Three-dimensional spheroid culture of canine hepatocyte-like cells derived from bone marrow mesenchymal stem cells

Nitta, Suguru; Hisasue, Masaharu; Horiguchi, Yu; Yamada, Yoko; Kikuchi, Kaoruko; Kubo, Takeaki; Igarashi, Hirotaka; Neo, Sakurako

doi:10.1016/j.reth.2020.09.002

Cited by 5 publications

(8 citation statements)

References 16 publications

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“…Larval material was obtained from euthanized BALB/c mice experimentally infected with homogenized larval tissue of E. multilocularis , which was originally isolated from a naturally infected plateau pika ( Ochotona curzoniae ) collected in Yushu, Qinghai Province, China. Molecular identification of the isolate of E. multilocularis was performed as described by Nitta et al [ 19 ]. E. multilocularis metacestodes were prepared and cultured as previously described [ 20 ].…”

Section: Methodsmentioning

confidence: 99%

“…First-strand complementary DNA (cDNA) was synthesized using a FastKing gDNA Dispelling RT SuperMix Kit (TianGen Biotech). PCR was performed using specific primers for amplification of E. multilocularis glyceraldehyde-3-phosphate dehydrogenase (GAPDH) [ 19 ] and mouse GAPDH (forward, 5′-CGTGGGGCAGCCCAGAACAT-3′; reverse, 5′-GAGCAATGCCAGCCCCAGCA-3′).…”

Section: Methodsmentioning

confidence: 99%

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In vivo and in vitro efficacy of crocin against Echinococcus multilocularis

et al. 2021

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Background Alveolar echinococcosis (AE) is a fatal zoonosis caused by the larvae of Echinococcus multilocularis. However, current chemotherapy treatment options are based on benzimidazoles [albendazole (ABZ) and mebendazole], which have limited efficacy. Therefore, novel drugs are necessary for the treatment of this disease. Methods The anthelmintic effects of crocin were tested on E. multilocularis metacestodes, germinal cells and protoscoleces in vitro. Human foreskin fibroblasts (HFFs) and Reuber rat hepatoma (RH) cells were used to assess cytotoxicity. The in vivo efficacy of crocin was investigated in mice following secondary infection with E. multilocularis. Furthermore, collagen deposition and degradation in host tissues around the metacestodes were evaluated. Results In vitro, crocin had a median effective concentration of 11.36 μM against cultured E. multilocularis metacestodes, while it reduced germinal cell viability at a median inhibitory concentration of 10.05 μM. Crocin was less toxic to HFFs and RH mammalian cell lines than to metacestodes. Transmission electron microscopy revealed that crocin treatment resulted in structural damage in the germinal layer. In addition, 60.33 ± 3.06% of protoscoleces were killed by treatment with 10 μM crocin for 7 days, indicating that crocin has a parasiticidal effect. In vivo, the metacestode weight was significantly reduced after the administration of crocin at 50 mg/kg and 100 mg/kg (55.1 and 68.1%, respectively). Metacestode pathology showed structural disruption of the germinal and laminated layers after crocin treatment. The crocin- and ABZ-treated groups presented significant increases in the levels of interleukin (IL)-2 and IL-4. Furthermore, crocin inhibited the expression of matrix metalloproteinases (MMPs) (MMP2 and MMP9) and promoted collagen deposition in the metacestode. Conclusions Crocin was demonstrated to exert parasiticidal activity against E. multilocularis in vitro and in vivo, and can be developed as a novel drug for the treatment of AE. Graphical abstract

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

In vivo and in vitro efficacy of crocin against Echinococcus multilocularis

et al. 2021

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“…Here, we indicated that LcHS enables easy and efficient large-scale hepatocyte culture in dogs and suggests that LcHS can be applied in the development of toxicity evaluation tools and transplantation medicine as a basic technique. In liver regenerative medicine research, many studies have been conducted on the formation of spheroids and their culture improvement in mice, humans, and dogs [ [11] , [12] , [13] , [14] ]. In humans, studies have been conducted in vitro to culture tissues that are physiologically and functionally closer to in vivo organs by producing spheroids by mixing multiple cell types.…”

Section: Discussionmentioning

confidence: 99%

“…However, canine allogeneic liver transplantation, including living-donor liver transplantation, has not been established in the field of veterinary medicine. In addition, a novel construct culture protocol must be established with a three-dimensional (3D) structure of canine artificial liver, given that hepatic function and metabolism such as albumin and cytochrome (CYP) expression are exerted in the liver tissue of a three-dimensional structure [ 11 ]. To solve this problem, research on constructing tissue-like structures using 3D cell culture is being actively conducted.…”

Section: Introductionmentioning

confidence: 99%

Establishment of large canine hepatocyte spheroids by mixing vascular endothelial cells and canine adipose-derived mesenchymal stem cells

Ichikawa

Neo

Nukui

et al. 2022

Regenerative Therapy

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Introduction Differentiation of hepatocytes and culture methods have been investigated in dogs as a tool to establish liver transplant and drug metabolism examination systems. However, mass culture techniques for canine hepatocytes (cHep) have not been investigated, and it is necessary to construct a suitable culture system. Recently, a protocol called Bud production has attracted attention, and a mixed culture of human and mouse hepatocytes, stem cells, and artificial blood vessels significantly improved the size and formation ratio of spheroids. The purpose of this study was to investigate and improve the in vitro culture of cHep by mixing canine adipose-derived mesenchymal stem cells (cASCs) and human umbilical vein endothelial cells (HUVECs). Methods Spheroid formation ratio and histological examination were evaluated among four culture methods, including cHep alone, two-mix (cHep + cASCs and cHep + HUVEC), and three-mix (cHep + HUVEC + cASCs), on days 0, 4, and 7. Expression levels of liver-related genes ( ALB , AFP , α1-AT , CDH1 , CYP2E1 , CYP3A12 , and TAT ) were evaluated by quantitative real-time polymerase chain reaction (RT-PCR). Protein expression of albumin, vimentin, and von Willebrand Factor (vWF) was investigated to confirm the location of the hepatocytes. Results The ratio of spheroid formation was 60.2% in the three-mix culture and was significantly improved compared with cHep alone (5.9%) and two-mix; cHep + cASCs (36.2%) and cHep + HUVEC (26.4%) (P < 0.001). Histological evaluation revealed that the three-mix spheroids formed large canine hepatocyte spheroids (LcHS), and hepatocytes were distributed in the center of the spheroids. Quantitative gene expression analysis of LcHS showed that liver-related genes expression were maintained the same levels with that of a culture of cHep alone from days 4–7. Conclusion These results revealed that the three-mix culture method using cHep, HUVECs, and cASCs was capable of promoting LcHS without impairing liver function in cHep, suggesting that LcHS could be used for the application of high-volume culture techniques in dogs.

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“…The first-strand cDNA was synthesized using the FastKing gDNA Dispelling RT SuperMix Kit (TianGen Biotech). PCR was performed using primers for the specific amplification of E. multilocularis GAPDH [ 27 ] and mouse GADPH (forward: 5′-CGTGGGGCAGCCCAGAACAT-3′; reverse: 5′-GAGCAATGCCAGCCCCAGCA-3′).…”

Section: Methodsmentioning

confidence: 99%

In vitro and in vivo efficacy of thiacloprid against Echinococcus multilocularis

Liu

Fan

et al. 2021

Parasites Vectors

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Background Alveolar echinococcosis (AE) is a chronic zoonosis caused by the larval form of Echinococcus multilocularis (E. multilocularis). Current chemotherapy against AE has relied on albendazole and mebendazole, which only exhibit parasitostatic and not parasiticidal efficacy. Therefore, novel compounds for the treatment of this disease are needed. Methods Phosphoglucose isomerase (PGI) assays were used for compound screening of seven neonicotinoids. The anti-parasitic effects of thiacloprid were then evaluated on E. multilocularis metacestode vesicles, germinal cells and protoscoleces in vitro. Human foreskin fibroblasts (HFF) and Reuber rat hepatoma (RH) cells were used to assess cytotoxicity. Glucose consumption in E. multilocularis protoscoleces and germinal cells was assessed by measuring uptake of 2-deoxyglucose (2-DG). Molecular docking was used to evaluate the potential binding sites of thiacloprid to acetylcholine receptors. In vivo efficacy of thiacloprid was evaluated in mice by secondary infection with E. multilocularis. In addition, ELISA and flow cytometry were used to evaluate the effects of cytokines and T lymphocyte subsets after thiacloprid treatment. Furthermore, collagen deposition and degradation in the host lesion microenvironment were evaluated. Results We found that thiacloprid is the most promising compound, with an IC50 of 4.54 ± 1.10 μM and 2.89 ± 0.34 μM, respectively, against in vitro-cultured E. multilocularis metacestodes and germinal cells. Thiacloprid was less toxic for HFF and RH mammalian cell lines than for metacestodes. In addition, thiacloprid inhibited the acetylcholinesterase activity in protoscoleces, metacestodes and germinal cells. Thiacloprid inhibited glucose consumption by protoscoleces and germinal cells. Subsequently, transmission electron microscopy revealed that treatment with thiacloprid damaged the germinal layer. In vivo, metacestode weight was significantly reduced following oral administration of thiacloprid at 15 and 30 mg/kg. The level of CD4+ T lymphocytes in metacestodes and spleen increased after thiacloprid treatment. Anti-echinococcosis-related cytokines (IL-2, IL-4, IL-10) were significantly increased. Furthermore, thiacloprid inhibited the expression of matrix metalloproteinases (MMPs 1, 3, 9, 13) and promoted collagen deposition in the host lesion microenvironment. Conclusions The results demonstrated that thiacloprid had parasiticidal activity against E. multilocularis in vitro and in vivo, and could be used as a novel lead compound for the treatment of AE. Graphical abstract

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Three-dimensional spheroid culture of canine hepatocyte-like cells derived from bone marrow mesenchymal stem cells

Cited by 5 publications

References 16 publications

In vivo and in vitro efficacy of crocin against Echinococcus multilocularis

In vivo and in vitro efficacy of crocin against Echinococcus multilocularis

Establishment of large canine hepatocyte spheroids by mixing vascular endothelial cells and canine adipose-derived mesenchymal stem cells

In vitro and in vivo efficacy of thiacloprid against Echinococcus multilocularis

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