Decellularization and Recellularization of Whole Livers
Summary
Perfusion decellularization is a novel technique to produce whole liver scaffolds that retains the organ’s extracellular matrix composition and microarchitecture. Herein, the method of preparing whole organ scaffolds using perfusion decellularization and subsequent repopulation with hepatocytes is described. Functional and transplantable liver grafts can be generated using this technique.
Abstract
The liver is a complex organ which requires constant perfusion for delivery of nutrients and oxygen and removal of waste in order to survive1. Efforts to recreate or mimic the liver microstructure with grounds up approach using tissue engineering and microfabrication techniques have not been successful so far due to this design challenge. In addition, synthetic biomaterials used to create scaffolds for liver tissue engineering applications have been limited in inducing tissue regeneration and repair in large part due to the lack of specific cell binding motifs that would induce the proper cell functions2. Decellularized native tissues such blood vessels3and skin4on the other hand have found many applications in tissue engineering, and have provided a practical solution to some of the challenges. The advantage of decellularized native matrix is that it retains, to an extent, the original composition, and the microstructure, hence enhancing cell attachment and reorganization5.
In this work we describe the methods to perform perfusion-decellularization of the liver, such that an intact liver bioscaffold that retains the structure of major blood vessels is obtained. Further, we describe methods to recellularize these bioscaffolds with adult primary hepatocytes, creating a liver graft that is functional in vitro, and has the vessel access necessary for transplantation in vivo.
Protocol
Discussion
The perfusion decellularization method described here produces a whole liver scaffold that has the same gross structure and the vascular microarchitecture of the native liver. The scaffold has an extracellular matrix composition similar to the native liver. The recellularization method achieves repopulation of the scaffold with cells at high efficiency and the cells remain viable and functional during the in vitro culture period tested. With the addition of nonparenchymal cells into the recellularized liver graf…
Declarações
The authors have nothing to disclose.
Acknowledgements
The authors would like to thank Jack Milwid for the design of the in vitro perfusion chamber. This work was supported by grants from the US NIH, R01DK59766 and R01DK084053 to M.Y., R00DK080942 to K.U., US NSF CBET- 0853569 to K.U. and the Shriners Hospitals for Children to B.E.U. (grant no. 8503). We also acknowledge support and the Shriners Hospitals for Children.
Materials
| Material Name | Tipo | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| Sodium dodecyl sulfate | Sigma-Aldrich | L4390 | ||
| Triton X-100 | Sigma-Aldrich | T8787 | ||
| Masterflex L/S Digital Drive | Cole-Parmer | EW-07523-80 | ||
| Masterflex L/S Standard pump head | Cole-Parmer | EW- 07013-81 | ||
| Bubble trap | Radnoti | 130149 |
Referências
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- Lutolf, M. P., Hubbell, J. A. Synthetic biomaterials as instructive extracellular microenvironments for morphogenesis in tissue engineering. Nature biotechnology. 23, 47-55 (2005).
- Dahl, S. L., Koh, J., Prabhakar, V., Niklason, L. E. Decellularized native and engineered arterial scaffolds for transplantation. Cell Transplant. 12, 659-666 (2003).
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- Gilbert, T. W., Sellaro, T. L., Badylak, S. F., F, S. Decellularization of tissues and organs. Biomaterials. 27, 3675-3683 (2006).
- Uygun, B. E. Organ reengineering through development of a transplantable recellularized liver graft using decellularized liver matrix. Nat Med. , (2010).
