- Email: [email protected]
CD34+ stromal vascular fraction was instrumental in mediating hepatic repair regarding gene expression profiles
Poster Abstract Presentations problems in tissue engineering. Therefore, several studies to fabricate tissue replacement with the antioxidant capability to protect the replaced organ from free radicals have been reported. Further, multifunctional bone grafting biomaterials with both antioxidants and angiogenic properties have earned increasing interest in regenerative medicine. This study focuses on the functionality of antioxidant and angiogenic efficacy of p-CA and COMP-Ang1 to promote bone and vascular growth into biomaterials. Methods, Results & Conclusion: Collagen scaffold were synthesized and loaded with p-CA and COMP-Ang1. Scaffold were divided into four groupsonly scaffold, scaffold with p-CA, scaffold with COMP-Ang1 and scaffold with p-CA + COMP-Ang1.To evaluate the potential of each scaffold in bone regeneration, a critical-sized defect was made at the mandible of SpragueDawley rats. The defects were filled with scaffold accordingly groups as mentioned above. At 2, 6 and 10 weeks post-implantation, bone growth around the defect was examined by histology and mCT. Results revealed that implanting a COMP-Ang1 plus p-CA impregnated scaffold into a bone defects synergistically enhanced the amount of new bone. Furthermore, osteoinductivity of the COMP-Ang1+ p-CA scaffold result in striking upregulation of osteogenesisrelated molecules, including osterix, osteocalcin and osteopontin with increased expression of angiogenic molecules, like- fibroblast growth factor-2 and vascular endothelial growth factor compared with only COMP-Ang1 and p-CA activated scaffold. These findings demonstrate that combined grafting of COMP-Ang1 with p-CA promotes bone formation in mandible defects, which is coupled with enhanced osteogenesis and angiogenesis and provides new insights for developing bone substitutes for tissue engineering and regenerative medicine. 322 MONITORING THE ONLINE LIVE CELL CONCENTRATION IN THE EXPANSION PHASE OF GENE AND CELL THERAPY APPLICATIONS J. Carvell1 & M. Rotundi2 1 aber instruments, Aberystwyth, United Kingdom, 2Biochemical Engineering, University College London, London, United Kingdom Background & Aim: In the cell expansion phase in cell processing, cells that have undergone genetic modification need to be expanded to target concentrations. Many of the new processes are completely closed in order to eliminate the risk of contamination and so there is a requirement for the online monitoring of the live cell concentration in real time. In mammalian cell culture, Radio-Frequency Impedance (RFI) spectroscopy probes are widely used for cell concentration monitoring. With the availability of scaled down single use probes, RFI can be used to monitor process irregularities, define when sufficient cells are available for or it might be the trigger point for providing additional feed to the bioreactor in perfused systems. Moreover, with stem cell cultures once embryoid bodies form it is both difficult to take a representative sample and to get a true cell count using a trypsin pre-treatment. The same problems occur if the cells are grown on micro-carriers. Methods, Results & Conclusion: In this work, we have evaluated the performance of RFI probes in bioreactors designed for applications in gene and cell therapy in regenerative medicine. This included bioreactors where the sensor is integrated into a small rocking motion bag, a cell expansion bag designed for adherent cells, a stirred tank bioreactor and a flow past cell that allows measurement in an external loop to the bioreactor. . It will also include the first published results of the online monitoring of the growth of T cells in a stirred tank bioreactor. The results will show how RFI can be used to monitor cell growth of Mesenchymal stem cells grown on micro-carriers from 50,000 cells/ml through to 300,000 cells/ml. The reference method based on trypsining the cells from the carriers and counting the nuclei with a Coulter Counter would only provide the total cells. A good correlation was found between the offline method and the RFI method. 324 CD34+ STROMAL VASCULAR FRACTION WAS INSTRUMENTAL IN MEDIATING HEPATIC REPAIR REGARDING GENE EXPRESSION PROFILES C. Ho1, Y. Chen1, S. Ho1, H. Chen1, C. Chien2, J. Chen3, C. Hsiao3, H. Chen1, R. Hu1, D. Shih3 & P. Lee1 1 National Taiwan University Hospital, Taipei, Taiwan, 2National Taiwan University, Taipei, Taiwan, 3Taipei Medical University, Taipei, Taiwan Background & Aim: Cell therapy for patients with acute-on-chronic liver failure (ACLF), which is associated with high short-term mortality, is a
e9
promising strategy. We reasoned that administering stromal vascular cell (SVF) therapy to achieve cell-mediated microenvironment modulation for liver regeneration and hepatocyte replenishment in ACLF is an attractive alternative strategy to liver transplantation. We evaluated the perturbation of microenvironment by CD34+/CD34¡ SVF cells in hepatocyte cotransplantation in a rat model of ACLF regarding selected gene expression. Methods, Results & Conclusion: Donor hepatocytes (1 £ 107/1mL) and SVF cells (1 £ 106/0.5mL) were isolated from Tg(UBC emGFP) rats and human adipose tissues, respectively, and were intraportally transplanted into ACLF rats. SVF cells were divided into CD34+ and CD34¡ groups. The surviving animals were sacrificed at 1 and 2 weeks after the transplantation. Gene expression levels, measured by quantitative real-time RT-PCR, were normalized against endogenous glyceraldehyde-3-phosphatedehydrogenase control. Histologically, prominent biliary ductular proliferation and significantly progression of fibrosis (P = 0.005) were observed in the CD34¡ group than in the CD34+ group. Genes directly related to the end result of fibrosis (collagen type I, MMP9, and TIMP1) showed significantly higher expression in the CD34¡ group than in the CD34+ group at 1 week after cell transplantation (P < 0.05). These findings were consistent with histological changes. Moreover, TGFb1 and IRF5 had the same expression pattern with significance (P < 0.05). Fibrosis-prone histopathology in the CD34¡ group was associated with TGFb1 and IRF5. On the other side, significantly higher gene expression of vascular endothelial growth factor was found in the CD34+ group compared to the CD34- group (P = 0.032), suggesting transplanted CD34+ SVF cells are key components and might lead to liver repair by modulating angiogenesis. In conclusion, hepatic gene expression in the rat model of ACLF after cytotherapy of SVF cells and hepatocytes were differed with CD34 positivity. CD34+ SVF was instrumental in mediating hepatic repair. 325 LABEL-FREE SPECTROSCOPY FOR CELL MONITORING D. Yong1, A. Abdul Rahim2, B. Wei3, Y. Wu2, C. Thwin2, S. Chen2 & M. Win Naing2 1 Precision Measurements Group, Singapore Institute of Manufacturing Technology, Singapore, Singapore, Singapore, 2Bio-Manufacturing Programme, Singapore Institute of Manufacturing Technology, Singapore, Singapore, 3School of Electrical and Electronic Engineering, Nanyang Technological University, Singapore, Singapore Background & Aim: Cell monitoring is essential to the quality assurance and control of cell-based products, especially those used in therapeutics. Conventionally, sampling is performed at various stages of the manufacturing process to probe for the condition of cells in terms of viability, potency, phenotype and other characteristics. This is achieved by either an indirect assessment of the cells’ health through measurements of contents in their environment or a direct but destructive measurement of the cells. Indirect measurements are not absolute and require reference either to earlier time points or a calibration curve, while destructive testing is not ideal as it results in wastage and increases the risk of contamination. Considering that cells are the product in cell-based therapeutics, it is necessary that direct measurements be made to access their quality. The high cost of such therapeutics further motivates the development of non-destructive measurement technologies, so as to maximize yield. Methods, Results & Conclusion: We developed a method of monitoring that is able to directly measure cell characteristics without sacrificing cell samples. The method is based on optical spectroscopy, where the collected signal is distributed according to its wavelength in a spectrum. Specifically, we made use of cell autofluorescence, which is the emission from native metabolic coenzymes when excited by ultraviolet A light. We studied the autofluorescence spectra for cultured cells at different levels of confluency, from 20% to 90% confluence in over 450 spectra. Spectral data were first signal processed and analysed using a spectral decomposition method, from which a redox ratio was computed. The redox ratio is a representative measurement of the metabolic state of cells. A strong correlation between cell confluence and redox ratio was noted, with statistical significance observed at 20% confluence intervals. In order to implement this correlation in monitoring, machine learning was applied to raw spectral data to train classification and regression models with supervision. The trained models achieved accuracies of up to 70% when predicting cell confluences from autofluorescence spectra. Following our measurements, assays were conducted and results validate that ultraviolet A light did not adversely affect cell viability and DNA integrity. In conclusion, we demonstrated the use of a label-free and non-destructive method in determining cell metabolic states with correlation to cell confluence.
e9
promising strategy. We reasoned that administering stromal vascular cell (SVF) therapy to achieve cell-mediated microenvironment modulation for liver regeneration and hepatocyte replenishment in ACLF is an attractive alternative strategy to liver transplantation. We evaluated the perturbation of microenvironment by CD34+/CD34¡ SVF cells in hepatocyte cotransplantation in a rat model of ACLF regarding selected gene expression. Methods, Results & Conclusion: Donor hepatocytes (1 £ 107/1mL) and SVF cells (1 £ 106/0.5mL) were isolated from Tg(UBC emGFP) rats and human adipose tissues, respectively, and were intraportally transplanted into ACLF rats. SVF cells were divided into CD34+ and CD34¡ groups. The surviving animals were sacrificed at 1 and 2 weeks after the transplantation. Gene expression levels, measured by quantitative real-time RT-PCR, were normalized against endogenous glyceraldehyde-3-phosphatedehydrogenase control. Histologically, prominent biliary ductular proliferation and significantly progression of fibrosis (P = 0.005) were observed in the CD34¡ group than in the CD34+ group. Genes directly related to the end result of fibrosis (collagen type I, MMP9, and TIMP1) showed significantly higher expression in the CD34¡ group than in the CD34+ group at 1 week after cell transplantation (P < 0.05). These findings were consistent with histological changes. Moreover, TGFb1 and IRF5 had the same expression pattern with significance (P < 0.05). Fibrosis-prone histopathology in the CD34¡ group was associated with TGFb1 and IRF5. On the other side, significantly higher gene expression of vascular endothelial growth factor was found in the CD34+ group compared to the CD34- group (P = 0.032), suggesting transplanted CD34+ SVF cells are key components and might lead to liver repair by modulating angiogenesis. In conclusion, hepatic gene expression in the rat model of ACLF after cytotherapy of SVF cells and hepatocytes were differed with CD34 positivity. CD34+ SVF was instrumental in mediating hepatic repair. 325 LABEL-FREE SPECTROSCOPY FOR CELL MONITORING D. Yong1, A. Abdul Rahim2, B. Wei3, Y. Wu2, C. Thwin2, S. Chen2 & M. Win Naing2 1 Precision Measurements Group, Singapore Institute of Manufacturing Technology, Singapore, Singapore, Singapore, 2Bio-Manufacturing Programme, Singapore Institute of Manufacturing Technology, Singapore, Singapore, 3School of Electrical and Electronic Engineering, Nanyang Technological University, Singapore, Singapore Background & Aim: Cell monitoring is essential to the quality assurance and control of cell-based products, especially those used in therapeutics. Conventionally, sampling is performed at various stages of the manufacturing process to probe for the condition of cells in terms of viability, potency, phenotype and other characteristics. This is achieved by either an indirect assessment of the cells’ health through measurements of contents in their environment or a direct but destructive measurement of the cells. Indirect measurements are not absolute and require reference either to earlier time points or a calibration curve, while destructive testing is not ideal as it results in wastage and increases the risk of contamination. Considering that cells are the product in cell-based therapeutics, it is necessary that direct measurements be made to access their quality. The high cost of such therapeutics further motivates the development of non-destructive measurement technologies, so as to maximize yield. Methods, Results & Conclusion: We developed a method of monitoring that is able to directly measure cell characteristics without sacrificing cell samples. The method is based on optical spectroscopy, where the collected signal is distributed according to its wavelength in a spectrum. Specifically, we made use of cell autofluorescence, which is the emission from native metabolic coenzymes when excited by ultraviolet A light. We studied the autofluorescence spectra for cultured cells at different levels of confluency, from 20% to 90% confluence in over 450 spectra. Spectral data were first signal processed and analysed using a spectral decomposition method, from which a redox ratio was computed. The redox ratio is a representative measurement of the metabolic state of cells. A strong correlation between cell confluence and redox ratio was noted, with statistical significance observed at 20% confluence intervals. In order to implement this correlation in monitoring, machine learning was applied to raw spectral data to train classification and regression models with supervision. The trained models achieved accuracies of up to 70% when predicting cell confluences from autofluorescence spectra. Following our measurements, assays were conducted and results validate that ultraviolet A light did not adversely affect cell viability and DNA integrity. In conclusion, we demonstrated the use of a label-free and non-destructive method in determining cell metabolic states with correlation to cell confluence.