Mod 3 Flashcards
What are the two groupings of models that are used experimentally to conduct toxicological experiments?
In vivo and in vitro
What is in vivo?
Experiments that occur within a body, such as those with animals
What is in vitro?
Experiments that occur outside a body, such as within a petri dish
What is is a cell culture, and what three materials are usually involved in it?
Where cells from an organism are maintained and propagated under specified conditions.
Three materials:
- Vessel (ie. dish or flask)
- Nutrient media (“feeds” the cells)
- Incubator (house the cells at ideal temp, humidity, and oxygen/CO2 levels)
What are the two types of cell cultures?
Primary cell culture and cell line
What are cell lines?
Cell lines are subcultures of cells derived from a primary cell culture that are transferred to new dishes to continue growing and proliferating after reaching confluency in the original dish.
What does confluency mean in the context of cell culture?
Confluency refers to the state when cells have grown to occupy all the available space in a culture dish, necessitating their transfer to a new dish to continue proliferating.
How are primary cells obtained?
Primary cells are extracted directly from a tissue or organism and then transferred to a culture vessel for maintenance and proliferation under controlled conditions.
What happens when primary cells reach confluency in a culture dish?
When primary cells reach confluency, they must be transferred to a new dish, becoming subcultures known as cell lines, to continue growing.
What is the difference between a primary cell culture and a cell line?
A primary cell culture consists of cells directly extracted from a tissue or organism, while a cell line refers to subcultures derived from primary cells, which are transferred to new dishes to continue proliferating.
What are continuous cell lines?
Continuous cell lines are derived from primary cell cultures but modified to prevent senescence, allowing them to replicate indefinitely.
These cells are often immortalized, sometimes through viruses or chemicals, and are frequently cancerous in nature.
How do continuous cell lines become immortalized?
Continuous cell lines become immortalized either spontaneously or by intentional alteration using viruses or chemicals, resulting in traits that prevent senescence and allow indefinite replication.
Why are many immortal cell lines cancerous?
Many immortal cell lines are cancerous because cancer cells inherently possess the ability to avoid senescence, enabling continuous replication.
What are finite cell lines?
Finite cell lines are derived from primary cell cultures and have a limited number of cell divisions before they senesce, losing their ability to proliferate. Unless modified, these cells eventually stop growing.
What is the key limitation of finite cell lines?
Finite cell lines are limited in the number of divisions they can undergo before senescence and can suffer from rapid dedifferentiation, making them short-lived in culture.
What does senesce mean?
Senesce refers to the deterioration of cells with age, leading to a loss of their ability to proliferate.
What is dedifferentiation?
Dedifferentiation occurs when cells regress from a specialized function to a more simplified, stem cell-like state.
What is one key factor to consider when choosing a species for a cell line in toxicology research?
The species chosen should align with the experimental purpose. For example, if researching the toxicity of a chemical in human breast cancer cells, human breast cancer cell lines should be used instead of non-human cell lines.
Why is the experimental purpose important when choosing a cell line?
The experimental purpose helps determine the type of cell line to use. For example, liver cells might be appropriate for studying liver toxicity, while cells derived from embryonic or fetal tissue could be used for developmental toxicity studies.
What are the benefits of using continuous cell lines?
Continuous cell lines are easier to propagate, maintain, and provide increased consistency in results. They are also well-characterized, which helps with predictability in experiments.
Why might a finite cell line be preferred in some experiments?
Finite cell lines may better express the correct cellular phenotypes and offer more options for cell types to culture, making them useful in studies requiring more in vivo-like characteristics.
What is the difference between normal and transformed cell lines?
Normal cell lines have not undergone significant changes, while transformed cell lines have altered phenotypes and increased growth rates, which may affect their response to experimental conditions.
What is a potential drawback of using transformed cell lines?
Transformed cell lines may have altered phenotypes that could affect how they respond to experimental toxicants, potentially impacting the validity of the results.
Why is feasibility an important consideration when choosing a cell line?
Feasibility considerations include access to the cell line, ease of storage, availability of stocks, and whether the cell line is well-characterized. These factors influence the practical execution of experiments
How do cell characteristics influence the choice of a cell line?
Cell characteristics, such as protein expression, growth rate, and confluency, must align with the research question. For example, a cell line should express the proteins necessary for studying a specific signaling pathway.
Why is the length of study important in cell line selection?
The length of time cells are exposed to a chemical in an experiment may affect cell viability and replication, influencing the results. This should be considered when choosing an in vitro model.
What is the primary advantage of using cell culture in toxicology experiments?
The primary advantage of using cell culture in toxicology is the consistency and reproducibility of results that can be obtained.
What is the purpose of a cell death assay in toxicology research?
A cell death assay is used to assess whether exposure to a chemical affects the extent or rate of cell death in a particular cell type, with the total number of cells decreasing over time after exposure to a toxicant.
What does a proliferation assay measure?
A proliferation assay measures whether exposure to a chemical affects the ability of cells to proliferate or differentiate, typically observing a decrease in cell proliferation over time.
What type of experimental question would be addressed by studying phenotypic changes in cell culture?
An experiment studying phenotypic changes might ask: “Does exposure to a chemical alter the phenotype or morphology of the cell population?”
What is the focus of mechanistic studies in cell culture experiments?
Mechanistic studies aim to understand biological processes or the mechanism of action of a drug or toxicant. They may investigate whether chemical exposure causes mitochondrial dysfunction, cellular stress, or changes in gene/protein expression or signaling pathways.
What type of chemical is 5-AZA-CR, and what is its role in cell culture experiments? - probably dont need to know this
5-AZA-CR is a chemical that inhibits DNA methylation and is used in experiments to study the effects of DNA methylation changes on cell populations, such as in phenotypic alteration studies.
T or F:
In vitro experiments help detect whether a toxicant causes mutations in cellular DNA.
T
What is the Ames Test used for?
The Ames Test is used to detect mutations caused by chemical exposure, indicating whether a toxicant causes DNA mutations.
In the Ames Test, what kind of bacteria are used?
Bacterial cells that are unable to produce histidine (his-) are used in the Ames Test.
How does a positive Ames Test result occur?
A positive result occurs when a chemical exposure causes a mutation that allows the bacteria to produce histidine (his+), enabling growth in the absence of histidine.
What is a unique technique in cell culture that is not possible in other toxicology models?
Transfection is a unique technique used in cell culture.
What is transfection in the context of in vitro cell culture models?
Transfection is the process of inserting DNA that has been amplified into a suitable vector to introduce it into a mammalian or other cell.
What are two methods commonly used to amplify DNA for transfection?
Polymerase Chain Reaction (PCR) and molecular cloning.
Why is it important to know the type of cell culture used before attempting transfection?
Not all cell types are amenable to transfection, so it is important to ensure the chosen cell type can be transfected.
What can transfection allow a cell to do?
Transfection can enable a cell to stably or transiently express gene products such as proteins, reporters, or inhibitors that it wouldn’t normally express.
How can transfection help in toxicology research?
By transfecting cells with specific genes, researchers can induce the expression of proteins, reporters, or inhibitors. This allows them to observe how these gene products interact with the toxicant, revealing how the toxicant affects cellular processes and providing insights into its mechanism of action.
What is molecular cloning?
Molecular cloning is the isolation and generation of recombinant DNA molecules that are placed in organisms for replication and study.
What are the key steps in the process of transfection?
First, the plasmid of interest is collected and applied to the cells. Then, the cell membrane’s permeability is increased chemically or electrically, allowing the plasmids to enter the cell and its nucleus. Once inside the nucleus, the cell expresses the gene product of the plasmid, often a protein.
What is one major limitation of cell culture experiments?
A key limitation is their lack of predictive ability. Since cells are cultured outside of their in vivo environment, the results from cell culture studies may not accurately predict outcomes in living organisms.
How does metabolism limit the predictive ability of cell culture experiments?
The metabolic capabilities of isolated cells may not reflect those of intact organisms. This is crucial in toxicology, as the lack of necessary enzymes in cell culture can lead to overlooked or misrepresented effects of toxicants. Research is focused on enhancing the metabolic relevance of cell culture assays to align better with in vivo data.
What are stem cells capable of building?
Stem cells are capable of building any tissue type that exists within the body.
What are the two unique features of stem cells?
- They can differentiate into a number of different specialized cell types.
- They can renew themselves indefinitely.
How can stem cells be defined?
Stem cells can be defined by the developmental stage of the animal from which they are derived and their biological characteristics based on their ability to differentiate.
What are the two types of stem cells based on their differentiation capabilities?
Pluripotent and multipotent stem cells.
What are pluripotent stem cells?
Pluripotent stem cells have the capacity to differentiate into all cell types within the body, across all germ lines (endoderm, mesoderm, and ectoderm).
What are multipotent stem cells?
Multipotent stem cells have the capacity to differentiate into any cell type within one of the germ lines (endoderm, mesoderm, or ectoderm).
How can the use of stem cells benefit the field of toxicology?
The unique properties of stem cells allow researchers to address questions that are not possible with conventional cell culture.
Why is stem cell toxicology considered more consistent with in vivo data?
Stem cell toxicology increases the predictive ability of the model over other types of cell culture, making it a useful in vitro model in toxicological research.
What are the three types of stem cells applicable in toxicological research?
- Embryonic stem cells
- Adult stem cells
- Induced pluripotent stem cells
What are embryonic stem cells and their characteristics?
Embryonic stem cells are harvested from an embryo that is less than 5 days old and are pluripotent, meaning they can differentiate into any cell type.
Where are adult stem cells harvested from, and what is their limitation?
Adult stem cells are harvested from adult tissues such as bone marrow, skin, cord blood, and brain.
They are limited in the types of cells they can differentiate into compared to embryonic stem cells.
What are induced pluripotent stem cells, and how are they created?
These stem cells are harvested from somatic cells and are induced to a state in which they can give rise to a number of different cell types via genetic transformation.
Functionally, they are also pluripotent
Which type of stem cells is considered the most useful in research due to fewer ethical constraints?
Induced pluripotent stem cells (iPSCs) are considered the most useful because they do not have the ethical constraints associated with embryonic stem cells, while also possessing pluripotent capabilities that adult stem cells lack.
What are some advantages of using stem cells in toxicological studies?
- Stem cells do not lose their replicative ability and do not require genetic manipulation.
- They allow for assessment of developmental toxicity testing in vitro.
- Toxicity tests in stem cells tend to have more consistency with in vivo data compared to conventional cell types.
What are some disadvantages of using stem cells in research?
- Stem cells are technically difficult to culture, and there is a lack of established protocols, making their use intimidating.
- A general lack of understanding means stem cells are often not well characterized.
What are co-culture systems used for in cell studies?
Co-culture systems are used to study interactions between different cell populations and are fundamental for investigating cell-cell interactions.
What defines a co-culture setup?
A co-culture setup involves two or more different populations of cells grown with some degree of contact between them.
How do recent advances in co-culture improve toxicity investigations?
Recent advances involve co-culturing different types of cells, making the models more predictive of human responses when investigating the toxicity of substances.
What are two promising models in co-culture systems?
The two promising models are the organotypic model and organ-on-a-chip.
What is the organotypic model in cell culture?
The organotypic model is a type of cell culture where two or more cell types from a complex tissue or organ are cultured together to mimic in vivo tissue, utilizing 3D culturing to allow for more natural cell interactions, including extracellular matrix formation.
How does the organotypic model improve the study of dermal toxicity?
For studying dermal toxicity, co-culturing keratinocytes and fibroblasts mimics the in vivo environment of the skin, potentially providing more accurate toxicity reflections compared to monocultures.
What is the organ-on-a-chip model?
The organ-on-a-chip model involves culturing cells on a specialized microchip designed to recapitulate the microenvironment of a human organ, providing a more accurate representation of the organ system of interest.
How does the organ-on-a-chip model differ from the organotypic model?
While both models aim to mimic in vivo conditions, the organ-on-a-chip model uses a microenvironment that better reflects the specific organ system being studied.
What physiological processes can be simulated in the organ-on-a-chip model for studying lung toxicity?
The organ-on-a-chip model allows for the culture of relevant lung cells and the simulation of physiological processes, such as air exposure and the stretch force of breathing, to better replicate the in vivo environment.
