|M.Sc Student||Nayshool Omri|
|Subject||Transplant of Hepatocytes Encapsulated in Hydrogel Polymers-|
Basis for Liver Tissue Engineering
|Department||Department of Medicine||Supervisor||Clinical Professor Yaacov Baruch|
|Full Thesis text - in Hebrew|
Hepatocyte transplantation is a promising alternative to OLT for the treatment of some liver-based metabolic disorders or liver failure; however bench-to-bedside hurdles, including shortage and poor engraftment of the transplanted cells, need to be overcome.
We have developed micro-polymer hydrogel constructs for intrahepatic or intraportal injection. They are made of fibrinogen conjugated with polyethylene glycol (PEG) to form a PEG-Fibrinogen (PF) hydrogel when mixed with cells and exposed to UV light.
In order to determine the polymers-cells interaction, we encapsulated HuH-7 cells in PF for 72H. MTT and BrdU assays revealed that the cells in PF are viable and can proliferate. Albumin production and CYP-1A1 activity of encapsulated cells was the same as free cells. Those results convinced us that the PF can be used as scaffold for hepatocytes transplantation.
HuH-7 cells suspended in PF were injected while polymerized into the spleen, liver and subcutaneous of rats. The animals were sacrificed 24, 48 and 72 hours, and fluorescent markers and live/dead assays in the spleen and the subcutaneous showed that the cells remain in the polymer and maintain their viability. However, Histologic sections revealed that the cells were appeared mostly in the margins of the polymer and disappeared from the center, indication to poor diffusion of oxygen and nutrients through the hydrogel.
In order to solve the diffusion hurdle, we developed a method to produce microcapsules (MC) using MCILWAIN TISSUE CHOPPER which adapted. FDA/PI assays showed that the cells reached up to ~73% viability after the microencapsulation and a better diffusion rate 72H post encapsulation in-vitro.
Transplantations of free and microencapsulated adult hepatocytes were done in a model which includes direct injection into selected lobes and a 34% partial hepatectomy. The animals were sacrificed 24, 72 and 168H after the surgery. RT-PCR results show that most cells were destroyed a few hours after the transplantation and that there is no difference between the control and experiment groups in cell's engraftment rate (~1%). Histologic sections show MC with cells in all time points.
The most significant result of this study was the ability to deliver into the liver via the portal vein a significant amount of adult hepatocytes, which were protected from shear stress and were able to survive within the polymer for a week. Intraportal injection of microcapsules is a novel model for polymer-encapsulated cell transplantation and it's an important step for the development of injectable tissue engineered liver analogs.