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A three-dimensional mouse liver organoid platform for assessing EDCs metabolites simulating liver metabolism.

Hepatic metabolism is an important process for evaluate the potential activity and toxicity of endocrine disrupting chemicals (EDCs) metabolites. Organization for Economic Co-operation and Development (OECD) has advocated the development of in vitro assays that mimic in vivo hepatic metabolism to eventually replace classical animal tests. In response to this need, we established a 3D mouse liver organoid (mLO) platform that mimics the animal model and is distinct from existing models. We evaluated the effects the activity of EDC metabolites generated through mLOs based on human cell-based reporter gene assays in addition to existing models. This study emphasizes the importance of hepatic ex-vivo and suggests the need a new metabolic model through a 3D mLOs platform. These results indicate that mLOs provides a novel biological method to screen for potential endocrine-disrupting activities of EDC metabolites.

Animals↗

A population of c-Kit(low)(CD45/TER119)- hepatic cell progenitors of 11-day postcoitus mouse embryo liver reconstitutes cell-depleted liver organoids.

Embryo liver morphogenesis takes place after gastrulation and starts with a ventral foregut evagination that reacts to factor signaling from both cardiac mesoderm and septum transversum mesenchyme. Current knowledge of the progenitor stem cell populations involved in this early embryo liver development is scarce. We describe here a population of 11-day postcoitus c-Kit(low)(CD45/TER119)- liver progenitors that selectively expressed hepatospecific genes and proteins in vivo, was self-maintained in vitro by long-term proliferation, and simultaneously differentiated into functional hepatocytes and bile duct cells. Purified c-Kit(low)(CD45/TER119)- liver cells cocultured with cell-depleted fetal liver fragments engrafted and repopulated the hepatic cell compartments of the latter organoids, suggesting that they may include the embryonic stem cells responsible for liver development.

Animals↗

Reconstruction of liver organoid using a bioreactor.

AIM: To develop the effective technology for reconstruction of a liver organ in vitro using a bio-artificial liver. METHODS: We previously reported that a radial-flow bioreactor (RFB) could provide a three-dimensional high-density culture system. We presently reconstructed the liver organoid using a functional human hepatocellular carcinoma cell line (FLC-5) as hepatocytes together with mouse immortalized sinusoidal endothelial cell (SEC) line M1 and mouse immortalized hepatic stellate cell (HSC) line A7 as non parenchymal cells in the RFB. Two x 10(7) FLC-5 cells were incubated in the RFB. After 5 d, 2 x 10(7) A7 cells were added in a similar manner followed by another addition of 10(7) M1 cells 5 d later. After three days of perfusion, some cellulose beads with the adherent cells were harvested. The last incubation period included perfusion with 200 nmol/L swinholide A for 2 h and then the remaining cellulose beads along with adherent cells were harvested from the RFB. The cell morphology was observed by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). To assess hepatocyte function, we compared mRNA expression for urea cycle enzymes as well as albumin synthesis by FLC-5 in monolayer cultures compared to those of single-type cultures and cocultures in the RFB. RESULTS: By transmission electron microscopy, FLC-5, M1, and A7 were arranged in relation to the perfusion side in a liver-like organization. Structures resembling bile canaliculi were seen between FCL-5 cells. Scanning electron microscopy demonstrated fenestrae on SEC surfaces. The number of vesiculo-vacuolar organelles (VVO) and fenestrae increased when we introduced the actin-binding agent swinholide-A in the RFB for 2h. With respect to liver function, urea was found in the medium, and expression of mRNAs encoding arginosuccinate synthetase and arginase increased when the three cell types were cocultured in the RFB. However, albumin synthesis decreased. CONCLUSION: Co-culture in the RFB system can dramatically change the structure and function of all cell types, including the functional characteristics of hepatocytes. Our system proves effective for reconstruction of a liver organoid using a bio-artificial liver.

Albumins↗

Intravacuolar proteolysis in Plasmodium falciparum digestive vacuoles is similar to intralysosomal proteolysis in mammalian cells.

Hemoglobin of intact human erythrocytes was labelled with [125I]iodide and these cells were inoculated with late-stage cultures of Plasmodium falciparum. Subcellular parasite particulate material containing intact digestive vacuoles filled with 125I-labelled hemoglobin was incubated in sucrose-buffer medium and degradation of labelled intravacuolar hemoglobin was measured by precipitation with trichloroacetic acid. Proteolysis was maximal at pH 5.0 or in the presence of MgATP at pH 8.0. The stimulatory effect of MgATP was probably due to energization of a proton pump activity as reported by others (Krogstad, D.J., Schlesinger, P.H. and Gluzman, I.Y. (1984) J. Cell Biol. 101, 2302-2309). Proteolysis was also inhibited by ionophores and antimalarials. These results suggest that P. falciparum digestive vacuoles have an ATP-dependent acidification mechanism similar to mammalian lysosomes but with some exceptions. The properties of this intravacuolar proteolysis were remarkably similar to intralysosomal proteolysis in mouse liver or kidney preparations.

Adenosine Triphosphate↗

Cytochemical studies on induced autophagocytosis in mouse exocrine pancreas.

1. The origin of the limiting membranes of autophagic vacuoles (AVs) in mouse pancreatic acinar cells was studied in vinblastine-induced autophagocytosis. 2. The marker enzymes used were adenosine triphosphatase, lipase, inosine diphosphatase and thiamine pyrophosphatase. The following impregnation techniques were used: unbuffered osmium tetroxide impregnation, imidazole-buffered osmium tetroxide impregnation and uranyl-lead-copper impregnation. 3. Only a weak lipase activity was observed between the limiting membranes of a few AVs. The AV membranes were stained heavily with all impregnation techniques used. 4. The origin of AV membranes seems to be same in mouse liver and exocrine pancreas in vinblastine-induced autophagocytosis.

Acid Anhydride Hydrolases↗