In vitro Toxicity Testing of Nanoparticles in 3D Cell Culture

Nanomaterials are used in a variety of fields and toxicity assessment is paramount for their development and application. Although most toxicity assessments have been performed in 2D (2-Dimensional) cell culture, the inability to adequately replicate the in vivo environment and toxicity is a limitation. To overcome the limitation, a 3D (3-Dimensional) cell culture method has been developed to make an environment closer to an in vivo system. In this study, 20 nm SiO2 nanoparticles were dispersed in serum-containing (SC) and serum-free (SF) media to compare 2D cell culture and 3D cell culture toxicity. The cells were subjected to a 3D cell culture method in which HepG2, a human-derived liver cancer cell line, was mixed on a scaffold. We found that nanoparticles induced toxicity in 2D cell culture, but toxicity was not observed in 3D cell culture similar to in vivo environment. However, differences in toxicity were observed between the three types of scaffolds in the absence of serum as the number of cells decreased.

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Establishment of long-term ostracod epidermal culture

In Vitro Cellular & Developmental Biology - Animal ◽

10.1007/s11626-020-00508-8 ◽

2020 ◽

Vol 56 (9) ◽

pp. 760-772

Author(s):

Siân R. Morgan ◽

Laura Paletto ◽

Benjamin Rumney ◽

Farhana T. Malik ◽

Nick White ◽

...

Keyword(s):

Cell Culture ◽

Cell Biology ◽

Toxicity Testing ◽

Culture Method ◽

Explant Culture ◽

In Vitro Toxicity ◽

Epidermal Layer ◽

Epidermal Tissue ◽

Novel Approach

Abstract Primary crustacean cell culture was introduced in the 1960s, but to date limited cell lines have been established. Skogsbergia lerneri is a myodocopid ostracod, which has a body enclosed within a thin, durable, transparent bivalved carapace, through which the eye can see. The epidermal layer lines the inner surface of the carapace and is responsible for carapace synthesis. The purpose of the present study was to develop an in vitro epidermal tissue and cell culture method for S. lerneri. First, an optimal environment for the viability of this epidermal tissue was ascertained, while maintaining its cell proliferative capacity. Next, a microdissection technique to remove the epidermal layer for explant culture was established and finally, a cell dissociation method for epidermal cell culture was determined. Maintenance of sterility, cell viability and proliferation were key throughout these processes. This novel approach for viable S. lerneri epidermal tissue and cell culture augments our understanding of crustacean cell biology and the complex biosynthesis of the ostracod carapace. In addition, these techniques have great potential in the fields of biomaterial manufacture, the military and fisheries, for example, in vitro toxicity testing.

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Large scale and integrated platform for digital mass culture of anchorage dependent cells

Nature Communications ◽

10.1038/s41467-019-12777-3 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 6

Author(s):

Kyoung Won Cho ◽

Seok Joo Kim ◽

Jaemin Kim ◽

Seuk Young Song ◽

Wang Hee Lee ◽

...

Keyword(s):

Cell Culture ◽

Cell Cultures ◽

Mass Culture ◽

Large Scale ◽

Cultured Cells ◽

Toxicity Testing ◽

Industrial Applications ◽

In Vitro Toxicity ◽

Scale Production

Abstract Industrial applications of anchorage-dependent cells require large-scale cell culture with multifunctional monitoring of culture conditions and control of cell behaviour. Here, we introduce a large-scale, integrated, and smart cell-culture platform (LISCCP) that facilitates digital mass culture of anchorage-dependent cells. LISCCP is devised through large-scale integration of ultrathin sensors and stimulator arrays in multiple layers. LISCCP provides real-time, 3D, and multimodal monitoring and localized control of the cultured cells, which thereby allows minimizing operation labour and maximizing cell culture performance. Wireless integration of multiple LISCCPs across multiple incubators further amplifies the culture scale and enables digital monitoring and local control of numerous culture layers, making the large-scale culture more efficient. Thus, LISCCP can transform conventional labour-intensive and high-cost cell cultures into efficient digital mass cell cultures. This platform could be useful for industrial applications of cell cultures such as in vitro toxicity testing of drugs and cosmetics and clinical scale production of cells for cell therapy.

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Testing Strategies of the In Vitro Micronucleus Assay for the Genotoxicity Assessment of Nanomaterials in BEAS-2B Cells

Nanomaterials ◽

10.3390/nano11081929 ◽

2021 ◽

Vol 11 (8) ◽

pp. 1929

Author(s):

Tereza Cervena ◽

Andrea Rossnerova ◽

Tana Zavodna ◽

Jitka Sikorova ◽

Kristyna Vrbova ◽

...

Keyword(s):

Cytochalasin B ◽

Culture Media ◽

Methodological Approach ◽

Micronucleus Assay ◽

Toxicity Testing ◽

In Vitro Toxicity ◽

Testing Strategies ◽

Transmission Electron ◽

The Impact

The evaluation of the frequency of micronuclei (MN) is a broadly utilised approach in in vitro toxicity testing. Nevertheless, the specific properties of nanomaterials (NMs) give rise to concerns regarding the optimal methodological variants of the MN assay. In bronchial epithelial cells (BEAS-2B), we tested the genotoxicity of five types of NMs (TiO2: NM101, NM103; SiO2: NM200; Ag: NM300K, NM302) using four variants of MN protocols, differing in the time of exposure and the application of cytochalasin-B combined with the simultaneous and delayed co-treatment with NMs. Using transmission electron microscopy, we evaluated the impact of cytochalasin-B on the transport of NMs into the cells. To assess the behaviour of NMs in a culture media for individual testing conditions, we used dynamic light scattering measurement. The presence of NMs in the cells, their intracellular aggregation and dispersion properties were comparable when tests with or without cytochalasin-B were performed. The genotoxic potential of various TiO2 and Ag particles differed (NM101 < NM103 and NM302 < NM300K, respectively). The application of cytochalasin-B tended to increase the percentage of aberrant cells. In conclusion, the comparison of the testing strategies revealed that the level of DNA damage induced by NMs is affected by the selected methodological approach. This fact should be considered in the interpretation of the results of genotoxicity tests.

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Mammary gland 3D cell culture systems in farm animals

Veterinary Research ◽

10.1186/s13567-021-00947-5 ◽

2021 ◽

Vol 52 (1) ◽

Author(s):

Laurence Finot ◽

Eric Chanat ◽

Frederic Dessauge

Keyword(s):

Cell Culture ◽

Mammary Gland ◽

Bacterial Infections ◽

Three Dimensional ◽

3D Cell Culture ◽

Farm Animals ◽

Culture Systems ◽

Cell Culture Systems

AbstractIn vivo study of tissue or organ biology in mammals is very complex and progress is slowed by poor accessibility of samples and ethical concerns. Fortunately, however, advances in stem cell identification and culture have made it possible to derive in vitro 3D “tissues” called organoids, these three-dimensional structures partly or fully mimicking the in vivo functioning of organs. The mammary gland produces milk, the source of nutrition for newborn mammals. Milk is synthesized and secreted by the differentiated polarized mammary epithelial cells of the gland. Reconstructing in vitro a mammary-like structure mimicking the functional tissue represents a major challenge in mammary gland biology, especially for farm animals for which specific agronomic questions arise. This would greatly facilitate the study of mammary gland development, milk secretion processes and pathological effects of viral or bacterial infections at the cellular level, all with the objective of improving milk production at the animal level. With this aim, various 3D cell culture models have been developed such as mammospheres and, more recently, efforts to develop organoids in vitro have been considerable. Researchers are now starting to draw inspiration from other fields, such as bioengineering, to generate organoids that would be more physiologically relevant. In this chapter, we will discuss 3D cell culture systems as organoids and their relevance for agronomic research.

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A survey of aerosol exposure systems relative to the analysis of cytotoxicity: A Cooperation Centre for Scientific Research Relative to Tobacco (CORESTA) perspective

Toxicology Research and Application ◽

10.1177/23978473211022267 ◽

2021 ◽

Vol 5 ◽

pp. 239784732110222

Author(s):

David Thorne ◽

Roman Wieczorek ◽

Toshiro Fukushima ◽

Han-Jae Shin ◽

Robert Leverette ◽

...

Keyword(s):

Scientific Research ◽

Toxicity Testing ◽

User Preferences ◽

In Vitro Toxicity ◽

Aerosol Generation ◽

Aerosol Exposure ◽

Comprehensive Survey ◽

Survey Results ◽

System Data

During a Cooperation Centre for Scientific Research Relative to Tobacco (CORESTA) meeting, the in vitro toxicity testing Sub-Group (IVT SG) met to discuss the evolving field of aerosol exposure research. Given the diversity of exposure parameters and biological endpoints being used, it was considered a high priority to investigate and contextualise the responses obtained. This is particularly driven by the inability to compare between studies on different exposure systems due to user preferences and protocol differences. Twelve global tobacco and contract research companies met to discuss this topic and formulate an aligned approach on how this diverging field of research could be appropriately compared. Something that is becoming increasingly important, especially in the light of more focused regulatory scrutiny. A detailed and comprehensive survey was conducted on over 40 parameters ranging from aerosol generation, dilution and data analysis across eight geographically independent laboratories. The survey results emphasise the diversity of in vitro exposure parameters and methodologies employed across the IVT SG and highlighted pockets of harmonisation. For example, many of the biological protocol parameters are consistent across the Sub-Group. However, variables such as cell type and exposure time remain largely inconsistent. The next steps for this work will be to map parameters and system data against biological findings and investigate whether the observed inconsistencies translate into increased biological variability. The results from the survey provide improved awareness of parameters and nuances, that may be of substantial benefit to scientists in intersecting fields and in the development of harmonised approaches.

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Mechanical Strain-Enabled Reconstitution of Dynamic Environment in Organ-on-a-Chip Platforms: A Review

Micromachines ◽

10.3390/mi12070765 ◽

2021 ◽

Vol 12 (7) ◽

pp. 765

Author(s):

Qianbin Zhao ◽

Tim Cole ◽

Yuxin Zhang ◽

Shi-Yang Tang

Keyword(s):

Cell Culture ◽

Shear Stress ◽

Cultured Cells ◽

Mechanical Strain ◽

3D Cell Culture ◽

Small Scale ◽

Mechanical Stimuli ◽

Human Organ ◽

Organ On A Chip

Organ-on-a-chip (OOC) uses the microfluidic 3D cell culture principle to reproduce organ- or tissue-level functionality at a small scale instead of replicating the entire human organ. This provides an alternative to animal models for drug development and environmental toxicology screening. In addition to the biomimetic 3D microarchitecture and cell–cell interactions, it has been demonstrated that mechanical stimuli such as shear stress and mechanical strain significantly influence cell behavior and their response to pharmaceuticals. Microfluidics is capable of precisely manipulating the fluid of a microenvironment within a 3D cell culture platform. As a result, many OOC prototypes leverage microfluidic technology to reproduce the mechanically dynamic microenvironment on-chip and achieve enhanced in vitro functional organ models. Unlike shear stress that can be readily generated and precisely controlled using commercial pumping systems, dynamic systems for generating proper levels of mechanical strains are more complicated, and often require miniaturization and specialized designs. As such, this review proposes to summarize innovative microfluidic OOC platforms utilizing mechanical actuators that induce deflection of cultured cells/tissues for replicating the dynamic microenvironment of human organs.

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In Vitro Toxicity Testing: Redefining our Objectives

Alternatives to Laboratory Animals ◽

10.1177/026119299202000411 ◽

1992 ◽

Vol 20 (4) ◽

pp. 571-574

Author(s):

Oliver Flint

Keyword(s):

Toxicity Testing ◽

In Vitro Toxicity

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Self-organization and culture of Mesenchymal Stem Cell spheroids in acoustic levitation

Scientific Reports ◽

10.1038/s41598-021-87459-6 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Nathan Jeger-Madiot ◽

Lousineh Arakelian ◽

Niclas Setterblad ◽

Patrick Bruneval ◽

Mauricio Hoyos ◽

...

Keyword(s):

Cell Culture ◽

3D Cell Culture ◽

Culture Conditions ◽

Acoustic Levitation ◽

Cell Sheets ◽

Cell Therapies ◽

Cellular Models ◽

Cell Spheroids

AbstractIn recent years, 3D cell culture models such as spheroid or organoid technologies have known important developments. Many studies have shown that 3D cultures exhibit better biomimetic properties compared to 2D cultures. These properties are important for in-vitro modeling systems, as well as for in-vivo cell therapies and tissue engineering approaches. A reliable use of 3D cellular models still requires standardized protocols with well-controlled and reproducible parameters. To address this challenge, a robust and scaffold-free approach is proposed, which relies on multi-trap acoustic levitation. This technology is successfully applied to Mesenchymal Stem Cells (MSCs) maintained in acoustic levitation over a 24-h period. During the culture, MSCs spontaneously self-organized from cell sheets to cell spheroids with a characteristic time of about 10 h. Each acoustofluidic chip could contain up to 30 spheroids in acoustic levitation and four chips could be ran in parallel, leading to the production of 120 spheroids per experiment. Various biological characterizations showed that the cells inside the spheroids were viable, maintained the expression of their cell surface markers and had a higher differentiation capacity compared to standard 2D culture conditions. These results open the path to long-time cell culture in acoustic levitation of cell sheets or spheroids for any type of cells.

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Time-lapse Phase-contrast Microphotography of Cell Populations as a Basis for Improvement of In Vitro Toxicity Assessment

Alternatives to Laboratory Animals ◽

10.1177/026119299202000223 ◽

1992 ◽

Vol 20 (2) ◽

pp. 302-306

Author(s):

Miroslav Červinka

Keyword(s):

Cell Proliferation ◽

Cell Morphology ◽

Video Recording ◽

Toxicity Testing ◽

Time Lapse ◽

Time Dependent ◽

In Vitro Toxicity ◽

Simple Modification ◽

Pertinent Data

Recent trends in the field of in vitro toxicology have centred around the validation of in vitro methods. The ultimate goal is to obtain pertinent data with the minimum of effort. In our laboratory, we have used toxicological methods based on the evaluation of cell morphology and cell proliferation. A method suitable for this purpose is time-lapse microcinematographic (or video) recording of cellular changes, which we used for many years. For practical in vitro toxicity testing, however, this method is far too complicated. Therefore, we have tried to develop a simple modification for the evaluation of cell morphology and cell proliferation, which would still allow for a basic time-dependent analysis. Comparison of detailed microcinematographic analysis with analysis according to our new proliferation assay is demonstrated with cisplatin as the toxicant. We believe that a time-dependent approach could improve the in vitro assessment of toxicity.

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