ABSTRACT
BACKGROUND: Recent study has demonstrated the important role of endothelial-mesenchymal interactions in 3-dimensional self-organization of immature progenitor populations with the use of mimicking of organogenesis. Here, we show that the same principle can be applicable to adult mature cells, ie, human adult hepatocytes (hAHs). METHODS: We cultivated hAHs with fluorescence-labeled human mesenchymal cells (hMSCs) and human umbilical vein endothelial cells (HUVECs) in micro-well culture plates and observed them for 9 days. Fluorescence microscopy imaging analyses were performed to evaluate the internal structures of generated 3-dimensional tissues. Maintenance of in vitro protein production capacity was examined with the use of enzyme-linked immunosorbent assay (ELISA). RESULTS: hAHs started to self-organize into 3-dimensional tissue with the use of coculturing with hMSCs and HUVECs. Live imaging analyses showed that endothelial cells started sprouting inside the generated tissues after 2 days of culture. ELISA showed that human albumin production capacity was improved with the use of coculture compared with hAHs-only culture after 9 days. CONCLUSIONS: We demonstrated that 3-dimensional vascularized hepatic tissue could be generated from hAHs by reconstituting endothelial-mesenchymal interactions. Future studies are needed to evaluate the therapeutic potential of vascularized hepatic tissue transplantation, and this may pave a new way to establish a new transplantation modality as an alternative to hepatocyte transplantation.
Subject(s)
Bioartificial Organs , Cell Communication , Hepatocytes/physiology , Human Umbilical Vein Endothelial Cells/physiology , Liver/blood supply , Liver/cytology , Neovascularization, Physiologic , Tissue Engineering/methods , Adult , Cell Culture Techniques , Cells, Cultured , Hepatocytes/metabolism , Hepatocytes/transplantation , Human Umbilical Vein Endothelial Cells/metabolism , Humans , Liver/metabolism , Liver Transplantation/methods , Luminescent Proteins/biosynthesis , Luminescent Proteins/genetics , Mesoderm/cytology , Microscopy, Fluorescence , Serum Albumin/metabolism , Serum Albumin, Human , Time Factors , Transfection , Red Fluorescent ProteinABSTRACT
We presented a new colorimetric bioassay of aminoglycoside antibiotics, which were represented by netilmicin (NTL) in this study, based on the discoloration of thymolphthalein (TP) in paper (indicator-disc) by carbon dioxide produced by Bacillus subtilis. To evaluate the amount of the carbon dioxide, the following experiment was carried out. One milliliter of B. subtilis suspension containing 4.5 x 10(7) colony forming units/ml, 1 ml of nutrient broth, 0.9 ml of 0.1 M phosphate buffer (pH 8.0) and 0.1 ml NTL sample solution were added to an incubation container, which was then placed in a water-bath (37 degrees C) for 3 hours. The oxygen concentration in the head space of flask was determined using gas-chromatograph. The dose-response curves showed good correlation between amounts of NTL and carbon dioxide produced by B. subtilis. The indicator-disc containing TP and sodium hydroxide was placed into the Reacti-flask and then incubated in the same manner as described above. After incubation, concentration of blue colored TP was determined using a TLC scanner. The discoloration of blue color to white showed the proportionality between NTL concentrations and the degrees of discoloration of TP. The method can accurately measure NTL levels down to 2.5 micrograms/ml in water using 0.1 ml samples, and should be adequate for rapid bioassay.
Subject(s)
Chromatography, Thin Layer , Netilmicin/analysis , Bacillus subtilis , Bacteriological Techniques , Carbon Dioxide/analysis , Thymolphthalein/chemistryABSTRACT
A simple method has been developed for the determination of the aminoglycoside antibiotic isepamicin (ISP) in whole blood, using dried blood spots (DBSs) on filter-paper. ISP in the DBSs were recovered most effectively in 0.5 M Na2HPO4-NaH2PO4 buffer (pH 7) by incubation for 30 minutes at 35 degrees C in an ultrafiltration tube. The eluates from the DBS papers were centrifuged at 3,000 x g for 10 minutes. The clear, colorless filtrates were transferred to an Abbott TDx cartridge for measurement by the fluorescence polarization immunoassay. The lower limit for the quantitative determination of ISP in the DBS was 2.5 micrograms per ml of whole blood. The method permits a simple collection of blood at a microliter level and should prove particularly useful for therapeutic drug monitoring of ISP in blood at effective levels in pediatric and geriatric patients.
Subject(s)
Gentamicins/blood , Animals , Blood Specimen Collection , Filtration/instrumentation , Fluorescence Polarization Immunoassay , Guinea Pigs , HumansABSTRACT
In general, collection of serial blood samples from small experimental animals is difficult in terms of sampling site and operation technique. To overcome these problems, a simple reproducible method has been improved by the use of filter papers. Whole blood obtained by venipuncture from the ear vein of guinea-pig was spotted onto a filter paper. Isepamicin in the dried blood spot was extracted with 0.5 M Na2HPO4 buffer by incubation and determined by fluorescence polarization immunoassay. Linearity was established over the range of 5-150 micrograms/ml by using only 100 microliters of whole blood. Consequently its accuracy and precision were good, with mean coefficient of variation of less than 5%. The method described here correlates well with a conventional sampling method and could be used for the pre-clinical study of isepamicin blood levels of individual guinea-pigs. This method is suitable for the simultaneous measurement of aminoglycoside antibiotics or physiological parameters after the administration of aminoglycoside antibiotics for the pharmacokinetics study.
