. A., Breuer, C. K., Niklason, L. E. Development of decellularized human umbilical arteries as small-diameter vascular grafts. Tissue Eng Pt A. 15 (9), 2665-2676 (2009).
  16. Li, T., Javed, R., Ao, Q. Xenogeneic decellularized extracellular matrix-based biomaterials For peripheral nerve repair and regeneration. Curr Neuropharmacol. 19 (12), 2152-2163 (2021).
  17. Crapo, P. M., Gilbert, T. W., Badylak, S. F. An overview of tissue and whole organ decellularization processes. Biomaterials. 32 (12), 3233-3243 (2011).

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