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Questions and Answers
What is the primary purpose of using special media in cell culture?
What is the primary purpose of using special media in cell culture?
Which of the following tasks is NOT involved in maintaining a cell culture?
Which of the following tasks is NOT involved in maintaining a cell culture?
What is a key difference between mammalian and bacterial cell cultures?
What is a key difference between mammalian and bacterial cell cultures?
Which of the following is an application of cell line culture?
Which of the following is an application of cell line culture?
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What is essential for maintaining a sterile environment in a cell culture laboratory?
What is essential for maintaining a sterile environment in a cell culture laboratory?
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What is the primary function of the CO2 incubator in a tissue culture laboratory?
What is the primary function of the CO2 incubator in a tissue culture laboratory?
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Which type of laminar flow hood provides airflow in a vertical direction?
Which type of laminar flow hood provides airflow in a vertical direction?
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What is the purpose of using 70% ethanol in a tissue culture hood?
What is the purpose of using 70% ethanol in a tissue culture hood?
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How long should a laminar flow hood be turned on before starting work?
How long should a laminar flow hood be turned on before starting work?
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Which type of microscope is commonly used for visualizing cells in culture?
Which type of microscope is commonly used for visualizing cells in culture?
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Which of the following is NOT a rule for maintaining a tissue culture hood?
Which of the following is NOT a rule for maintaining a tissue culture hood?
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What is the recommended temperature setting for CO2 incubators?
What is the recommended temperature setting for CO2 incubators?
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What is the effect of UV light in a tissue culture hood?
What is the effect of UV light in a tissue culture hood?
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What is a disadvantage of isolating cells for culture?
What is a disadvantage of isolating cells for culture?
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What is a distinguishing feature of adherent cells in culture?
What is a distinguishing feature of adherent cells in culture?
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What type of cells can grow without the need for attachment?
What type of cells can grow without the need for attachment?
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Which advantage is associated with immortalized cell lines?
Which advantage is associated with immortalized cell lines?
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What is one key component of cell culture media that supports cell growth?
What is one key component of cell culture media that supports cell growth?
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What is a disadvantage of using foetal calf/bovine serum in cultures?
What is a disadvantage of using foetal calf/bovine serum in cultures?
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During which phase of growth curve is there enhanced cellular activity with no apparent cell increase?
During which phase of growth curve is there enhanced cellular activity with no apparent cell increase?
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What is one of the main roles of growth factors in cell culture?
What is one of the main roles of growth factors in cell culture?
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What is the primary purpose of using 70% ethanol in a laboratory setting?
What is the primary purpose of using 70% ethanol in a laboratory setting?
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Which type of culture is obtained directly from tissues following enzymatic dissociation?
Which type of culture is obtained directly from tissues following enzymatic dissociation?
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What is the maximum number of times finite cell cultures can divide before their growth rate declines?
What is the maximum number of times finite cell cultures can divide before their growth rate declines?
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Why should sterile pipettes be used only once?
Why should sterile pipettes be used only once?
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Which practice is recommended to minimize contamination during sterile procedures?
Which practice is recommended to minimize contamination during sterile procedures?
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What should be done immediately after an accidental spillage in a sterile workspace?
What should be done immediately after an accidental spillage in a sterile workspace?
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What is a characteristic of continuous cell cultures?
What is a characteristic of continuous cell cultures?
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Which statement is true about the sharing of bottles of media and reagents?
Which statement is true about the sharing of bottles of media and reagents?
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What is a characteristic feature of the log phase in cell culture?
What is a characteristic feature of the log phase in cell culture?
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Which factor does NOT affect the duration of the log phase?
Which factor does NOT affect the duration of the log phase?
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What is one purpose of passaging cells in culture?
What is one purpose of passaging cells in culture?
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Why is cryopreservation important in cell culture?
Why is cryopreservation important in cell culture?
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What is the primary function of DMSO in cryopreservation?
What is the primary function of DMSO in cryopreservation?
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Which of the following describes a cell culture contaminant?
Which of the following describes a cell culture contaminant?
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What is crucial for maintaining sterile conditions during bacterial cell culture?
What is crucial for maintaining sterile conditions during bacterial cell culture?
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What happens to cells once they reach the stationary phase?
What happens to cells once they reach the stationary phase?
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Study Notes
Biotechniques (BMS 34010A)
- Course offered Fall semester 2024-2025
- Instructor: Dr. Tania Tahtouh
- Email: [email protected]
Cell Culture Techniques
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Learning Outcomes:
- Describe cell culture tasks (media prep, maintaining animal cells, contamination control, cryopreservation).
- Differentiate between mammalian and bacterial cultures.
- Identify and resolve common cell culture problems.
- Utilize Gibco Cell culture Virtual Lab (https://www.thermofisher.com/ae/en/home/global/forms/cell-culture-basics/cell-culture-basics-virtual-lab.html)
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What is Cell Culture?:
- Cells grown in artificial environments (plastic/glass) in incubators at body temperature.
- Special media (nutrients) allow cell growth and division.
- In vivo (in the body); In vitro (in lab conditions).
Applications of Cell Lines
- Studying cells: Normal, pathological, isolation.
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Biological products:
- Viral vaccines
- Antibodies
- Therapeutic proteins
- Recombinant proteins
- Drug testing: Metabolism, cytotoxicity, artificial tissue generation (e.g., skin).
- Stem cell therapy: Treating diseases.
- Gene function study.
- Genetic engineering.
Setting up a Cell Culture Laboratory
- Sterile environment: Reagents, media, and consumables in designated tissue culture zones.
- Aseptic technique: Essential at all times.
- Limited entry/exit: Freezers, centrifuges, and storage areas for medium and supplements.
- Routine maintenance: Daily cleaning of surfaces and immediate waste disposal.
Equipment for Tissue Culture Laboratory
- Tissue culture hood: Cell handling in controlled environments
- CO2 incubators: Maintaining optimal cell growth conditions, precisely controlled temperature (37°C) and CO2 levels (5% or 10%). Frequent cleaning is crucial.
- Microscopes: Visualizing cell morphology and their state.
- Centrifuges: Necessary for cell separation.
- Water baths: Thawing frozen cells and warming media.
- Fridges and freezers: Storage for media and other materials.
Cell Culture Hoods
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Laminar flow hoods: Create sterile airflow, preventing contamination.
- Types: Vertical and horizontal.
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Three Classes:
- Class I: Basic protection for the operator.
- Class II: Most common, providing protection for both the operator and the cells.
- Class III: Highest level, completely sealed, for highly pathogenic materials.
Cell Culture Hood Rules
- Cleanliness: Necessary to prevent contamination.
- Disinfectant: 70% Ethanol used for cleaning and disinfecting.
- Maintenance: Routinely running, only turning off for extended periods.
- Air stabilization: Allow 10 minutes for the air to stabilize after turning on.
- HEPA filters: High-efficiency particulate air filters to remove contaminants.
- UV light: Avoid using during cell culture.
CO2 Incubators
- Function: Controlling pH (7.2-7.4) by regulating CO2 levels (5% or 10%).
- Temperature: Maintain at 37°C.
- Cleaning: Crucial for preventing contamination.
Microscope
- Inverted phase-contrast: Routinely used for visualizing cells in culture.
- Location: Light source above, objective lenses below the specimen.
- Information: Provides useful information about cell morphology and state.
Aseptic Handling
- Handwashing: Essential before, during, and after handling cells.
- Surface disinfection: Disinfect work areas and equipment with 70% ethanol.
- Equipment use: Minimize cross-contamination by using different pipettes for each task.
- Arrangement: Optimal organization and accessibility of equipment.
- Handling media: Sterile techniques must be followed when working with media.
- Openings facing down and caps on: To prevent contamination.
Aseptic Techniques
- Crucially important to prevent microbial contamination of cultures and cross-contamination of cell cultures.
Types of Cell Culture
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Primary cultures: Derived from fresh tissues, maintain donor tissue characteristics. Limited lifespan. Labor intensive and produce a heterogeneous population of cells.
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Secondary cultures: Established from primary cultures.
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Continuous cultures: Immortalized cells, capable of infinite growth.
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Finite cultures: Have a limited lifespan.
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Indefinite cultures: Have the ability to proliferate indefinitely
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Both are useful for research purposes.
Continuous Cultures
- Single cell: That maintains the ability to continue growing indefinitely.
- Immortalization: Spontaneous genetic mutation or from transformation vectors.
- Disadvantages: Lose some original in vivo characteristics.
- Advantages: Fewer serum requirements and a shorter doubling time for growth. Can grow without attachment to the flask surface.
Systems for Growing Cells in Culture
- Adherent cells (anchorage dependent): Require attachment to a solid substrate. These cells are immobile and grow in monolayers.
- Suspension cells (anchorage independent): Do not require attachment grow in a fluid culture environment.
Examples of Cell Lines
- Multiple cell lines from commercial sources, such as the European Collection of Animal Cell Cultures (ECACC), with specific morphologies and origins.
Cell Morphologies
- Cell shapes vary based on cell type (fibroblastic, endothelial, epithelial, neuronal).
Cell Culture Media
- Nutrients: Amino acids, carbohydrates, vitamins.
- Inorganic salts: Minerals (Magnesium, sodium, potassium, etc.)
- pH indicator (phenol red).
- Antibiotics, Antimycotics: Controlling microbial growth (penicillin, etc.).
- Buffers (bicarbonate, HEPES): Maintaining pH.
- Supplements: Growth factors, hormones, non-essential amino acids, etc.
Fetal Calf/Bovine Serum
- Function: Provides essential growth factors and hormones.
- Advantages: Enhances cell attachment.
- Disadvantages: Risk of infectious agents, variable composition, and expense.
How to Culture Cells
- Reviving frozen cells
- Isolating tissues
- Aseptic culturing (maintaining the cultures)
- Cell passaging (multiplying)
- Counting cells
- Cryopreservation
Growth Curve
- Lag phase: Early period of cellular activity, preparatory phase.
- Log phase: Exponential cell division.
- Stationary phase: Population growth plateaus due to external factors
- Decline phase: Population starts declining.
Passaging Cells
- Purpose: Maintaining cells in culture and increasing cell numbers.
- Procedure: Removing medium, washing with PBS, using trypsin and EDTA, and then transferring to a new flask.
Cell Counting
- Manual method (haemocytometer): Directly counting cells using a specialized counting chamber.
- Automated method (automated cell counters): Utilizing various instruments for automated cell counting
- Relevant formulas for calculation.
Cryopreservation
- Purpose: Maintaining a cell population for later use.
- Mechanism: Using cryoprotectants to prevent ice crystal formation.
- Procedure: Freezing and storing cells at ultra-low temperatures and then thawing it for later use.
Source of Contamination
- Potential sources of chemical and biological contamination in cell cultures (media, serum, water, detergents, bacteria, viruses, and fungi)
Bacterial Cell Culture
- Safety: Aseptic techniques, protective measures required, sterilizing instruments.
- Culture procedures: Utilizing liquid and solid culture media.
Plating Techniques
- Serial dilutions: Creating a dilution series for determining the number of bacteria per unit volume for cultures.
- Colony counts: To determine bacteria/ml ratios in the sample
- Critical calculations related to serial dilutions.
DNA Transfer into Cells
- Transformation (prokaryotes): Non-viral methods
- Transduction (prokaryotes and eukaryotes): Viral methods, vectors
- Transfection (eukaryotes): Non-viral methods.
DNA Cloning
- Technique for creating copies of DNA sequences.
- Cloning overview, overview of the components
- Cloning Steps and Diagram discussion
Extraction of Plasmids
- Isolating and purifying plasmid DNA from bacterial cells, based on differential characteristics.
References
- Given reference, Sanders ER ( 2012), Aseptic laboratory techniques: plating methods. J Vis Exp.
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Description
Test your knowledge on cell culture techniques including media preparation, contamination control, and cryopreservation. This quiz will help differentiate between mammalian and bacterial cultures while addressing common issues faced in cell culture. Dive into the applications and significance of cell lines in biological studies.
