Center for Engineering in Medicine, Massachusetts General Hospital, Harvard Medical School, Shriners Hospitals for Children
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Uygun, B. E., Price, G., Saeidi, N., Izamis, M., Berendsen, T., Yarmush, M., et al. Decellularization and Recellularization of Whole Livers. J. Vis. Exp. (48), e2394, doi:10.3791/2394 (2011).
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.
1. Liver Decellularization
2. Recellularization of Decellularized Liver Matrix
3. In Vitro Culture of the Recellularized Liver Graft
4. Representative Results:
The complete decellularization of a rat liver takes about 72 hours using the described protocol. The resulting matrix retains 100% of the fibrillar collagen, 50% of the glycosaminoglycans and only 5% of the DNA of the native liver (Table 1)6. The vascular structure of the matrix is preserved as evidenced by corrosion casting and scanning electron microscopy analysis (Figure 4)6. The presence of the vascular microarchitecture within the DLM facilitates its repopulation with cells with an efficiency of 96% and its subsequent perfusion for in vitro culture. The recellularized liver graft may be cultured up to 10 days in vitro and displays proper liver functions as confirmed via albumin, urea and total bile acid secretion (Figure 5)6.
Figure 1. Decellularized liver matrix at the end of the decellularization process. (a) the whole liver (b) the median lobe after resection.
Figure 2. Schematic representation of the recellularization of the DLM.
Figure 3. Perfusion system setup for in vitro culture of the recellularized liver graft.
Figure 4. The microvascular structure is retained in the decellularized liver matrix. Corrosion cast images of a) a normal liver b) a decellularized liver, portal (red) and venous (blue) vasculature. Scanning electron microscopy images of the DLM c) a vessel, d) a section featuring bile duct-like small vessels (arrows), Scale bars (a,b) 5 mm (c,d) 20 μm.
Figure 5. Liver specific functions of the recellularized liver graft during the in vitro perfusion culture. a) albumin secretion (p = 0.5249), b) urea production (p = 0.5271) and c) total bile acid secretion (p = 0.0114). Statistical analysis of the difference between the experiment and the control was done over the 10 d culture period by Friedman's test at a = 0.01. Error bars represent s.e.m. (n = 3).
|Fresh livera||Decellularized liver matrixa||p-values||% of fresh liver|
|n = 4||n = 8|
|Collagen||0.07 ± 0.01||0.08 ± 0.03||0.56||114%|
|(mg per g liver)|
|Glycosaminoglycans||73.1 ± 6.7||34.2 ± 2.9||0.004||47%|
|(mg per g liver)|
|DNA||14.9 ± 5.6||0.44 ± 0.08||3.3 10-5||2.9%|
|(mg per g liver)|
Table 1. The biochemical composition of the decellularized liver matrix compared to the native liver.
aValues are represented as mean ± s.e.m.
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 graft, we envision that the recellularized liver graft may be used as a platform to study various liver functions such as drug metabolism, regeneration and development. Furthermore, the recellularized graft may find clinical application, since it can potentially be transplanted through connection to the blood circulation of a recipient animal thanks to the presence of vascular structure in the graft6.
No conflicts of interest declared.
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.
|Sodium dodecyl sulfate||Sigma-Aldrich||L4390|
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|Masterflex L/S Standard pump head||Cole-Parmer||EW- 07013-81|
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Formal Correction: Erratum: Decellularization and Recellularization of Whole Livers
Posted by JoVE Editors on 03/14/2011. Citeable Link.
A correction was made to Decellularization and Recellularization of Whole Livers. There was an error with an author's name. The author's last name had a typo and was corrected to: