preliminary study of cell viability at three cell

Hoshino et al., Nano Biomedicine 12(2), 83-88, 2020

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Introduction Embryonic stem (ES) cells and induced pluripo-tent stem (iPS) cells may differentiate when cul-tured for a long period of time, and change their characteristics such as proliferation rate and morphology. Undifferentiated ES cells and iPS cells typically have round or oval shapes. In or-der to maintain such shapes, several factors are supplemented in culture medium to suppress the differentiation of ES and iPS cells. Indeed, pre-vious studies demonstrated that cells cultured in leukemia inhibitory factor (LIF)[1-3] -free me-dium start to become more elongated and fibro-blast-like after 4 days, and continue to differen-tiate for up to 2 weeks. Using this technique, fibroblast-like cell lines can be established after multiple passages. As such, differentiated and

undifferentiated cells may have different sensi-tivities to chemicals. However, little is known about whether the degree of differentiation af-fects the sensitivity of cells to chemicals. Similar to bacteria and viruses, nano- and sub-micron-sized materials are phagocytosed into cells [4-10]. For the purpose of our study, nano-sized titanium dioxide (nano-TiO2) was selected because its toxicity profile has been well characterized.

In the present study, we used ES-D3 cells, which are commonly used to evaluate develop-mental and reproductive toxicity, to compare the viability of undifferentiated and differentiated cells at different passages after exposure to nano-TiO2.

Preliminary Study of Cell Viability at Three Cell Differentiation Levels of Mouse ES Cells

using Nano-sized Titanium Dioxide

Yumi HOSHINO1, and Yasuhiko MATSUSHIMA2

1Kanagawa Dental University Junior College, Kanagawa, Japan 2Matsushima Dental Clinic, Osaka, Japan

Synopsis The viability of cells exposed to chemicals may differ depending on the degree of differentiation.In the present study, we used a murine embryonic stem (ES) cell line, ES-D3, which does notrequire feeder cells in culture, to compare the viability of undifferentiated and differentiated cellsfollowing exposure to a chemical. Differentiated cells were maintained at the first (P1) and secondpassages (P2), and cells were exposed to nano-sized titanium dioxide (nano-TiO2). There were nosignificant differences in the viability of cells among the three differentiation stages; however,undifferentiated cells had slightly lower viability than differentiated cells. There was no significantdifference in cell viability between differentiated cells at P1 and P2. Our findings were based on theresponse of cells to nano-TiO2; thus, it should be noted that other chemicals may have differenteffects. Our study also suggests that undifferentiated ES cells are more sensitive to chemicals;therefore, they may be useful to evaluate the cytotoxicity of nano- and sub-micron-sized chemicals.

Key words: cell viability, cell differentiation, ES-D3 cells, Nano, titanium dioxide

ORIGINAL ARTICLE

Hoshino et al., Cell Viability at Three Cell Differentiation Levels of Mouse ES Cells, Nano Biomed 12(2), 83-88, 2020

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Fig.1 Inverted phase-contrast micrographs of five ES-D3 cellsLeft: undifferentiated ES-D3 cells, center: ES-D3 cells in 1 passage, Right: ES-D3 cells in 2 passages Vertical: The numbers of cell culture bottles are listed.

100 μm


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Materials and Methods Cells The embryonic stem cells from the D3 mouse cell line (ES-D3 cells). Feeder cells are not used for this ES-D3 cell culture. Assay medium Preheat treated 20% (v/v) Fetal Calf Serum (FCS, HyClone®, UT, USA) was added to 1% (v/v) non-essential amino acids (NAA, Invitrogen, CA, USA), 0.1 mM β-mercapto- ethanol (Invitrogen), 2 mM L-glutamine (Invitrogen), and Dulbecco's Modified Eagle Medium (DMEM, Gibco, Thermo Fisher Scien-tific, MNC) with penicillin / streptomycin (Invitrogen). Cell culture The following groups of cells were prepared: undifferentiated ES-D3 cells (UD), and ES-D3 cells maintained in static culture for 1 week at the first (P1) and second (P2) passages. For UD cell culture, the medium was supplemented with 1,000U/mL leukemia inhibitory factor (LIF, Gibco). Figure 1 shows an inverted phase- contrast microscope (Olympus Corp., Tokyo) image of each differentiation stage. In this ex-periment, the mean and standard deviation were calculated using five ES-D3 cells. Then, the cell culture of each ES cell differentiation stage is shown in Figure 2.

Test solution Assay medium (50 mL) was added to test tubes containing 50 μg of TiO2 (TT-55(A), Rutile type, Diameter of particles; 0.03-0.05 μm, TiO2; 96.0%, Ishihara Sangyo Kaisha Ltd., Osaka). After ensuring that the test tube was sealed properly, the solution was mixed on a vortex for 5 minutes. A dilution series of the TiO2 solution was prepared for subsequent experiments. The solution was not clear when the culture medium was added to TiO2. To prevent the precipitation of TiO2, solutions at each dilution were mixed on a vortex prior to use. Experiment UD, P1, and P2 cells were seeded on 96-well plates (Iwaki, AGC Techno Glass Co., Ltd, Shizuoka, Japan) at 100 μL/well, and cultured for 24 hours in a CO2 incubator. After the incu-bation period, an inverted phase-contrast micro-scope was used to confirm that the cells were attached to the bottom of the dish. Subsequently, the culture media was replaced with the dilutions of TiO2 solution (100 μL/well) using multichan-nel pipettes. Cells grown in culture medium were used as the control. Cells were incubated for 4 days in a CO2 incubator and cell viability was measured using the MTT assay at 570 nm. The control was a TiO2-free culture medium.

UD: Undifferentiated ES-D3 cells

Medium: Assay medium + 1,000U/mL LIF Cell culture: 7 day culture in 37℃ CO2 incubator

P1: First passage undifferentiated ES-D3 cells

Medium: Assay medium Cell culture: 7 day culture of undifferentiated ES-D3 cells in 37℃ CO2 incubator

P2: Second passage Medium: Assay medium

Cell culture: 7 day cutture of first passage cells in 37℃ CO2 incubator

Fig. 2 Undifferentiated ES-D3 cells and two subcultured cells


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Results The viability of UD, P1, and P2 cells is shown in Figure 3. For all groups, cell viability increased as the concentration of TiO2 decreased. The overall viability was lower in UD cells than in P1 and P2 cells, but there was no significant dif-ference between P1 and P2 cells. Three types of differentiated cells in the 96-well-multidish were observed with an inverted phase-contrast micro-scope, and almost no difference in morphologi-cal changes was observed. Discussion The mouse ES-D3 cell line was originally derived from blastocysts. ES-D3 cells are pluri-potent and can differentiate into various somatic cells. In general, feeder cells are required to cul-ture ES cells [11-16]. However, ES-D3 cells are used in ES tests of developmental and reproduc-tive toxicity, and do not require feeder cells for culture. In addition to serum and other supple-ments typically used for culturing ES cells, murine ES-D3 cells require LIF to suppress spontaneous differentiation and maintain the undifferentiated state [17-21]. LIF is a member of the IL-6 cytokine family, and is effective at suppressing the differentiation of murine ES and

iPS cells. Once LIF is removed from the culture medium, cells immediately start to differentiate. Our study demonstrated that TiO2 was the most toxic to undifferentiated ES-D3 cells, although the difference in cell viability between undiffer-entiated and differentiated cells was not sig-nificant. Furthermore, there was no significant difference in cell viability between P1 and P2 cells. Thus, although the cell turnover was slightly delayed in P2, cell passages did not have a significant influence on viability.

DNA degradation and damage have little impact on senescent cells. Indeed, similar to cells in the D0 cycle, senescent cells are not sen-sitive to chemical exposure. Compared with P1 and P2 cells, the viability of senescent cells may significantly differ from that of undifferentiated cells.

The two types of cell death, apoptosis and necrosis, are induced differently and character-ized by different morphological features [21]. Unlike apoptosis, also known as programmed cell death, necrosis is passive and is induced by exposure to chemicals. However, the mecha-nisms underlying necrotic cell death may differ depending on the type of chemicals used. Thus, the effects of nano-TiO2 may not be observed

Fig.3 Comparison of cell viability by exposure to TiO2 by three levels ofdifferentiation ES-D3 cells


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with other chemicals. Our study suggests that undifferentiated ES cells can be used effectively to evaluate the in vitro cytotoxicity of nano- and sub-micron-sized chemicals. Conflicts of Interest All authors declare no conflicts of interest regarding the publication of this paper. References 1) Ohtsuka S, Nakai-Futatsugi Y, Niwa H. LIF

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(Received: November 25, 2020/ Accepted: December 16, 2020)

Corresponding author: Yumi Hoshino, Ph.D. Kanagawa Dental University Junior College 82 Inaoka-cho, Yokosuka, Kanagawa 238-8580, Japan Tel: +81-46-822-8781 E-mail: [email protected]

preliminary study of cell viability at three cell

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