Biotechniques BMS 34010A: Cell Culture Quiz

Biotechniques (BMS 34010A)

• Course offered Fall semester 2024-2025
• Instructor: Dr. Tania Tahtouh
• Email: [email protected]

Cell Culture Techniques

• 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)

• 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.
• 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

• Laminar flow hoods: Create sterile airflow, preventing contamination.

  • Types: Vertical and horizontal.

• 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

• Primary cultures: Derived from fresh tissues, maintain donor tissue characteristics. Limited lifespan. Labor intensive and produce a heterogeneous population of cells.

• Secondary cultures: Established from primary cultures.

• Continuous cultures: Immortalized cells, capable of infinite growth.

• Finite cultures: Have a limited lifespan.

• Indefinite cultures: Have the ability to proliferate indefinitely

• 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.