Cell Transformation and Immortalization
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Questions and Answers

What is the definition of transformation of cultured cells?

A spontaneous or induced permanent phenotypic change resulting from a heritable change in DNA and gene expression.

What are the three major classes of phenotypic change associated with transformation?

  • Immortalization, Aberrant growth control, Malignancy (correct)
  • Cell division, Apoptosis, Senescence
  • Genetic instability, DNA repair, Protein synthesis
  • Differentiation, Metastasis, Proliferation

Immortalization refers to the acquisition of an infinite life span by cells.

True (A)

What is aberrant growth control in the context of transformation?

<p>The loss of contact inhibition of cell motility, density limitation of cell proliferation, and anchorage dependence.</p> Signup and view all the answers

Malignancy is characterized by the growth of invasive tumors in vitro.

<p>False (B)</p> Signup and view all the answers

What is the primary reason for using immortalized cell lines?

<p>To provide a consistent and inexhaustible source of cells for research purposes.</p> Signup and view all the answers

What are two major examples of common immortalized cell lines?

<p>All of the above (D)</p> Signup and view all the answers

What is a major advantage of using immortalized cell lines?

<p>All of the above (D)</p> Signup and view all the answers

What is the SV40 LT gene?

<p>A viral gene often used to induce immortalization in cultured cells.</p> Signup and view all the answers

How does the T antigen influence cell cycle control?

<p>It binds to the p53 protein, a key tumor suppressor, preventing it from halting the cell cycle in response to DNA damage.</p> Signup and view all the answers

Immortalization using viral genes like SV40 LT can lead to an increase in genomic instability.

<p>True (A)</p> Signup and view all the answers

What is the primary function of a biosafety cabinet in a tissue culture laboratory?

<p>To protect the operator and the surrounding environment (A)</p> Signup and view all the answers

Which factor is NOT considered when choosing a culture vessel for cell cultures?

<p>Color of the vessel (A)</p> Signup and view all the answers

What is the main advantage of using poly-D-lysine (PDL) coated flasks?

<p>They ensure cell attachment for primary cells that struggle to grow on surfaces. (A)</p> Signup and view all the answers

Which component of a tissue culture facility is essential for maintaining a controlled environment?

<p>Heating, ventilation, and air conditioning (HVAC) system (C)</p> Signup and view all the answers

Which type of air filter is typically used in laminar airflow cabinets?

<p>High efficiency particulate air (HEPA) filter (D)</p> Signup and view all the answers

Which of the following materials is NOT a recommended interior finish for a tissue culture laboratory?

<p>Wooden fixtures (D)</p> Signup and view all the answers

What role does the CO2 incubator play in a tissue culture facility?

<p>Regulates CO2 levels essential for cell growth (C)</p> Signup and view all the answers

What type of cells do multi-well plates typically accommodate?

<p>Any type, depending on experimental needs (C)</p> Signup and view all the answers

What role do glutamine and glutamate serve in cell culture media?

<p>They serve as sources of energy and carbon. (C)</p> Signup and view all the answers

Which vitamin is NOT typically found in Eagle’s MEM medium?

<p>Vitamin A (B)</p> Signup and view all the answers

What is a primary advantage of using serum in cell culture medium?

<p>It supplies specific growth factors essential for cell proliferation. (B)</p> Signup and view all the answers

What is a significant disadvantage of using serum in cell culture media?

<p>It introduces batch-to-batch variation in compositions. (D)</p> Signup and view all the answers

Which mineral found in serum is important for promoting cell attachment?

<p>Calcium (Ca2+) (A)</p> Signup and view all the answers

Polyamine oxidase in serum reacts with which substances to form cytotoxic compounds?

<p>Polyamines (C)</p> Signup and view all the answers

How does one of the benefits of cell culture aid in biological research?

<p>By allowing researchers to alter environmental factors to study their effects. (B)</p> Signup and view all the answers

What is a common limitation associated with the use of fetal calf serum in cell culture?

<p>It presents a high risk of contamination with pathogens. (C)</p> Signup and view all the answers

What is the primary role of phenol red when added to balanced salt solutions?

<p>To check pH variations (A)</p> Signup and view all the answers

What is a primary source of contamination in cell culture?

<p>Airborne particles laden with microorganisms (C)</p> Signup and view all the answers

Which serum is most commonly used for cell culture due to its rich composition?

<p>Fetal bovine serum (FBS) (B)</p> Signup and view all the answers

Why is it important to screen batches of serum used in cell culture?

<p>To assess serum's ability to support cell growth (D)</p> Signup and view all the answers

Which of the following is NOT a component of culture medium for animal cells?

<p>Heavy metals (C)</p> Signup and view all the answers

What defines artificial culture media?

<p>Has a designed composition to meet nutrient needs (A)</p> Signup and view all the answers

During the development of complete culture media, what did Eagle focus on determining?

<p>The nutrient requirements for mammalian cell cultures (C)</p> Signup and view all the answers

What is essential for maintaining proper culture conditions for animal cells?

<p>Controlled temperature and appropriate growth medium (A)</p> Signup and view all the answers

What is the purpose of heat inactivating serum at 56º C?

<p>To eliminate immune factors that may harm cells (B)</p> Signup and view all the answers

What essential components must be added to the medium for optimal cell culture?

<p>Both essential and non-essential amino acids (C)</p> Signup and view all the answers

Which factor is important for cell attachment in culture?

<p>The presence of a suitable substrate (B)</p> Signup and view all the answers

Which of the following is NOT a type of balanced salt solution?

<p>FBS (C)</p> Signup and view all the answers

What is the purpose of using serum in cell culture medium?

<p>To provide growth factors and hormones (D)</p> Signup and view all the answers

What is the significance of serum in basal media for cell cultures?

<p>It serves as a source of hormones and growth factors (C)</p> Signup and view all the answers

Which characteristic does NOT apply to artificial culture media?

<p>Sterilization is optional (D)</p> Signup and view all the answers

What defines natural media in cell culture?

<p>Derived from biological resources like serum or tissue extracts (C)</p> Signup and view all the answers

What is the desirable pH range for culture media?

<p>7.0 - 7.4 (C)</p> Signup and view all the answers

How does the carbonate-bicarbonate buffer system affect pH?

<p>It maintains pH by reacting with hydrogen ions. (A)</p> Signup and view all the answers

What is a common outcome of exposing mammalian cells to higher oxygen concentrations?

<p>High oxidative stress that adversely affects the cells (B)</p> Signup and view all the answers

What role does serum supplementation play in culture media?

<p>It contains vital antioxidants that relieve oxidative stress. (A)</p> Signup and view all the answers

Which of the following components can be added to Balanced Salt Solutions (BSS)?

<p>Glucose and HEPES (B)</p> Signup and view all the answers

What is the significance of the phenol red indicator in culture media?

<p>It serves as a pH indicator by changing color. (B)</p> Signup and view all the answers

What defines a Balanced Salt Solution (BSS)?

<p>Solutions containing inorganic salts with similar composition to body fluids. (A)</p> Signup and view all the answers

What effect does increasing the CO2 concentration in the gas phase have on culture media?

<p>It increases the concentration of carbonic acid and decreases pH. (B)</p> Signup and view all the answers

Flashcards

Cell Transformation

A permanent change in a cell's characteristics due to alterations in DNA and gene expression. These changes can be spontaneous or induced.

Immortalization

The ability of a cell to divide indefinitely.

Passaging/Splitting

The process of transferring a portion of a cell culture to a new container to allow for continued growth.

Immortalized Cell Lines

Cells that have an unlimited capacity for division and can be cultured indefinitely.

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HEK-293T cells

A type of cell line derived from human embryonic kidney cells.

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HeLa cells

A type of cell line derived from cervical cancer cells.

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Aberrant Growth Control

The loss of contact inhibition, density limitation, and anchorage dependence.

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Malignancy

The ability of cells to grow and invade surrounding tissues.

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SV40 LT Gene

A gene from the Simian Virus 40 (SV40) that is commonly used to induce immortalization in cells.

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T Antigen

The protein product of the SV40 LT gene, known to bind to and inactivate p53.

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p53

A tumor suppressor protein that plays a critical role in regulating cell cycle and preventing cancer.

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Culturing Cells

The process of maintaining a cell culture in a controlled environment to facilitate growth.

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Primary Cultures

Cells derived directly from a living organism, often with a limited lifespan in culture.

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Cell Line

A type of cell culture where the cells have undergone extensive propagation and are considered immortalized.

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Proliferation

A term for cell proliferation.

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Contact Inhibition

A characteristic of normal cells where they stop dividing when they come into contact with each other.

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Density Limitation

A characteristic of normal cells where they stop dividing when they reach a certain density.

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Anchorage Dependence

A characteristic of normal cells where they require a solid surface to attach to in order to grow.

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Telomerase

A key enzyme involved in maintaining telomeres and preventing chromosome shortening during cell division.

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Telomeres

Protective caps on the ends of chromosomes that help prevent degradation.

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Telomere Shortening

A process that involves the loss of telomeres, leading to cell senescence and eventual death.

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Cell Cycle Override

The ability of SV40 T antigen to disrupt the cell cycle and promote immortalization.

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Genomic Instability

A process that contributes to the accumulation of genetic mutations, leading to uncontrolled cell growth.

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Cell Senescence

A state where cells are no longer capable of dividing and enter a state of dormancy.

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Phenotypic Change

A state of change or difference, particularly in cell behavior.

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Homogeneity

A property of immortalized cells that are genetically identical and allow for reproducible experiments.

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Reproducibility

The ability to obtain consistent results in repeated experiments.

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In Vivo Conditions

Conditions that mimic a normal living organism.

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Aseptic Technique

The practice of using techniques and procedures to prevent contamination of cell cultures by microbes.

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Cell Culture Media

Materials used to grow cells in a laboratory setting. They provide nutrients, support, and a controlled environment for cell survival and proliferation.

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Natural Media

A type of cell culture medium derived from natural sources like animal tissue extracts, body fluids, or biological products.

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Artificial Media

A type of cell culture medium with a precisely defined and controlled composition, made from synthetic components.

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Balanced Salt Solution

A solution that maintains a stable pH and osmotic balance in cell cultures.

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Laminar Air Flow Cabinet

A microbial-free environment created within a sealed unit by HEPA filters, which remove almost all airborne particles exceeding 0.3 microns, including bacteria and dust.

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Biosafety Cabinet

A biosafety cabinet protects not only the sample but also the operator and surrounding environment from contamination, unlike laminar airflow cabinets.

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HEPA filter

This type of air filter removes at least 99.97% of dust, pollen, mold, bacteria, and other airborne particles 0.3 microns or larger.

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CO2 Incubator

This controlled environment mimics the conditions needed for cell growth and proliferation, often with regulated temperature, CO2 levels, and humidity.

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Inverted Microscope

This specialized microscope allows for the observation of cells from the bottom, aiding in the visualization of cell behavior and morphology.

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Culture Vessels

These vessels, like flasks, multi-well plates, and Petri dishes, provide a controlled environment for cell growth and culture, offering various sizes and functionality for different purposes.

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Poly-D-lysine (PDL) and Collagen I Coated Flasks

Coated flasks enhance cell adhesion by creating a positive charge on the surface, facilitating the growth of primary cells that might otherwise struggle to attach.

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Infrastructural Requirements for Tissue Culture

A set of interconnected mechanical, plumbing, and engineering systems that ensure optimal conditions and safety within the facility.

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Ideal pH for cell culture

The ideal pH range for most cell cultures, where cells thrive best. A pH outside this range can negatively impact cell growth and function.

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Phenol red

A color indicator commonly used in cell culture media to visually monitor the pH. Its color changes with pH variations.

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Carbonate-Bicarbonate Buffer

A buffer system used to control the pH of cell culture media. It relies on the balance between bicarbonate ions and carbon dioxide.

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Oxygen in cell culture

A crucial element required for aerobic respiration in cells, where oxygen is converted to energy. Cells need the right amount for optimal function.

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Balanced Salt Solutions (BSS)

Solutions mimicking body fluids that maintain proper pH and osmotic pressure for cells, providing essential salts and nutrients.

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Ringer's Solution

A type of balanced salt solution formulated by Sydney Ringer, similar in composition to body fluids.

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Supplements in cell culture

Substances added to cell culture media to enhance cell growth by providing vital nutrients that are otherwise deficient in the medium.

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Serum in cell culture

A component of serum essential for cell growth. It provides a rich mix of growth factors, amino acids, and other vital molecules.

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What is a Balanced Salt Solution (BSS)?

A simple solution containing inorganic salts and sometimes glucose, used to maintain cells and tissues alive in vitro for short periods.

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What is serum in cell culture?

A vital component of cell culture media that provides essential nutrients, growth factors, and other components needed for cell growth and function.

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What is serum heat inactivation?

The practice of heating serum at 56°C for 30 minutes to inactivate complement proteins that can harm cells.

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What are essential amino acids in cell culture?

Amino acids that cannot be synthesized by cells and must be added to the culture medium.

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What are non-essential amino acids in cell culture?

Amino acids that can be synthesized by cells but are often added to the medium to ensure their availability.

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What is a complete culture medium?

A specialized type of cell culture medium designed to support the growth of specific cell types.

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What is EMEM?

Eagle’s Minimum Essential Medium; a widely used complete culture medium for mammalian cell culture.

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What is DMEM?

Dulbecco’s Modified Eagle’s Medium; a commonly used complete culture medium, especially for fibroblast and other adherent cell cultures.

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Glutamine/Glutamate in cell culture media

Glutamine and glutamate are often added to cell culture media because they provide a readily available energy source and carbon for cell growth.

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Vitamin types in culture media

Eagle's MEM contains only water-soluble vitamins, while M199 contains both water-soluble and fat-soluble vitamins.

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Advantages of Serum in cell culture

Serum provides essential nutrients, growth factors, hormones, and attachment factors for cell culture.

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Role of Insulin and Transferrin in serum

Insulin promotes cell growth, while transferrin binds iron and helps with cell function.

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Disadvantages of Serum in cell culture

Serum is expensive, can vary from batch to batch, and can be contaminated.

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Advantages of cell culture

Culturing cells allows researchers to control specific conditions and study cell behavior in a controlled environment.

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Studying Cell Culture Conditions

By manipulating the pH, temperature, gas concentrations, and osmotic pressure of the media, researchers can investigate their effects on cells.

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Analyzing cell functions through culture

Cell culture allows for detailed studies of cell metabolism, physiology, and biochemistry.

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Study Notes

Transformation and Immortalization of Cultured Cells

  • Transformation of cultured cells is a spontaneous or induced permanent phenotypic change. This change results from a heritable alteration in DNA and gene expression.
  • Transformation is linked to genetic instability and involves three main phenotypic alterations:
    • Immortalization: The acquisition of an unlimited lifespan,
    • Aberrant growth control: Loss of contact inhibition, density limitation of cell proliferation (and anchorage dependence), and abnormal cell motility.
    • Malignancy: Evidenced by the growth of invasive tumors in vivo. The term "transformation" encompasses all three processes.

Immortalization

  • Immortalized cell lines are either:
    • Tumorous: They don't stop dividing,
    • Artificially manipulated: They can proliferate indefinitely.
  • Immortalized cells continuously divide, filling the culture dish. Scientists "split" or "passage" the cells to create space for continued growth, transferring fractions of proliferating cells to new containers.
  • Common immortalized cell lines include human embryonic kidney 293T (HEK-293T) cells and HeLa cells.

Immortalization: Advantages and Disadvantages

  • Advantages:*

  • Well-characterized and homogeneous, with genetically identical cells, allowing for consistent and reproducible results.

  • Easier to culture than primary cells.

  • Grow quickly and continuously.

  • Disadvantages:*

  • Cannot be considered normal cells in in vivo conditions.

  • Cell characteristics can change and become significantly different from normal cells after several passages.

Primary Cells

  • Advantages:*

  • Similar chromosome number as the parent tissue.

  • Possess unique biochemical properties that reflect the parent tissue (e.g., growth factors and hormone secretions).

  • Serve as excellent experimental models for in vivo situations.

  • Disadvantages:*

  • Finite lifespan and a limited number of cell divisions.

  • Considerable cell-to-cell variability in populations and between preparations.

  • Difficult to maintain and can be challenging to obtain, due mainly to donor availability issues.

Immortalized Cells

  • Advantages:*

  • Exhibit a tendency to grow faster and can reach higher cell densities compared to primary cells.

  • Often characterized by a uniform cell type (mostly clonal).

  • Typically derived from the same donor, decreasing batch-to-batch variation.

  • Maintain most cellular characteristics from their parent cell.

  • Save money and time for prolonged projects.

  • Useful for in vitro experiments.

  • Disadvantages:*

  • May differentiate over time in culture.

  • Cellular behavior in vitro might not perfectly represent in vivo scenarios

  • Potential alterations in cell phenotype or function.

Immortalization with Viral Genes

  • The SV40 LT gene is frequently used to induce immortalization.
  • The LT gene's product (T antigen) interferes with the cell cycle regulation, notably binding to the p53 protein, overriding the cell cycle, which makes the cells proliferate indefinitely.
  • This process extends the proliferative lifespan and restricts the DNA surveillance mechanisms, notably the p53 gene activity, thereby allowing for increased genomic instability and promoting further mutations like telomerase upregulation or downregulation of telomerase inhibitors.

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Description

This quiz explores the processes of cell transformation and immortalization, focusing on the genetic changes that lead to distinct phenotypic characteristics in cultured cells. Understand the implications of these changes in cell biology, particularly in relation to tumor growth and cell proliferation. Test your knowledge on how these concepts are applied in scientific research.

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