Cultured animal and human cells have been used in biomedical research for many decades (Rodríguez-Hernández et al., 2014). Cultured cells are commonly used in toxicology experiments to identify the biological effects of chemicals and drugs (mechanisms of action) and in screening assays to determine the relative toxicity of substances. The goal of in vitro toxicology is to develop a range of cellular and organotypic models and assays capable of replacing animals in various toxicity tests that are used to predict toxic effects to humans from exposure to chemicals and product ingredients.
Methods such as growing cells as a single layer on plastic plates are usually insufficient for replicating the in vivo behavior and responses of cells and tissues. Therefore, each cell-based test method involves (a) the development of a cellular/organotypic model that replicate the in vivo tissue (i.e., that is biologically-relevant), and (b) the use of this biologically-relevant model in one or more biologically-relevant assay(s)/test system(s) to assess the toxicity response(s) to the substance being tested. Since tests on whole animals involve significant biological complexity, more than one in vitro model/assay are typically needed to replicate the key biological toxicity responses and provide sufficient data to replace any of the human health endpoints assessed for regulatory testing.
In some cases, in vitro methods will be part of a testing scheme integrated with other methods such as -omics (genomics, proteomics, metabolomics), (quantitative) structure activity relationship or (Q)SAR analysis, other in silico methods and computational approaches, and/or decision support analysis. Eventually all of the information will be integrated in a systems biology approach for hazard assessment decision making.
Each new test method and/or test system for regulatory testing must be evaluated systematically to demonstrate its validity for predicting human adverse responses. In the 1990’s test method validation criteria and processes were established by the Organisation for Economic Co-operation and Development (OECD) and the European and U.S. validation centers (ECVAM and ICCVAM), and were consolidated into one agreed guidance in 2005, the OECD Guidance Document on the Validation and International Acceptance of New or Updated Test Methods for Hazard Assessment (No. 34) (OECD, 2005). These procedures have been adequate for establishing the validity of some stand-alone assays, but their reliance on using animal data as the comparative data for assessing new test method performance has always been problematic. For example, the animal data is considered proprietary by most companies and so is not readily available. What is available is of limited use due to differences in the animal test protocols, variability in the results, changes in the purity and/or manufacturing processes of the previously tested substances, and other issues. Additionally, there is no way to validate a new method that performs better than an animal test method, but that does not agree with the animal test results. Methodology to validate test batteries and integrated test methods also needs further consideration. These are among the many challenges awaiting innovative and entrepreneurial scientists willing to tackle this challenging field that is no less biologically complex than the BRAIN Initiative or the Cancer Moonshot.
Author: Sherry L. Ward, AltTox Contributing Editor
Cells – What is a cell?
Cells are the basic building block of all organisms. Various types of human cells are the building blocks of all human tissues and organs. The total number of cells in the human body is estimated to be 37.2 trillion (3.72 x 1013) cells (Bianconi et al., 2013).
Human cells are microscopic, and range in size from 8-130 µm. The interactive image, “Cell Size and Scale,” can be used to compare the size of several types of cells to other common objects (use the slider beneath the image).
The basic parts of a cell are:
Human cells can be isolated from various human tissues and grown in the research laboratory for the purpose of conducting experiments to study human physiological processes. Cells that are isolated and grown in the laboratory can also be frozen and stored for later use, a technique called cryopreservation. Aseptic technique must be used in all steps where cells are isolated and maintained, and involves using equipment and processes that prevent bacteria and other microorganisms from contaminating the cells. Examples of aseptic technique include the use of sterilized tools and cultureware that come into contact with the cells, and the use of laminar airflow hoods to conduct procedures where the sterile equipment must be opened. Specialized equipment, supplies, and cell culture reagents have been developed and are commercially available so that cells can be isolated and maintained without contamination.
Cells grown in the laboratory are often cultured on plastic plates where they form a layer of cells (sometimes several layers depending on the cell type), and the cells are overlaid with a liquid medium containing the nutrients needed for their maintenance and growth. This technique is called monolayer (or 2-dimensional (2D)) cell culture, and it allows for the rapid expansion of cells so that large numbers are available for experiments. To maintain cultures of cells over time in the laboratory, cabinets that control environmental factors (temperature, humidity, oxygen levels, etc.) are used.
Until relatively recently, the cells/cell lines used to generate human cell-based models have been either primary or immortalized cells. Primary cells are those isolated directly from a tissue and then grown in the laboratory. Immortalized cell lines, on the other hand, contain gene modifications that increase their ability to proliferate and survive in culture. Both of these types of cell lines have their advantages and disadvantages (see section below, Primary Cells versus Cell Lines for In Vitro Toxicology). Both primary and immortalized cell lines can change over time in culture, so the number of times a particular cell line can be “passaged” (i.e., grown and split into new cultures to expand the numbers of cells available for experiments) must be defined when a cell line is characterized. An emerging capability is to utilize a third type of cell, stem cells, to develop cell-based models. Basic information on the types of stem cells and their uses in toxicity testing are discussed in another section (see section below, Stem Cell Basics).
Many types of cells have a characteristic morphology (their shape and appearance), which can be viewed by magnifying the cells using a microscope. However, to specifically identify the type of cells in a culture, the cells must be further characterized, and then the same methods can be used later to verify the identity of the cells. Techniques to characterize cells include: morphology, identification of cell-specific proteins within and/or on the surface of the cells, genetic analysis, and functional analysis.
There are also a number of quality control issues to address when isolating and/or using cells for research or testing, which are covered by good laboratory practice (GLP) and good cell culture practice (GCCP) guidelines (see section below, Quality requirements: GLPs, GCCPs).
Microscopy – How to visualize cells
Individual mammalian cells are not visible to the unaided human eye. However, when cells are cultured in the laboratory, optical instruments called microscopes can be used to magnify cells so they can be visually observed.
Microscopy is used to follow the growth of live cells in tissue culture plates that are being propagated for experiments. As part of experiments, cells or tissues are often fixed and stained prior to viewing microscopically. Most scientists are familiar with using light and phase-contrast microscopy. Methods called scanning and transmission electron microscopy (SEM and TEM, respectively) are used for even greater magnification, but can be used only with fixed, non-live samples.
New methodologies in imaging are allowing us to move beyond static cell images and obtain data on functional molecules in 3-dimensional (3D) viable cell cultures over time. Changes within cells and their responses to stimuli can be observed in real time using live cell imaging (Molecular Expressions™, 2015; Weitzman, 2016). Live cell imaging can be performed using various types of microscopy, including confocal (Molecular Expressions™, 2015), and requires an environmentally controlled chamber on the microscope stage for maintaining the cell’s environment as they are viewed. Live cell imaging is an extremely powerful tool that goes beyond visualization of cell structures, and has contributed to our knowledge of the location, function, and interactions of proteins in cells. Peter O’Toole tells us in the Making Light Work video why “microscopy is the number one technology that all biologists should be using.”
For microscopic visualization, cells and cellular components are typically labeled in some manner. For live cell imaging, concerns about labeling include whether the technique affects cell viability and/or perturbs cellular function. The workhorse of cellular labeling has been the fluorescent-tagged protein with its ease of use and wide availability, but there are with technical limitations (autofluorescence and photobleaching) and the potential for phototoxic damage to the cells. Molecular biology techniques can be used to transfect cDNA into cultured cells that signal the cells to express specific proteins tagged with the green fluorescent protein (GFP). Cells expressing GFP-tagged proteins can be imaged using real-time live cell microscopy. Limitations to this technique include potential artifacts and possible loss of function of the tagged protein. Even so, GFP labeling, digital imaging, and advances in microscopy instrumentation have revolutionized our ability to visualize and understand the functioning of living cells.
Cell-based Assays – How are cells used in experiments?
The growing investments made by pharmaceutical companies in drug discovery, replacement of in vivo toxicity testing by in vitro testing methods, and the increasing adoption rate of cell assay based high throughput screening methods of drug compound screening are some of the major growth drivers for the market.
A bioassay can be defined as “a procedure for determining the concentration, purity, and/or biological activity of a substance by measuring its effect on an organism, tissue, cell, enzyme, or receptor preparation compared to a standard preparation.” Thus, a cell-based assay is a procedure for determining some biological effect of a test substance using a cell-based system. Cell-based assays involve the use of a detection method for measuring the biological effect, which typically involves some type of spectroscopic detection (e.g., colorimetry, luminescence, fluorescence).
Various types of cytotoxicity assays are the most common type of cell-based assay. They involve treating cultured cells with a test substance, and then using a detection method to quantify the cell’s responses at different doses and/or times after exposure to the test substance. Assays can also be developed to measure changes in a specific cellular component such as the expression of a protein. Additional types of cell-based assays include: metabolism, receptor binding, oxidative stress, cell proliferation, cell morphology, cell signaling, cell adhesion, and cell migration.
The video, Culture Preparation and Plating, illustrates some of the common steps in preparing cells for use in a cell-based assay. Various companies offer reagents, instrumentation, cells, and even complete kits for conducting cell-based assays, and many contract testing laboratories offer cell-based testing services. Many of these companies also provide publications, videos, and/or consulting services to assist in the selection of the best method(s) for the information needed.
Companies producing regulated products, including drugs, chemicals, and certain consumer products, tend to use cell-based screening methods supported with databases of reference test data. However, for regulatory submissions the test method must either have been accepted as valid by the agency, or sufficient data would need to be submitted to convince the agency that the method is valid and sufficient for the indicated use.
Emerging methods employing cultured cells include high-throughput and high-content screening assays and microfluidic cell culture devices and assays. Cell imaging technologies are being combined with high-throughput and high-content cell screening assays as multi-parameter assays, sometimes referred to as cell-based safety assessments, for the early identification of problem drugs or chemicals.
Before use as a research or testing model, an in vitro cell-based model should be thoroughly characterized for how well it replicates key physiological parameters of the same tissue in vivo; especially those features involved with the endpoint being assessed (Ward et al., 2003). These characterization parameters will vary by the cell/tissue type and possibly by the intended use of the in vitro model. The key parameters determined to be useful/necessary in characterizing a cell-based model can then serve as the baseline for evaluating changes to the cell’s microenvironment, as culture conditions are optimized. Rigorous characterization is often shortchanged due to cost and time; however, the data derived from poorly conceived/characterized models can provide irrelevant, non-reproducible, or even misleading results.
Cell-based Toxicity Tests – Using cells to identify toxic substances
The 2007 report by the U.S. National Research Council, Toxicity Testing in the 21st Century: A Vision and Strategy, forecasted a shift in the direction of toxicity testing away from in vivo methods and towards in vitro and other non-animal methods. Cell-based assays, as key components of test batteries and/or tiered or integrated test schemes to predict human toxicity endpoints, will be a key technology used to achieve this goal. The role of cell-based assays in toxicology and toxicity testing has steadily increased over the past several decades, however, their validation and acceptance for regulatory testing purposes has been slow and problematic. Only stand-alone cell-based assays have been evaluated thus far in validation studies, and, although some have been recommended for use as screening assays or as a component in a tiered or integrated test scheme, few have been endorsed as full replacements for an animal test method.
Assays to assess cytotoxicity or cell death are the most common type of cell-based assay used in toxicity testing. They involve treating cultured cells with increasing doses of a test substance, and then using one of the many methods available to quantify the percent of live and/or dead cells at each dose. The endpoint at some percentage of cell death or viability is then used to compare the relative effect of different test substances. Common assays to measure cell cytotoxicity include lactate dehydrogenase (LDH) leakage, reduction of the tetrazolium dye MTT, and neutral red uptake (NRU) (DB-ALM; Fotakis & Timbrell, 2006; Grant et al., 1992). Some functional cell assays, such as fluorescein permeability across cell layers (TEP) and disruption of cellular tight junctions (TER) have also been found to correlate with cell viability in dose-response assessments (Ward et al., 1997). The video, New techniques for continuous real time monitoring of viability and cytotoxicity, explains some emerging methodologies for cell cytotoxicity assessment.
There are several mechanisms of cell death. Necrosis is the most common mechanism of cell death from in vitro chemical exposures, and involves rapid cell lysis due to the loss of cell membrane integrity. Cells can also just stop proliferating following certain exposures, or can undergo a type of programmed cell death called apoptosis.
Cells are considered to be the basic ‘building blocks’ of living organisms. However, the identity, behavior, and survival of a cell depend not only on neighboring cells, but on many molecular pathways and biological processes occurring within the entire organism. This is the premise – the biological complexity and inter-connectedness of the pathways – which is used by whole animal toxicologists to suggest that cell-based assays will never be sufficient to predict human toxicity. On the other hand, it is precisely this complexity of the whole organism as well as the need to use animal models to obtain whole organism toxicity data that are problematic when it comes to understanding human and mechanistic toxicity responses. This ‘battle’ over ‘the best’ test model has existed since cell biologists, biochemists, and toxicologists developed their respective research specialties and preferences (S. Ward, personal observation).
There is a great need for physiologically relevant human cell-based assays that can provide the biomedical and toxicity data that cannot be obtained from animal models or humans. With the current growth in new technologies, knowledge, and methods for complex data analysis, it now appears feasible that data from reductionist methods (of which cellular assays are an important component) will be able to be reassembled into models that can predict human toxicity even better than animal models.
Pre-market regulatory testing, although country and agency-specific, requires toxicity data showing the effect of a substance on a number of human health endpoints. Except for genotoxicity, the data primarily used for these regulatory determinations has been animal test data. Other human health endpoints in need of animal alternative approaches for regulatory submissions include: acute and repeated-dose systemic toxicity, toxicokinetics, carcinogenicity, genotoxicity, eye and skin irritation/corrosion, skin sensitization, reproductive toxicity, endocrine disruption, photosafety, and immuotoxicity. Human cell-based studies that provide mechanistic understanding are often used to support regulatory submissions, but are typically submitted in addition to rather than as replacements for the animal test methods.
Since the late 1990’s, international validation authorities have conducted and/or reviewed validation studies for a number of new toxicity test methods, many being cell-based assays. The methods endorsed as valid by international authorities can be found in various tables and lists, including AltTox’s Table of Validated & Accepted Alternative Methods.
The Next Frontier – The Human Cell Atlas
The estimated 37.2 trillion cells in the human body are commonly grouped into around 200-300 different major cell types. New methods of characterizing cells, however, show that even within what appears to be a homogenous population, there is great variability. Recent studies on single cells suggest that “the assumption that all cells of a particular ‘type’ are identical” is incorrect, and that “individual cells within the same population may differ dramatically, and these differences can have important consequences for the health and function of the entire population.”
Mapping the diversity of human cell types has been compared to the mapping of the human genome, and was identified as the next frontier to developing a full understanding of how biological organisms function. According to the U.S. National Institutes of Health, an early funder of the Human Cell Atlas initiative, “we have the opportunity to identify the foundational principles underlying cellular organization in human tissues that could lead to a new level of understanding in many scientific areas including developmental and aging processes, emergence of pathological states, and how to engineer complex functional tissue.”
Although it is neither necessary or desirable to engineer multiple types of extremely complex tissues to replace each animal bioassay, the greater understanding obtained from this new research initiative in mapping all human cell types and their interactions can only facilitate the development of better in vitro models/methods for toxicity testing.
EURL ECVAM DataBase service on ALternative Methods to animal experimentation DB-ALM
Promega, Culture Preparation and Plating
Rodríguez-Hernández, et al. (2014). Cell Culture: History, Development and Prospects. Int.J.Curr.Res.Aca.Rev. 2(12), pp. 188-200.
Sigma-Aldrich Cell Culture Videos
ThermoFisher Scientific, Introduction to Cell Culture
Worthington Biochemical Corporation, Tissue Dissociation Guide
Author: Albert P. Li, Ph. D., In Vitro ADMET Laboratories Inc., 9221 Rumsey Road, Suite 8, Columbia, MD 21045, USA
Mammalian cells in culture represent a key in vitro system to define xenobiotic toxicity. Current practice involves both primary cells (cells cultured from specific organs) and immortalized cell lines. The advantages and limitations of primary cells versus cell lines are reviewed.
It is important to firstly clarify the purpose of in vitro toxicity evaluation. The term “alternatives,” meaning “alternatives to animal testing” is used which, while appropriate in defining the goal of reduction of the use of animals, undermine a major advantage of in vitro evaluation, namely, the definition of human-specific toxicity. In vitro experimental systems using human-derived cells or tissue fractions can be used to define human-specific xenobiotic properties which, due to species-difference, cannot be readily obtained using animals in vivo. Human-based in vitro experimental systems are now fully embraced by the pharmaceutical industry and the corresponding governmental regulatory agencies. A clear example is that the United States Food and Drug Administration, European Medical Agency, Japanese Health Ministry, Sino Food and Drug Administration, all have issued guidance documents requiring the use of human-based in vitro system to define key drug properties such as metabolic fate and drug-drug interactions. Further, animal models used in safety evaluation are required to be substantiated with information on human relevance, especially for drug metabolism, based on human in vitro results. A key adverse drug property, drug-drug interaction potential, requires to be demonstrated using human in vitro metabolism systems and not nonhuman animal systems. In other words, human in vitro systems are believed to be relevant, while nonhuman animals are not as relevant, for the definition of drug-drug interaction potential.
For the definition of human-specificity, the following are key requirements of an in vitro experimental system:
With the above requirements, one can logically and scientifically select the appropriate experimental systems for the evaluation of xenobiotic toxicity. We now can proceed to compare primary cells and cell lines in this application:
Advantages of primary cells:
Disadvantages of primary cells:
Advantages of cell lines:
Disadvantages of cell lines:
In conclusion, primary cells, especially human-derived primary cells (e.g. human hepatocytes) can be used routinely for accurate definition of human-specific toxicity. Cell lines, especially well defined engineered cell lines, can be used for mechanistic definition of a specific toxicological pathways. When used appropriately within their limitations, both primary cells and cell lines are useful in vitro approaches for the definition of xenobiotic toxicity.
Den Hartogh, S.C., & Passier, R. (2016). Concise Review: Fluorescent reporters in human pluripotent stem cells: Contributions to cardiac differentiation and their applications in cardiac disease and toxicity. Stem Cells, 34(1), pp.13-26.
European Medicines Agency (2012). Guideline on the investigation of drug interactions. http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2012/07/WC500129606.pdf
Hendriks, G., Derr, R.S., Misovic, B., Morolli, B., Calléja, F.M., & Vrieling, H. (2016). The Extended ToxTracker Assay discriminates between induction of DNA damage, oxidative stress, and protein misfolding. Toxicological Sciences, 150(1), pp.190-203.
Japanese Pharmaceuticals and Devices Agency (2001). Guideline on methods of drug interaction study. http://www.nihs.go.jp/phar/pdf/DiGlEngFinal011209.pdf http://www.nihs.go.jp/phar/pdf/DiGlEngFinal011209.pdf%20
Kugler, J., Luch, A., & Oelgeschläger, M. (2016). Transgenic mouse models transferred into the test tube: New perspectives for developmental toxicity testing in vitro? Trends in Pharmacological Sciences, 37(10), pp. 822-830.
Li, A.P. (2004). Accurate prediction of human drug toxicity: a major challenge in drug development. Chemico-Biological Interactions, 150(1), pp.3-7.
Li, A.P. (2001). Screening for human ADME/Tox drug properties in drug discovery. Drug Discovery Today, 6(7), pp.357-366.
Schwartz, M.P., Hou, Z., Propson, N.E., Zhang, J., Engstrom, C.J., Costa, V.S., Jiang, P., Nguyen, B.K., Bolin, J.M., Daly, W., & Wang, Y. (2015). Human pluripotent stem cell-derived neural constructs for predicting neural toxicity. Proceedings of the National Academy of Sciences, 112(40), pp.12516-12521.
U. S. FDA (2016). Drug development and drug interactions. http://www.fda.gov/Drugs/DevelopmentApprovalProcess/DevelopmentResources/DrugInteractionsLabeling/ucm080499.htm
Zhang, J., Doshi, U., Suzuki, A., Chang, C.W., Borlak, J., Li, A.P., & Tong, W. (2016). Evaluation of multiple mechanism-based toxicity endpoints in primary cultured human hepatocytes for the identification of drugs with clinical hepatotoxicity: Results from 152 marketed drugs with known liver injury profiles. Chemico-Biological Interactions, 255, pp. 3-11.
Author: Sherry L. Ward, AltTox Contributing Editor
The Principles of Good Laboratory Practice (GLP) are quality measures used to ensure the implementation of standardized procedures and record keeping for non-clinical health and environmental studies intended to be used in submissions to regulatory authorities. Many national regulatory authorities have specified guidance related to the use of GLPs for non-clinical health and environmental safety testing for regulated products. Laboratories may seek GLP-accreditation for their testing services, and regulatory authorities may conduct compliance monitoring/inspections of the laboratories that are accredited.
GLPs were first developed by government authorities in the 1970’s, “in response to fraudulent scientific safety studies,” as a means “to ensure future safety studies would be of acceptable quality and integrity.” The Organisation for Economic Cooperation and Development (OECD) developed their first internationally harmonized GLPs in 1981. OECD developed Principles of GLP to “ensure the generation of high quality and reliable test data related to the safety of industrial chemical substances and preparations….created in the context of harmonising testing procedures for the Mutual Acceptance of Data (MAD).” OECD provides access to a number of OECD consensus, guidance, and advisory documents on GLPs, and links to many national organizations and their GLP guidance.
Good Cell Culture Practice (GCCP) is the expanded application of the Principles of GLP to cell-culture based studies. GCCP includes specifications for procedures that may not be encountered with traditional GLP studies. Good cell culture practices also have a major role in reducing the problem of cell line misidentification (Lorge et al., 2016; Mims et al., 2010).
Key organizations involved in assessing the validation status of toxicity test methods, including OECD, the European Centre for the Validation of Alternative Methods (EURL ECVAM), and the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM), have all participated in the development of the GCCP principles for regulatory testing procedures.
The OECD Advisory Document, The application of the principles of GLP to in vitro studies (2004), provides an interpretation of GLPs for in vitro studies conducted for regulatory purposes. Special considerations relevant to GCCP are described in the presentation “Critical aspects in implementing the OECD Monograph 14 ‘The application of the principles of GLP to in-vitro studies’.” Examples include: special attention for aseptic conditions, and the characterization, maintenance, viability, and responsiveness of the test system.
Some of the references below provide updated GLP/GCCP considerations for specific test types such as those for genotoxicity testing (Lorge et al., 2016) and for the development and use of stem cell-based models (Pamies et al., 2017).
Coecke, S., Balls, M., Bowe, G. et al. (2005). Guidance on Good Cell Culture Practice. A report of the second ECVAM Task Force on Good Cell Culture Practice. Altern.Lab.Anim. 33, 261-287.
Hartung, T., et al. (2002). Good Cell Culture Practice. ECVAM Good Cell Culture Practice Task Force Report. Altern.Lab.Anim. 30, 407-414.
Lorge, E., Moore, M.M., Clements, J., et al. (2016). Standardized cell sources and recommendations for Good Cell Culture Practices in genotoxicity testing. Mutat. Res. 809, 1-15.
Organisation for Economic Cooperation and Development (OECD) Home Page. (2016). OECD Series on Principles of Good Laboratory Practice (GLP) and Compliance Monitoring and Good Laboratory Practice (GLP) and Links to National Websites on Good Laboratory Practice
OECD. (2004). The application of the principles of GLP to in vitro studies. OECD Series on Principles of Good Laboratory Practice and Compliance Monitoring. Number 14.
OECD. (2005). Guidance document on the validation and international acceptance of new or updated test methods for hazard assessment. OECD Series on Testing and Assessment, Guidance Document. Number 34.
OECD. (2016). OECD Position Paper regarding the relationship between the OECD Principles of GLP and ISO/IEC 17025. OECD Series on Principles of Good Laboratory Practice and Compliance Monitoring, Number 18.
Pamies, D., Bal-Price, A., Simeonov, A., et al. (2017). Good Cell Culture Practice for stem cells and stem-cell-derived models. ALTEX 34(1), 95-132.
This section is under development – check back soon.
Authors: Emanuela Corsini1 and Helena Kandarova2
1 Laboratory of Toxicology, Department of Pharmacological and Biomolecular Sciences, Università degli Studi di Milano, Milan, Italy
2 In Vitro Life Science Laboratories, Bratislava, Slovakia
In the past decade the progress in tissue engineering, bioprinting and microtechnologies has enabled a rapid growth in the area of 3-dimensional (3D) model systems. Bioprinting technology enables precise control over spatial and temporal distribution of cells and extracellular matrix, which can be used to engineer artificial tissues and organs (Arslan-Yildiz et al., 2016). Human 3D models as well as dynamic on-a-chip platforms have the potential to significantly impact biomedical applications including drug development, toxicology screening, and disease modeling (Gordon et al., 2015; Skardal et al., 2016).
3D tissue models are going to change the use of in vitro testing for drug discovery and chemical safety assessment from traditional 2D monolayer cell cultures to more complex 3D systems characterized by greater physiological relevance. 2D cell cultures have been and still are invaluable tools in cell biology, experimental toxicology, and pharmacology, however, they tend to lose their phenotypical characteristics and fail to recreate the in vivo physiological environment. 3D cell culture models more closely resemble in vivo conditions and are believed to better predict in vivo effects, which have, however, to be evaluated thoroughly. 3D organ-on-a-chip devices, also referred to as ‘body-on-a-chip systems’, that can recapitulate in vivo tissue architectures, and the physiological fluid conditions, including fluid shear and mechanical stresses that support normal tissues, represent the next challenge and ultimate goal. If successful, these models will influence drug development by reducing costs and improving predictivity; they will increase the success of drug candidates in clinical trials (Skardal et al., 2016). Furthermore, these models have the potential to significantly decrease animal tests. At present, there are only a few models in which multiple tissues have been integrated in a single platform, and several projects are ongoing, e.g. ECHO, ATHENA, DARPA, and NIH initiatives (Skardal et al., 2016).
Cells grown on plastic dishes differ in many ways from the original tissue: lack of 3D architecture, surface stiffness, oxygen tension, biochemical composition, and cell density to name some. One of the faults of 2D models is the lack of longevity and tissue-level complexity, which may limit their utility in predictive toxicology. In this context, it is quite interesting a recent work published by Luckert et al. (2016) on hepatocytes demonstrated that “the previously reported increase of metabolic competence of hepatocarcinoma cell line HepG2 cells is not primarily the result of 3D culture but a consequence of the duration of cultivation. HepG2 cells grown for 21 days in 2D monolayer exhibit comparable biochemical characteristics, CYP activities and gene expression patterns as all 3D culture systems used.”
The performances of the 3D models of epithelial barriers, including reconstituted epidermis (RhE) models, are decidedly superior compared to 2D models (Gordon et al., 2015; Hayden et al., 2015). These models have reached an impressive levels of technical and scientific sophistication, and to a different extent have been implemented by industries. As an example, RhE models are being explored to resolve the in vitro assessment of chemical allergen potency (Corsini et al., 2016), which is required for a full replacement of animals. RhE models have several advantages over traditional cell cultures, and are expected to have broader applicability domain. Topical application in relevant vehicles (e.g. solvents used in animal tests or cosmetic/dermatological formulations) is only possible in RhE models, which mimic in vivo bio-availability of a chemical more closely, and which may therefore lead to improved assessment of sensitizer potency (Van der Veen et al., 2014). We previously demonstrated the possibility of combining the epidermal equivalent potency assay, based on the irritation potential, with the assessment of IL-18 release (RhE IL-18 potency assay) to provide a single test for identification and classification of skin sensitizing chemicals, including chemicals of low water solubility or stability (Gibbs et al., 2013).
|ADVANTAGES OF 3D MODELS||LIMITATIONS OF 3D MODLES|
|Long-term culture||High technology equipment required|
|Better recapitulation of in vivo function and response||Low-throughput setting|
|Less handling compared to 2D models|
3D models and on-a-chip technologies have gained a significant momentum in recent years, and hold promise for many applications in research and development. Additional advancements to better mimic human physiology and responses to drugs and toxicants are needed. Furthermore, studies are still necessary to establish the absolute advantage of 3D methods compared to simpler and less expensive 2D models. In relation to the final purpose, 2D methods could do just as well!
Arslan-Yildiz, A., El Assal, R., Chen, P., Guven, S., Inci, F., & Demirci, U. (2016). Towards artificial tissue models: past, present, and future of 3D bioprinting. Biofabrication 8, 014103. DOI:10.1088/1758-5090/8/1/014103.
Corsini, E., Roggen, E.L., Galbiati, V., & Gibbs, S. (2016). Alternative Approach for Potency Assessment: In Vitro Methods. Cosmetics 3, 7. DOI: 10.3390/cosmetics3010007.
Gibbs, S., Corsini, E., Spiekstra, S.W., Galbiati, V., Fuchs, H.W., Degeorge, G., Troese, M., Hayden, P., Deng, W., & Roggen, E. (2013). An epidermal equivalent assay for identification and ranking potency of contact sensitizers. Toxicol. Appl. Pharmacol., 272, 529-541. DOI: 10.1016/j.taap.2013.07.003.
Gordon, S., Daneshian, M., Bouwstra, J., Caloni, F., Constant, S., Davies, D.E., Dandekar, G., Guzman, C.A., Fabian, E., Haltner, E., Hartung, T., Hasiwa, N., Hayden, P., Kandarova, H., Khare, S., Krug, H.F., Kneuer, C., Leist, M., Lian, G., Marx, U., Metzger, M., Ott, K., Prieto, P., Roberts, M.S., Roggen, E.L., Tralau, T., van den Braak, C., Walles, H., & Lehr, C.M. (2015). Non-animal models of epithelial barriers (skin, intestine and lung) in research, industrial applications and regulatory toxicology. ALTEX 32, 327-378. DOI: 10.14573/altex.1510051.
Hayden, P.J., Bachelor, M., Ayehunie, S., Letasiova, S., Kaluzhny, Y., Klausner, M., & Kandarova, H. (2015). Application of MatTek in vitro reconstituted human skin models for safety, efficacy screening, and basic preclinical research. Appl. In Vitro Toxicol. 1, 226-233. DOI: 10.1089/aivt.2015.0012.
Luckert, C., Schulz, C., Lehmann, N., Thomas, M., Hofmann, U., Hammad, S., Hengstler, J.G., Braeuning, A., Lampen, A., & Hessel, S. (2016). Comparative analysis of 3D culture methods on human HepG2 cells. Arch. Toxicol. DOI: 10.1007/s00204-016-1677-z.
Skardal, A., Shupe, T., & Atala, A. (2016). Organoid-on-a-chip and body-on-a-chip systems for drug screening and disease modeling. Drug Discov. Today. DOI: 10.1016/j.drudis.2016.07.003.
Van der Veen, J.W., Soeteman-Hernández, L.G., Ezendam, J., Stierum, R., Kuper, F.C., & van Loveren, H. (2014). Anchoring molecular mechanisms to the adverse outcome pathway for skin sensitization: Analysis of existing data. Crit. Rev. Toxicol. 44, 590-599. DOI:10.3109/10408444.2014.925425.
Author: Sherry L. Ward, AltTox Contributing Editor
Organoids, or cultured mini-organs that are barely visible to the eye, are developed by the self-assembly of cultured stem cells, often using induced pluripotent stem cells (iPSCs). Even though organoids represent an early developmental stage of the organ, they develop intrinsic organization and begin to signal, differentiate, and form some organ-like structures (Ravven, 2016).
There is a growing interest in generating organoids as research and testing models (Lancaster & Knoblich, 2014). Researchers have developed 3-dimensional (3-D) organoid models for various types of human tissues, including the intestine (Wells and Spence, 2014; Zachos et al., 2015), gastric/stomach (McCracken et al., 2014), lung (Dye et al., 2015), kidney (Morizane et al., 2015), liver (Bell et al., 2016; Sirenko et al., 2016), and brain (Camp et al., 2015; Hartley & Brennand, 2016; Jo et al., 2016; Luo et al., 2016; Pamies et al, 2014).
Methods vary for the creation of each type of organoid, but all “use growth factors or nutrient combinations to drive the acquisition of organ precursor tissue identity,” followed by culture in an environment conducive to 3-D tissue formation (Lancaster & Knoblich, 2014). Dye, et al. (2015) explain how specific signaling pathways in human iPSCs were manipulated sequentially to generate lung organoids. “Scientists activated two important development pathways that are known to make endoderm form three-dimensional tissue. By inhibiting two other key development pathways at the same time, the endoderm became tissue that resembles the early lung found in embryos….to make these structures expand and develop into lung tissue…the team exposed the cells to additional proteins that are involved in lung development.”
Organoid cultures are being developed for many proposed applications. Some examples of their use in human safety assessments include: drug discovery and development (Liu et al., 2016), drug induced liver injury (Bell et al., 2016), and testing toxic compounds Sirenko et al., 2016. An important recent application of mini-brain organoids was their use in identifying how the Zika virus damages human fetal brains (Cugola et al., 2016), and for screening of potential treatments (Xu et al., 2016).
Future developments could include multi-organ organoids used in microfluidic platforms to model even more complex biological systems outside the living organism. “Exciting new platforms are currently being developed…devices where microfabrication and microfluidic technology are combined to support organoid culture and fluid flow, enabling high-throughput testing, environmental sampling, and biosensing” (Simian & Bissell, 2017).
Emanuela Corsini, Ph.D.
Laboratory of Toxicology, Department of Pharmacological and Biomolecular Sciences, Università degli Studi di Milano, Milan, Italy
Albert P. Li, Ph.D.
In Vitro ADMET Laboratories, Inc., 9221 Rumsey Road, Suite 8, Columbia, MD 21045, USA
Sherry L. Ward, Ph.D., MBA
Contributing Editor, AltTox
Consultant, BioTred Solutions