- 學位論文
Microfluidic Bioreactors for Long-Term Culture and Histopathological Studies of Transgenic Mice Liver Tissue
肝組織長期培養之微流控生物反應器研發
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摘要
Nurturing thick liver tissue for long-term allowing regeneration within 3D environment with perfusion system to improve clinical predictivity of preclinical testing is described in this thesis. At first, we have tried to prolong the culture period of primary tissue sample >2mm that were well supported on 3D microfluidic architecture or scaffold thereby aiding tissue samples to meet high oxygen demands of liver tissue mimicking 3D microenvironment present in vivo. Secondly to promote regeneration in tissue section we have added growth factors like hepatocyte growth factor (HGF) and insulin-like growth factor (IGF) and were successful in culturing tissue that could survive for at least 16 days allowing one round of ex vivo regeneration.. In order to supply the tissues with in vivo microenvironment there is a need for pumping system, reservoirs; microfluidic structures/hydrogels with appropriate porosity to meet the oxygen demand. The bioreactor comprised of microstructures or hydrogels that were bio-compatible to support the tissue and minimize the shear stress that was caused due to medium perfusion at high flow rates to meet the oxygen demand. The bioreactor comprises of a pumping device, supporting device, breathers, and reservoirs to meet the essential requirements of the tissue sample. Initiation of hepatocyte proliferation ii was observed in the ex vivo liver tissues between the eighth and twelfth days, which could be demonstrated by proliferating cell nuclear antigen (PCNA) staining. Furthermore we tried to observe the decrease in viable cells after 16days and found that cells were adopting extrinsic apoptotic mechanism for cell death when stained with different antibodies for intrinsic and extrinsic apoptosis. Once we found that there could be possibility of regeneration, we wanted to optimize the growth factor concentration and test with a drug Sorafenib which would be effective when combined with another drug or the growth factor. Therefore we have utilized a multi layered device enabling two different functions (encapsulation and gradient generation) on the same chip. Here we determined the efficacy of combined Sorafenib and HGF treatment against hepatoma tumorogenesis and progression using the transgenic mice model as a pilot study. The long-term culture and re-growth of cells within the primary liver tissue samples provided a promising model to study liver pathophysiology or to develop an efficient, safer and effective drug filling the gap between in vivo and cell culture models. The development of these in vitro cell and tissue culture models can contribute substantially to the reduction, refinement, and possibly to the replacement of animal experiments. The efficacy and tolerability of combined Sorafenib and HGF may provide rationale to use in treatment of HCC models.
參考文獻
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