Tissue bioengineering is the creation of functional tissues or whole organs by manufacturing body parts ex vivo, seeding cells on a supporting scaffold. The final goal of organ bioengineering is the use of the bioengineered organs as ‘replacement parts’ for the human body. The need for bioengineered livers is significant: currently, the only effective treatment for end-stage liver failure is orthotopic liver transplantation. However, the shortage of organ donors results every year in the death of many patients in the waiting list. Moreover, the advantage of this technology is the use of autologous cells that eliminates the need for post-transplant immunosuppression. In the present study, we decellularized pig livers and then repopulated them with allogeneic porcine mesenchymal stromal cells (pMSCs) to study the interaction between pMSCs and liver specific ECM. The final aim was to understand if ECM can influence and/or promote pMSCs toward differentiation into hepatocytes or hepatocyte-like cells without specific growth factor in culture medium. In our experimental project, porcine livers were obtained by a surgical technique similar to the one used for explant in a human cadaveric donor. Liver samples were cut and then decellularized through agitation with 0.15% SDS. The quality of the decellularization was evaluated both qualitatively and quantitatively, with histological staining and DNA quantification respectively. pMSCs were isolated from the porcine bone marrow (BM) and expanded in vitro. pMSC were characterized by assessment of morphology, proliferation capacity, immunophenotype and their differentiation ability. Then, pMSCs were used for seeding the scaffolds with static culture method. The repopulation of the recellularized scaffold was evaluated at 3, 7, 14 and 21 days after seeding with H&E stain, DAPI, MTT assay and SEM analysis, showing an increase in the cell number with increasing culture days. In order to determinate whether culture on liver ECM-scaffold could promote/address differentiation of pMSC towards hepatocyte, the transcriptional levels of some hepatic genes were tested. In particular, we evaluated six genes (ALB, AFP, HNF4a, Cyp1a1, Cyp7a1 and Krt18) associated to different phases of the hepatic development. A comparison with the expression profile was made with both porcine primary hepatocyte and pMSC. The observations obtained so far allow us to state that: i) our decellularization protocol is effective in the removal of the cells from native liver, respecting the parameters for decellularization without damage the structure of ECM; ii) pMSCs obtained from porcine BM have characteristic phenotypically and functionally comparable to those of their human counterparts and therefore they can be used as a model for experimental studies such as for liver ECM recellularization; iii) the static seeding strategy of pMSCs on the scaffold resulted to be effective in terms of ECM cell attachment, cell proliferation and migration inside the specimen, iv) the genic profile of cells seeded on ECM scaffold without any growth factors is more similar to pMSC suggesting that the only contact with liver specific ECM is not strong enough to induce a complete differentiation in HLCs. Despite this, we observed that Cyp7a1 gene, expressed in hepatocyte but not in MSC, was present in pMSC seeded scaffolds at each time points. In conclusion, we can observe that our results are in accordance with data reported in literature and sustain the possibility to use decellularizated organs as biological scaffold to create functional organs. We believe that our results may provide new insights toward a better understanding of early HLCs development on ECM-scaffolds. However, a more detailed decellularization process, a better cell differentiation capacity and a more detailed understanding of the interaction between cells and ECM could represent crucial steps in the progression of this research field.
|Titolo:||Mesenchymal stromal cells on bioscaffold for liver bioengineering|
|Data di pubblicazione:||18-mar-2020|
|Appare nelle tipologie:||8.01 Tesi di dottorato|