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Development and application of packed-bed bioartificial liver system
Abstracts / New Biotechnology 33S (2016) S1–S213
mized using design of experiment. Identified factors influencing the transduction are the host cell concentration, the virus concentration or incubation time. Optimizing both the genetic and practical setup lead to an increased expression of GFP in human mesenchymal stem cells. http://dx.doi.org/10.1016/j.nbt.2016.06.754
O2-4 Wrinkle-like structures in reconstructing stem cell niches Klaudia Trembecka-Wójciga 1,∗ , Roman Major 2 , Jurgen Lackner 3 , Marek Sanak 4 , Bogusław Major 5 1 Institute of Metallurgy and Materials Science Polish Academy of Sciences, Poland 2 Institute of Metallurgy and Materials Science Polish Academy of Science, Poland 3 Joanneum Research Forschungsges mbH, Poland 4 Institute of Surface Technologies and Photonics, Poland 5 Functional Surfaces, Poland
Stem cells are the subject of great interest due to their biological properties and clinical application. Each cell, which grows and matures, has its own niche which has a spatial structure of cells and an extracellular matrix. The stem cell niche refers to an anatomical and functional structure, including cellular and extracellular components, local and systemic factors that are integrated to regulate the stem cell proliferation, differentiation, survival and localization. The main purpose of the research is the scientific development of the surface activating cells, especially stem cells from the blood flow. The work lead to understand of the phenomena occurring between material surface and cell and the design dedicated surface which could effectively capture specific cells. In this study a medical-grade polyurethane applied in the heart assist systems was covered by thin a-C:H and a-C:N coatings and modified in the form of niches like structures by the micro and nanowrinkles. For the surface modification physical methods were used, including ion gun to modify biocompatible materials in order to achieve three dimensional structures. Transmission Electron Microscope (TEM), High resolution Electron Microscopy (HREM) were used for analyses. Atomic Force Microscopy (AFM) was used to assess coatings topography. The adhesion and differentiation of commercially available progenitor cells were investigated. For analysis Confocal Laser Fluorescence Microscopy and four monoclonal antibodies were used. The effectiveness of endothelial monolayer formation was evaluated by ZO-1 tight junctions antibody and DAPI. The progenitor cells differentiation into endothelial cells was evaluated by CD31 and CD62E antibodies. http://dx.doi.org/10.1016/j.nbt.2016.06.755
O2-5 Development and application of packed-bed bioartificial liver system Jung-Keug Park 1,∗ , Doo-Hoon Lee 2 , Hee-Hoon Yoon 3 , Ji-Hyun Lee 2 , Suk-Koo Lee 3 1
Dongguk University, Republic of Korea Biomedical Research Institute, Republic of Korea 3 LifeLiver Inc, Republic of Korea 2
To treat acute hepatic failure (AHF) patients, various extracorporeal bioartificial liver (BAL) systems have been developed. The
S9
performance of the BAL depends on the functional activities of the hepatocytes immobilized in the bioreactor. A packed-bed bioreactor with gel beads has higher surface area to volume ratio and cell capacity than those of a hollow fiber bioreactor. Therefore, this type of bioreactor may be a better alternative to hollow fiber-based bioreactor. In this study, we developed a stable packed-bed BAL system in which bioreactor containing porcine hepatocyte spheroids-encapsulated gel beads is perfused with down-ward flow that is generated by difference of a media or plasma level between the reservoir and outlet chamber placed before and after the bioreactor, respectively. This BAL system showed good efficacy and safety in preclinical study using anhepatic pigs or beagle dogs. This system is now under phase I/IIa clinical study and recently is designated as orphan drug in Korea. And also to replace with human hepatocytes in the near future, we have checked which cell (stem/progenitor cell, direct converted cell) is suitable to BAL system with respect to massive production, function and cost. http://dx.doi.org/10.1016/j.nbt.2016.06.756
O2-6 Withdrawn
http://dx.doi.org/10.1016/j.nbt.2016.06.757
O2-7 HO-1 and Nrf-2 deficiency affects generation and differentiation of induced pluripotent stem cells 1,∗ , Alicja Józkowicz 2 , Józef Dulak 1 ˛ Jacek Stepniewski 1 2
Jagiellonian University, Faculty of Biochemistry, Poland Faculty of Biophysics and Biotechnology, Poland
The aim of this study was to evaluate the role of heme oxygenase-1 (HO-1, encoded by Hmox1 gene) and Nrf2 (encoded by Nfe2l2 gene), two major cytoprotective factors, in the generation and differentiation of iPSCs. Hmox1−/− fibroblasts demonstrated elevated level of p53 and p53-regulated miR-34a and 14-3-3s protein followed with inhibition of cell cycle in G2/M phase. Importantly, Hmox1−/− cells generated lower number of iPSCs colonies upon reprogramming than control cells whereas stimulation of fibroblasts with cobalt protoporphyrin (CoPPIX) (HO-1 activator) increased the efficiency of this process. Interestingly, valproic acid shown previously to enhance reprogramming, in our hands decreased expression of HO1 in murine fibroblasts, what was followed by less efficient iPSCs generation. Similarly, impaired reprogramming was observed in Nrf2-lacking fibroblasts, which demonstrated lower level of HO1 than control cells. Additionally, sulforaphan, an Nrf2 activator, increased the number of iPSCs generated from murine fibroblasts. Hmox1+/+ and Hmox1−/− iPSCs, as well as Nfe2l2+/+ and Nfe2l2−/− cells demonstrated similar expression of pluripotency markers. Additionally, Hmox1+/+ and Hmox1−/− iPSCs were able to spontaneously differentiate to cells originating from three germ layers. However, lower number of contracting clusters was observed in HO-1-lacking cells. Interestingly, no teratomas were formed by Hmox1−/− iPSCs upon subcutaneous injection into immunocompromised mice in contrast to their control counterparts. Similar
mized using design of experiment. Identified factors influencing the transduction are the host cell concentration, the virus concentration or incubation time. Optimizing both the genetic and practical setup lead to an increased expression of GFP in human mesenchymal stem cells. http://dx.doi.org/10.1016/j.nbt.2016.06.754
O2-4 Wrinkle-like structures in reconstructing stem cell niches Klaudia Trembecka-Wójciga 1,∗ , Roman Major 2 , Jurgen Lackner 3 , Marek Sanak 4 , Bogusław Major 5 1 Institute of Metallurgy and Materials Science Polish Academy of Sciences, Poland 2 Institute of Metallurgy and Materials Science Polish Academy of Science, Poland 3 Joanneum Research Forschungsges mbH, Poland 4 Institute of Surface Technologies and Photonics, Poland 5 Functional Surfaces, Poland
Stem cells are the subject of great interest due to their biological properties and clinical application. Each cell, which grows and matures, has its own niche which has a spatial structure of cells and an extracellular matrix. The stem cell niche refers to an anatomical and functional structure, including cellular and extracellular components, local and systemic factors that are integrated to regulate the stem cell proliferation, differentiation, survival and localization. The main purpose of the research is the scientific development of the surface activating cells, especially stem cells from the blood flow. The work lead to understand of the phenomena occurring between material surface and cell and the design dedicated surface which could effectively capture specific cells. In this study a medical-grade polyurethane applied in the heart assist systems was covered by thin a-C:H and a-C:N coatings and modified in the form of niches like structures by the micro and nanowrinkles. For the surface modification physical methods were used, including ion gun to modify biocompatible materials in order to achieve three dimensional structures. Transmission Electron Microscope (TEM), High resolution Electron Microscopy (HREM) were used for analyses. Atomic Force Microscopy (AFM) was used to assess coatings topography. The adhesion and differentiation of commercially available progenitor cells were investigated. For analysis Confocal Laser Fluorescence Microscopy and four monoclonal antibodies were used. The effectiveness of endothelial monolayer formation was evaluated by ZO-1 tight junctions antibody and DAPI. The progenitor cells differentiation into endothelial cells was evaluated by CD31 and CD62E antibodies. http://dx.doi.org/10.1016/j.nbt.2016.06.755
O2-5 Development and application of packed-bed bioartificial liver system Jung-Keug Park 1,∗ , Doo-Hoon Lee 2 , Hee-Hoon Yoon 3 , Ji-Hyun Lee 2 , Suk-Koo Lee 3 1
Dongguk University, Republic of Korea Biomedical Research Institute, Republic of Korea 3 LifeLiver Inc, Republic of Korea 2
To treat acute hepatic failure (AHF) patients, various extracorporeal bioartificial liver (BAL) systems have been developed. The
S9
performance of the BAL depends on the functional activities of the hepatocytes immobilized in the bioreactor. A packed-bed bioreactor with gel beads has higher surface area to volume ratio and cell capacity than those of a hollow fiber bioreactor. Therefore, this type of bioreactor may be a better alternative to hollow fiber-based bioreactor. In this study, we developed a stable packed-bed BAL system in which bioreactor containing porcine hepatocyte spheroids-encapsulated gel beads is perfused with down-ward flow that is generated by difference of a media or plasma level between the reservoir and outlet chamber placed before and after the bioreactor, respectively. This BAL system showed good efficacy and safety in preclinical study using anhepatic pigs or beagle dogs. This system is now under phase I/IIa clinical study and recently is designated as orphan drug in Korea. And also to replace with human hepatocytes in the near future, we have checked which cell (stem/progenitor cell, direct converted cell) is suitable to BAL system with respect to massive production, function and cost. http://dx.doi.org/10.1016/j.nbt.2016.06.756
O2-6 Withdrawn
http://dx.doi.org/10.1016/j.nbt.2016.06.757
O2-7 HO-1 and Nrf-2 deficiency affects generation and differentiation of induced pluripotent stem cells 1,∗ , Alicja Józkowicz 2 , Józef Dulak 1 ˛ Jacek Stepniewski 1 2
Jagiellonian University, Faculty of Biochemistry, Poland Faculty of Biophysics and Biotechnology, Poland
The aim of this study was to evaluate the role of heme oxygenase-1 (HO-1, encoded by Hmox1 gene) and Nrf2 (encoded by Nfe2l2 gene), two major cytoprotective factors, in the generation and differentiation of iPSCs. Hmox1−/− fibroblasts demonstrated elevated level of p53 and p53-regulated miR-34a and 14-3-3s protein followed with inhibition of cell cycle in G2/M phase. Importantly, Hmox1−/− cells generated lower number of iPSCs colonies upon reprogramming than control cells whereas stimulation of fibroblasts with cobalt protoporphyrin (CoPPIX) (HO-1 activator) increased the efficiency of this process. Interestingly, valproic acid shown previously to enhance reprogramming, in our hands decreased expression of HO1 in murine fibroblasts, what was followed by less efficient iPSCs generation. Similarly, impaired reprogramming was observed in Nrf2-lacking fibroblasts, which demonstrated lower level of HO1 than control cells. Additionally, sulforaphan, an Nrf2 activator, increased the number of iPSCs generated from murine fibroblasts. Hmox1+/+ and Hmox1−/− iPSCs, as well as Nfe2l2+/+ and Nfe2l2−/− cells demonstrated similar expression of pluripotency markers. Additionally, Hmox1+/+ and Hmox1−/− iPSCs were able to spontaneously differentiate to cells originating from three germ layers. However, lower number of contracting clusters was observed in HO-1-lacking cells. Interestingly, no teratomas were formed by Hmox1−/− iPSCs upon subcutaneous injection into immunocompromised mice in contrast to their control counterparts. Similar