System for evaluation of oxidative stress on in-vitro-produced bovine embryos

Assis, Patrícia Monken de; Castro, Luiz Guilherme Martins; Siqueira, Adriano Felipe Perez; Delgado, Juliana de Carvalho; Hamilton, Thais Rose dos Santos; Goissis, Marcelo Demarchi; Mendes, Clayton Quirino; Nichi, Marcílio; Visintin, José Antônio; Assumpção, Mayra Elena Ortiz D’Ávila

doi:10.1016/j.rbmo.2015.06.014

Cited by 16 publications

(11 citation statements)

References 5 publications

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“…However, embryos that appeared cleaved and developed to the blastocyst stage quicker than their counterparts did not have an increased number of cells in the inner cell mass [16]. Another indicator of embryo quality or competence is the accumulation of reactive oxygen species, which can be indicators of cellular stress and altered metabolism [17][18][19]. Furthermore, cellular integrity, determined by cytoskeleton analysis and cell organization, is suggested to be an indicator of embryo quality in other species [20,21].…”

Section: Introductionmentioning

confidence: 99%

Improved cryopreservation of in vitro produced bovine embryos using FGF2, LIF, and IGF1

et al. 2021

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In vitro embryo production systems are limited by their inability to consistently produce embryos with the competency to develop to the blastocyst stage, survive cryopreservation, and establish a pregnancy. Previous work identified a combination of three cytokines [fibroblast growth factor 2 (FGF2), leukemia inhibitory factor (LIF), and insulin-like growth factor 1 (IGF1)], called FLI, that we hypothesize improve preimplantation development of bovine embryos in vitro. To test this hypothesis, FLI was supplemented into oocyte maturation or embryo culture medium. Embryos were produced in vitro using abattoir-derived oocytes and fertilized with sperm from a single bull known to have high fertility. After an 18–20 h fertilization period, putative zygotes were cultured in synthetic oviductal fluid (SOF) for 8 days. The addition of FLI to the oocyte maturation medium increased (P < 0.05) the dissociation of transzonal projections at 12, 18, and 24 h of maturation, as well as, the proportion of oocytes that reached the metaphase II stage of meiosis. Additionally, lipid content was decreased (P < 0.05) in the blastocyst stage embryo. The addition of FLI during the culture period increased development to the blastocyst stage, cytoskeleton integrity, and survival following slow freezing, as well as, decreased post thaw cell apoptosis (P < 0.05). In conclusion, the supplementation of these cytokines in vitro has the potential to alleviate some of the challenges associated with the cryo-survival of in vitro produced bovine embryos through improving embryo development and embryo quality.

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Section: Introductionmentioning

confidence: 99%

Improved cryopreservation of in vitro produced bovine embryos using FGF2, LIF, and IGF1

et al. 2021

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“…Depletion of intracellular glutathione (Cho et al, 1997) and mitochondrial impairment (Tolosa et al, 2012) by menadione have been reported to be the major mechanisms of menadione-induced toxicity in vitro (Niemczyk et al, 2004). Menadione is a vitamin K3 supplement investigated widely as an oxidative stress inducer (De Assis et al, 2015) and for its efficacy in the treatment of cancers (Niemczyk et al, 2004; Delwar et al, 2012; Akiyoshi et al, 2009). Its cytotoxic effect via generation of ROS has been reported in several studies (Castro et al, 2008; Ip et al, 2004).…”

Section: Discussionmentioning

confidence: 99%

High-content imaging assays on a miniaturized 3D cell culture platform

Joshi

Datar

et al. 2018

Toxicology in Vitro

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The majority of high-content imaging (HCI) assays have been performed on two-dimensional (2D) cell monolayers for its convenience and throughput. However, 2D-cultured cell models often do not represent the in vivo characteristics accurately and therefore reduce the predictability of drug toxicity/efficacy in vivo. Recently, three-dimensional (3D) cell-based HCI assays have been demonstrated to improve predictability, but its use is limited due to difficulty in maneuverability and low throughput in cell imaging. To alleviate these issues, we have developed miniaturized 3D cell culture on a micropillar/microwell chip and demonstrated high-throughput HCI assays for mechanistic toxicity. Briefly, Hep3B human hepatoma cell line was encapsulated in a mixture of alginate and fibrin gel on the micropillar chip, cultured in 3D, and exposed to six model compounds in the microwell chip for rapidly assessing mechanistic hepatotoxicity. Several toxicity parameters, including DNA damage, mitochondrial impairment, intracellular glutathione level, and cell membrane integrity were measured on the chip, and the IC values of the compounds at different readouts were determined to investigate the mechanism of toxicity. Overall, the Z' factors were between 0.6 and 0.8 for the HCI assays, and the coefficient of variation (CV) were below 20%. These results indicate high robustness and reproducibility of the HCI assays established on the miniaturized 3D cell culture chip. In addition, it was possible to determine the predominant mechanism of toxicity using the 3D HCI assays. Therefore, our miniaturized 3D cell culture coupled with HCI assays has great potential for high-throughput screening (HTS) of compounds and mechanistic toxicity profiling.

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“…Mitochondrial impairment is known to be the main mechanism of tamoxifen‐induced toxicity (Lee, Kang, Lee, & Kim, ; Tabassum et al., ; Tolosa et al., ; Tolosa, Carmona, Castell, Gómez‐Lechón, & Donato, ). Likewise, menadione is a vitamin K3 supplement effectively used in the treatment of cancer (Akiyoshi, Matzno, Sakai, Okamura, & Matsuyama, ; Delwar et al., ; Niemczyk et al., ) and also known to be an oxidative stress inducer (De Assis et al., ). Major mechanisms of menadione‐induced toxicity have been reported to be the depletion of intracellular glutathione (Cho, Kim, Lee, & Chung, ) and mitochondrial impairment (Niemczyk et al., ; Tolosa et al., ) via generation of ROS (Castro, Mariani, Panek, Eleutherio, & Pereira, ; Ip, Woo, Lau, & Che, ).…”

Section: Commentarymentioning

confidence: 99%

High‐Throughput Assessment of Mechanistic Toxicity of Chemicals in Miniaturized 3D Cell Culture

et al. 2018

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High-content imaging (HCI) assays on two-dimensional (2D) cell cultures often do not represent in vivo characteristics accurately, thus reducing the predictability of drug toxicity/efficacy in vivo. On the other hand, conventional 3D cell cultures are relatively low throughput and possess difficulty in cell imaging. To address these limitations, a miniaturized 3D cell culture has been developed on a micropillar/microwell chip platform with human cells encapsulated in biomimetic hydrogels. Model compounds are used to validate human cell microarrays for high-throughput assessment of mechanistic toxicity. Main mechanisms of toxicity of compounds can be investigated by analyzing multiple parameters such as DNA damage, mitochondrial impairment, intracellular glutathione level, and cell membrane integrity. IC 50 values of these parameters can be determined and compared for the compounds to investigate the main mechanism of toxicity. This paper describes miniaturized HCI assays on 3D-cultured cell microarrays for high-throughput assessment of mechanistic profiles of compound-induced toxicity. C 2018 by John Wiley & Sons, Inc.Keywords: 3D cell culture r assay miniaturization r high-content imaging r mechanistic toxicity r microarray chip platform -Y (2019).High-throughput assessment of mechanistic toxicity of chemicals in miniaturized 3D cell culture.This protocol provides detailed steps involved in high-throughput cell printing with a bioprinter (S+ Microarrayer) for the generation of miniaturized 3D cell culture on a micropillar/microwell chip platform and the measurement of dose responses of model compounds in the miniaturized 3D culture of human cells using an automated fluorescent microscope (S+ Scanner) for HCI assays. Incorporating 3D cell growth on the chip platform in combination with HCI assays can be established as a reliable technique for testing toxicity of compounds in an early stage of preclinical drug discovery and better predict in vivo toxicity in clinical trials. The procedures provided below enable users to efficiently determine the mechanism of toxicity of unknown compounds in high-throughput and without the excessive use of cells, hydrogels, and other reagents. MaterialsHep3B human hepatoma cell line (ATCC# HB-8064, RRID:CVCL_0326) as a model cell line Complete RPMI-1640 medium for culturing Hep3B cells (see recipe) 1× phosphate-buffered saline (PBS; see recipe)Joshi et al.

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System for evaluation of oxidative stress on in-vitro-produced bovine embryos

Cited by 16 publications

References 5 publications

Improved cryopreservation of in vitro produced bovine embryos using FGF2, LIF, and IGF1

Improved cryopreservation of in vitro produced bovine embryos using FGF2, LIF, and IGF1

High-content imaging assays on a miniaturized 3D cell culture platform

High‐Throughput Assessment of Mechanistic Toxicity of Chemicals in Miniaturized 3D Cell Culture

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