الفهرس 
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Abstract
Liver diseases are rising worldwide creating a global health burden. Deaths due to different liver diseases in Egypt reached 10.45% of total deaths according to WHO published data in 2017. Even though the liver has a great capacity for regeneration, in ESLD this capacity is lost leaving the hope for liver transplantation or HT to rejuvenate the diseased tissue.
Besides, formation of liver organoids can create an in-vitro model tailored for experimental studies that would save money and lives of many experimental animals and could give detailed results equivalent to in vivo studies.
Liver tissue engineering need adequate scaffold to be seeded with hepatocytes. Whole organ decellularization and recellularization is considered a promising modality for creation of an auxillary liver tissue. Decellularization is the process of removal of parenchymatous cell from the surrounding stroma of the donor organ producing a 3D scaffold that has the same ultra-structure and vascular networks for gas and nutrient exchange. Whereas recellularization is the seeding of hepatocytes with or without other non-parenchymatous cells to create an artificial liver.
This study was designed to obtain natural decellularized liver matrix and culturing AdHs in vitro in culture flasks and in DLM followed by evaluating the structural integrity of the natural scaffold and the histological characters of reseeded FH and AdH.
This experiment was conducted on fifty adult male albino rats with average weight of 170 grams and ten 2-3 day old rats which were purchased from the animal house of MASRI. The animals were divided into three main groups;
- group I (control group): consists of 10 rats, to study normal histological structure of the liver.
- group II (decellularization group): consists of 10 rats whose livers were decellularized.
- group III (recellularization group): consists of 40 rats in which:
o group IIIA: 10 adult male albino rats and 10 (2-3 day old) rats were considered donor for FH and another 10 rats’ livers were harvested and subjected to decellularization process then it was recellularized using donor FH.
o group IIIB: 20 adult male albino rats’ livers were harvested. 10 of them were subjected to decellularization process. The other 10 were considered AdH donor. Then the whole DLM was recellularized using donor AdH.
Operative procedures were done to extract the liver while preserving maximum length of PV to be cannulated for decellularization. The PV was then cannulated, and the cannulated liver transferred from animal house to stem cell lab to start decellularization process. After completion of decellularization process, the DLM was either processed for analysis of matrix integrity or recellularizated with primary hepatocytes isolated from donor liver. The recellularized scaffold was connected to dynamic culture for five days inside the Co2 incubator.
Primary AdHs were cultured in vitro for 21 days till confluency to assess cell survival and proliferation. They appeared as polygonal cells with granular cytoplasm and showed adequate mitotic activity throughout the experiment.
Examination of H&E stained sections of group II (decellularization group) showed total loss of cellular and nuclear material, whereas Mallory and PAS stained sections showed preservation of collagen and glycoprotein content in DLM. Whereas H&E and Mallory section of group IIIA and IIIB (recellularization group) showed FHs and AdHs aggregations in the portal branches. Hepatocytes were sometimes attached to the vessel wall.
PAS-stained sections showed satisfactory preservation of extracellular proteoglycans and glycoproteins and adequate amount of glycogen inside reseeded hepatocytes in comparison with control liver indication adequate metabolic function of reseeded cells. Immunohistochemical stained sections showed high AFP content inside reseeded hepatocytes indicating origin as well as high mitotic activity of the cells. Whereas positive PCNA reaction in the cells indicated adequate mitotic activity.
Conclusion
It was concluded that the present study managed to present efficient decellularization for the rat livers forming natural scaffold that can accommodate FHs and AdHs in addition to induce their proliferation while preserving integrity of most of the structural components of the ECM. This study found that AdH were superior to FH in the initial viable cell count obtained from each of 2-3day old or adult donor liver which directly affected the reseeding cell count and the overall results of liver recellularization. Moreover, the present study concluded that adult hepatocyte culturing in culture media flasks can be managed to proliferate to confluency with the presence of other cocultured cell types.
Recommendation
- More work is required to examine the functional effectiveness of the formed recellularized liver scaffolds in vivo and in vitro.
- Liver tissue engineering needs more experimentation as other cell types (endothelial cells, kupffer cells, ito cells, oval cells, cholangiocytes) need to be delivered through various routes to ensure attachment and ability to perform normal physiological liver function.
- In order to extend in vitro time a sealed tissue bioreactor should be created to protect against bacterial and fungal contamination.
- Connecting the recellularized tissue to a peristaltic pump that can be adjusted at perfusion rate ranging from 10-20 ml/min instead of syringe pump with rate of 1-2 ml/ hr to get advantage of adequate perfusion as well as circulating GFs formed by the AdH in the scaffold
- Further studies on human primary hepatocytes and cancer cell lines are recommended to extend our understanding about adult human hepatocytes.
- A biotechnology engineer needs to be added to the tissue culture team to facilitate the creation of new tools for new studies.
- Primary Fetal/neonatal livers should be reevaluated as a candidate for recellularization of whole adult DLM due to lower number of yielded hepatocytes as well as lower chance to get a healthy human donor fetal/neonatal liver.