Long-term survival and functional maintenance of hepatocytes by using a microfabricated cell array

Ikeda, Yutaka; Jomura, Tomoko; Horiuchi, Umeko; Saeki, Junko; Yoshimoto, Keitaro; Ikeya, Tsuyoshi; Nagasaki, Yukio

doi:10.1016/j.colsurfb.2012.04.022

Cited by 18 publications

(5 citation statements)

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“…The hepatocytes were isolated by the two-step collagenase perfusion method, and viable hepatocytes were purified by Percoll centrifugation, as described in Methods. The hepatocytes were seeded in the 96-well format Cell-able TM , a micropatterned 3D culture system [25], in which 3T3-Swiss albino cells were seeded as feeder cells 1 day before hepatocyte seeding. The culture medium was refreshed every 2 to 3 days, unless otherwise noted, and the culture was continued for approximately 1 month.…”

Section: Resultsmentioning

confidence: 99%

Continuous long-term cytotoxicity monitoring in 3D spheroids of beetle luciferase-expressing hepatocytes by nondestructive bioluminescence measurement

et al. 2017

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BackgroundThree-dimensional (3D) spheroids are frequently used in toxicological study because their morphology and function closely resemble those of tissue. As these properties are maintained over a long term, repeated treatment of the spheroids with a test object is possible. Generally, in the repeated treatment test to assess cytotoxicity in the spheroids, ATP assay, colorimetric measurement using pigments or high-content imaging analysis is performed. However, continuous assessment of cytotoxicity in the same spheroids using the above assays or analysis is impossible because the spheroids must be disrupted or killed. To overcome this technical limitation, we constructed a simple monitoring system in which cytotoxicity in the spheroids can be continuously monitored by nondestructive bioluminescence measurement.ResultsMouse primary hepatocytes were isolated from transchromosomic (Tc) mice harboring a mouse artificial chromosome (MAC) vector expressing beetle luciferase Emerald Luc (ELuc) under the control of cytomegalovirus immediate early enhancer/chicken β-actin promoter/rabbit β-globin intron II (CAG) promoter, and used in 3D cultures. We confirmed that both luminescence and albumin secretion from the spheroids seeded in the 96-well format Cell-ableTM were maintained for approximately 1 month. Finally, we repetitively treated the luminescent 3D spheroids with representative hepatotoxicants for approximately 1 month, and continuously and nondestructively measured bioluminescence every day. We successfully obtained daily changes of the dose-response bioluminescence curves for the respective toxicants.ConclusionsIn this study, we constructed a monitoring system in which cytotoxicity in the same 3D spheroids was continuously and sensitively monitored over a long term. Because this system can be easily applied to other cells, such as human primary cells or stem cells, it is expected to serve as the preferred platform for simple and cost-effective long-term monitoring of cellular events, including cytotoxicity.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-017-0374-1) contains supplementary material, which is available to authorized users.

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Section: Resultsmentioning

confidence: 99%

Continuous long-term cytotoxicity monitoring in 3D spheroids of beetle luciferase-expressing hepatocytes by nondestructive bioluminescence measurement

et al. 2017

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“…Furthermore, the increase in hepatocyte nuclear density is consistent with a reactive mechanism to tissue injury of the hepatic parenchyma, in which, events such as polyploidy and nuclear division may represent the hepatocytes attempting to compensate for metabolic deficits in macromolecular synthesis as well as replacing the function of dead cells (Ikeda et al . ).…”

Section: Discussionmentioning

confidence: 97%

Long‐lasting morphofunctional remodelling of liver parenchyma and stroma after a single exposure to low and moderate doses of cadmium in rats

Cupertino

Costa

Santos

et al. 2013

Int J Experimental Path

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Frequent exposure to cadmium (Cd) in low doses is common; however, the long-lasting effects of this exposure are still poorly understood. Therefore in this study we have evaluated long-lasting hepatic morphofunctional adaptations in rats exposed to low and moderate doses of Cd. Five experimental groups were tested: control (0.9% saline) and other four receiving single intraperitoneal doses of 0.67, 0.74, 0.86 and 1.1 mg of Cd/kg. The animals were killed after eight weeks and the following parameters were analysed: biometrics, oedema, Cd bio-accumulation, collagen, glycogen, lipid droplets, superoxide dismutase (SOD) and catalase (CAT), serum transaminases, liver histopathology and stereology. In all groups exposed to Cd there was significant increase in SOD and CAT activities, ALP levels, proportion of binucleated hepatocytes, nuclei/cytoplasm ratio, macrophages (Kupffer cells) and collagen fibres. In these groups, glycogen accumulation by hepatocytes and the proportion of sinusoidal capillaries were significantly reduced compared with controls. The liver somatic index was increased, and liver oedema was evident in animals exposed to higher dose of Cd. Areas of necrosis were found in animals exposed to the three highest doses. These results indicate that Cd is an extremely toxic bioactive heavy metal, which even at low doses is able to disrupt liver homeostasis. At low and moderate doses, Cd exposure induces morphofunctional pathological remodelling of the hepatic stroma and parenchyma, which remain active after eight weeks. In response to injury, the liver tissue triggers a reactive process by enhancing activation of antioxidant enzymes and collagenogenesis.

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“…All of the wells are coated with poly(ethylene glycol) (PEG) hydrogel and have 800 circular noncoated domains with 100 μm in diameter that are spaced, edge to edge, at 100 μm intervals on the culture surface. The seeded cells accumulate on circular noncoated domains and form spherical aggregates because the region of PEG hydrogel on the micropatterned surface inhibits cell adhesion. ,− Constructed cell aggregates adhere to the surface and are not easily peeled off.…”

Section: Materials and Methodsmentioning

confidence: 99%

Osteogenic Lineage Commitment of Adipose-Derived Stem Cells Is Predetermined by Three-Dimensional Cell Accumulation on Micropatterned Surface

Furuhata¹,

Yoshitomi²,

Kikuchi³

et al. 2017

ACS Appl. Mater. Interfaces

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Lineage commitment of stem cells is mainly regulated by their microenvironments, which comprise soluble growth factors, extracellular matrix, mechanical forces, and cell density. Although numerous studies have investigated stem cell response to these factors in two-dimensional (2D) culture, little is known about that in 3D culture. Here, we studied effects of 3D cell accumulation levels on the differentiation behavior of mesenchymal stem cells (MSCs) by using a micropatterned surface. After induction of 3D-cultured MSCs on the surface, their osteogenic differentiation was significantly promoted, while adipogenic differentiation was not. This differentiation behavior of densely packed MSCs in 3D culture is unlike that in 2D culture. Moreover, to determine the contributing factor of this commitment, the relationship between 3D cell accumulation levels and their differentiation potential was studied before differentiation induction. A series of MSCs with varied 3D accumulation levels were constructed on the micropatterned surface, where the accumulated MSCs were not in hypoxic environment. Interestingly, with increasing 3D accumulation levels, MSCs enhanced their osteogenic potential but repressed adipogenic potential in the gene expression level. These results suggest that preconditioned 3D microenvironments with high cell accumulation levels promote osteogenic differentiation of MSCs and their accumulation levels help in regulating MSC differentiation.

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Long-term survival and functional maintenance of hepatocytes by using a microfabricated cell array

Cited by 18 publications

References 5 publications

Continuous long-term cytotoxicity monitoring in 3D spheroids of beetle luciferase-expressing hepatocytes by nondestructive bioluminescence measurement

Continuous long-term cytotoxicity monitoring in 3D spheroids of beetle luciferase-expressing hepatocytes by nondestructive bioluminescence measurement

Long‐lasting morphofunctional remodelling of liver parenchyma and stroma after a single exposure to low and moderate doses of cadmium in rats

Osteogenic Lineage Commitment of Adipose-Derived Stem Cells Is Predetermined by Three-Dimensional Cell Accumulation on Micropatterned Surface

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