Recent cancer research has demonstrated the existence of circulating tumor cells(CTCs)in cancer patient’s blood.Once identified,CTC biomarkers will be invaluable tools for clinical diagnosis,prognosis and treatment.I...Recent cancer research has demonstrated the existence of circulating tumor cells(CTCs)in cancer patient’s blood.Once identified,CTC biomarkers will be invaluable tools for clinical diagnosis,prognosis and treatment.In this review,we propose ex vivo culture as a rational strategy for large scale amplification of the limited numbers of CTCs from a patient sample,to derive enough CTCs for accurate and reproducible characterization of the biophysical,biochemical,gene expressional and behavioral properties of the harvested cells.Because of tumor cell heterogeneity,it is important to amplify all the CTCs in a blood sample for a comprehensive understanding of their role in cancer metastasis.By analyzing critical steps and technical issues in ex vivo CTC culture,we developed a cost-effective and reproducible protocol directly culturing whole peripheral blood mononuclear cells,relying on an assumed survival advantage in CTCs and CTC-like cells over the normal cells to amplify this specified cluster of cancer cells.展开更多
Background： Large-scale muscle tissue engineering remains a major challenge. An axial vascular pedicle and perfusion bioreactor are necessary for the development and maintenance of large-scale engineered muscle to en...Background： Large-scale muscle tissue engineering remains a major challenge. An axial vascular pedicle and perfusion bioreactor are necessary for the development and maintenance of large-scale engineered muscle to ensure circulation within the construct. We aimed to develop a novel experimental model of a large-scale engineered muscle flap from an existing rat groin fat flap. Methods： A fat flap based on the superficial interior epigastric vascular pedicle was excised from rats and placed into a perfusion bioreactor. The flaps were kept in the bioreactor for up to 7 weeks, and transdifferentiation of adipose to muscle tissue could have taken place. This system enabled myogenic-differentiation medium flow through the bioreactor at constant pH and oxygen concentration. Assessment of viability was pertbrmed by an immunofluorescence assay, histological staining, a calcein-based live/dead test, and through determination of RNA quantity and quality after 1, 3, 5, and 7 weeks. Results： Immunofluorescence staining showed that smooth muscle around vessels was still intact without signs of necrosis or atrophy. The visual assessment of viability by the calcein-based live/dead test revealed viability of the rat adipose tissue preserved in the bioreactor system with permanent perfusion. RNA samples from different experimental conditions were quantified by spectrophotometry, and intact bands of 18S and 28S rRNA were detected by gel electrophoresis, indicating that degradation of RNA was minimal. Conclusions： Flow perfusion maintains the long-term viability of a rat groin engineered muscle flap in vitro, and a large-scale vascularized muscle could be engineered in a perfusion bioreactor.展开更多
基金This work is supported by US NIH/NCI research grant(2PO1CA098912)edars-Sinai Medical Center Board of Governors Cancer Research Chair(LWKC),and US NIH/NCI(UO1CA198900)(HRT/EMP).
文摘Recent cancer research has demonstrated the existence of circulating tumor cells(CTCs)in cancer patient’s blood.Once identified,CTC biomarkers will be invaluable tools for clinical diagnosis,prognosis and treatment.In this review,we propose ex vivo culture as a rational strategy for large scale amplification of the limited numbers of CTCs from a patient sample,to derive enough CTCs for accurate and reproducible characterization of the biophysical,biochemical,gene expressional and behavioral properties of the harvested cells.Because of tumor cell heterogeneity,it is important to amplify all the CTCs in a blood sample for a comprehensive understanding of their role in cancer metastasis.By analyzing critical steps and technical issues in ex vivo CTC culture,we developed a cost-effective and reproducible protocol directly culturing whole peripheral blood mononuclear cells,relying on an assumed survival advantage in CTCs and CTC-like cells over the normal cells to amplify this specified cluster of cancer cells.
文摘Background： Large-scale muscle tissue engineering remains a major challenge. An axial vascular pedicle and perfusion bioreactor are necessary for the development and maintenance of large-scale engineered muscle to ensure circulation within the construct. We aimed to develop a novel experimental model of a large-scale engineered muscle flap from an existing rat groin fat flap. Methods： A fat flap based on the superficial interior epigastric vascular pedicle was excised from rats and placed into a perfusion bioreactor. The flaps were kept in the bioreactor for up to 7 weeks, and transdifferentiation of adipose to muscle tissue could have taken place. This system enabled myogenic-differentiation medium flow through the bioreactor at constant pH and oxygen concentration. Assessment of viability was pertbrmed by an immunofluorescence assay, histological staining, a calcein-based live/dead test, and through determination of RNA quantity and quality after 1, 3, 5, and 7 weeks. Results： Immunofluorescence staining showed that smooth muscle around vessels was still intact without signs of necrosis or atrophy. The visual assessment of viability by the calcein-based live/dead test revealed viability of the rat adipose tissue preserved in the bioreactor system with permanent perfusion. RNA samples from different experimental conditions were quantified by spectrophotometry, and intact bands of 18S and 28S rRNA were detected by gel electrophoresis, indicating that degradation of RNA was minimal. Conclusions： Flow perfusion maintains the long-term viability of a rat groin engineered muscle flap in vitro, and a large-scale vascularized muscle could be engineered in a perfusion bioreactor.
