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Questions and Answers
What is the primary purpose of using enzymatic separation methods in cell isolation?
What is the primary purpose of using enzymatic separation methods in cell isolation?
Which cell type is characterized by its spindle-like shape and attachment to solid surfaces?
Which cell type is characterized by its spindle-like shape and attachment to solid surfaces?
Which technique allows for the counting of cells in a suspension?
Which technique allows for the counting of cells in a suspension?
What differentiates a continuous cell line from primary cell culture?
What differentiates a continuous cell line from primary cell culture?
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How can transformation of normal cells occur?
How can transformation of normal cells occur?
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Which characteristic is NOT typically associated with transformed cells?
Which characteristic is NOT typically associated with transformed cells?
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What type of cells can be cultured in suspension?
What type of cells can be cultured in suspension?
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What defines the morphological appearance of epithelial cells?
What defines the morphological appearance of epithelial cells?
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What is the most likely reason cells stop growing during culture?
What is the most likely reason cells stop growing during culture?
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Which method is utilized to separate adherent cells from their culture surface?
Which method is utilized to separate adherent cells from their culture surface?
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What is a critical step when sub-culturing cells?
What is a critical step when sub-culturing cells?
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Which statement about germ cell types is correct?
Which statement about germ cell types is correct?
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Which organization provides a stock of commonly used cell lines?
Which organization provides a stock of commonly used cell lines?
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What is the primary role of enzymatic digestion in cell preparation from testes?
What is the primary role of enzymatic digestion in cell preparation from testes?
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Which substance is primarily responsible for digesting collagen in the enzymatic preparation of Sertoli cells?
Which substance is primarily responsible for digesting collagen in the enzymatic preparation of Sertoli cells?
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What is the end product after isolating cells via mechanical methods from testes?
What is the end product after isolating cells via mechanical methods from testes?
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Which of the following attachment factors is derived from human blood plasma?
Which of the following attachment factors is derived from human blood plasma?
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At which step is trypsin used in the preparation of Sertoli cells?
At which step is trypsin used in the preparation of Sertoli cells?
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What type of cells can be purified using the mechanical method described?
What type of cells can be purified using the mechanical method described?
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Which method is commonly used for cell dissociation in tissues like spleen and thymus?
Which method is commonly used for cell dissociation in tissues like spleen and thymus?
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What is the purpose of DNase in the isolation of Sertoli cells?
What is the purpose of DNase in the isolation of Sertoli cells?
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What are the primary germ cells purified through the mechanical method described?
What are the primary germ cells purified through the mechanical method described?
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In serum-free conditions, which medium is typically used to resuspend Sertoli cells?
In serum-free conditions, which medium is typically used to resuspend Sertoli cells?
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Study Notes
Course Information
- Course Title: BIOL3402 Cell Biology and Cell Technology
- Instructor: Dr. W.Y. Lui
- Lectures: 6
- Office: Rm4N09 KBSB
- Email: [email protected]
Learning Outcomes
- Describe and explain media formulation
- Explain cell dissociation and separation techniques
- Distinguish different types of cell culture (primary vs. cell lines)
- Describe the characteristics of transformed cells
- Explain methods for modifying cells in culture and selection methods
- Explain how cell modification in culture helps answer scientific questions
- Describe essential laboratory facilities for cell culture
- Describe the method of cryopreservation
Why Tissue/Cell Culture?
- Study cell physiology
- Synthesize valuable cell products (e.g., recombinant protein)
Metabolism
- Food (polymeric molecules) are broken down into cellular molecules (small molecules).
- Energy is needed for cellular physiology, biosynthesis, and activation of biomolecules.
- Intermediate compounds in energy pathways are cellular molecules.
Obtaining Energy
- Stage 1: Breakdown of large macromolecules (proteins, polysaccharides, fats) into simple subunits (amino acids, simple sugars, fatty acids)
- Stage 2: Breakdown of simple subunits to acetyl CoA, producing limited ATP and NADH
- Stage 3: Complete oxidation of acetyl CoA to H2O and CO2, producing large amounts of NADH and ATP
Intermediate Products in Cellular Respiration
- Intermediate products serve as precursors for essential molecules (e.g., cell walls, membrane lipids, proteins, etc.)
Culture Medium (EMEM, MEM)
- Used for a wide variety of cell lines (e.g., HeLa cells)
- Contains glucose, amino acids, vitamins, and salts
- May include antibiotics and serum (e.g., FBS)
- Various formulations exist, like Ham's F12, customized with required growth factors for consistency and study specific requirements.
Culture Medium (Disadvantages of Serum-Supplemented Media)
- Serum is chemically undefined, leading to inconsistent results between batches.
- Serum is expensive, accounting for a significant portion of media costs.
- Serum-containing media is frequently contaminated by viruses.
- Serum-supplemented media is frequently contaminated by viruses.
Growth Factors-Supplemented Serum-Free Media
- Growth factors are polypeptides that promote cell growth, not based on their nutrition.
- They are essential for certain cell types but not involved in metabolic pathways.
- They can achieve significant effects at very low concentrations.
Common Supplements
- Insulin (glucose uptake and metabolism, amino acid uptake)
- Transferrin (iron-carrying protein)
- Epidermal growth factor (mitogen for epithelial and fibroblastic cells)
- Other growth factors (e.g., Nerve growth factor (NGF), Transforming growth factor (TGF))
Attachment Factors/Extracellular Matrix
- Required by some cells to attach to the substratum (plastic culture dish).
- Mimic in vivo conditions
- Common factors include Collagen, Fibronectin, Laminin, and Poly-lysine.
Cell Dissociation and Separation Techniques
- Perfusion: Removing blood from tissues (e.g., liver, kidney).
- Mechanical methods (e.g., spleen, thymus): Dissociating tissues physically.
- Enzymatic methods (e.g., Trypsin, collagenase, hyaluronidase): Dissociating tissues using enzymes
Isolation of Testicular Cells
- Methods outline steps for isolating cells from rats
Cell Counting
- Coulter Counter: Measures cell volume by counting the electrical changes.
- Hemocytometer: Manually counts cells in a gridded chamber.
Five Cell Types from Animal Tissue
- Epithelial (layer, polygonal/rectangular)
- Fibroblasts (spindle-shaped)
- Muscle (capable of fusion to muscle fiber)
- Nerve (highly differentiated, do not divide)
- Blood (suspension culture)
From Primary Cell Culture to Cell Lines
- Primary culture: Cells taken directly from tissue, limited life span.
- Continuous cell line: Immortalized and maintain continuous growth through sub-culture.
Transformation of Cells
- Transformation: Normal cells lose sensitivity to growth control stimuli;
- Transformation enables cells to become immortal.
- Spontaneous transformation, Mutagens, viral infections are the cause of immortality.
Characteristics of Transformed Cells
- May lose anchorage dependence
- May lose contact inhibition (uncontrolled growth)
- Chromosome fragmentation and alterations from the normal diploid state
- High capacity for increased growth
Where to Obtain Cell Lines
- American Type Culture Collection (ATCC)
- European Collection of Animal Cell Culture (ECACC)
Growth Parameter
- Inoculate cells into growth medium, at optimal cell density.
- Subculturing (passaging) is necessary when cells stop growing due to resource depletion & other factors.
Four Phases of a Culture
- Lag phase: Initial phase, no apparent cell increase.
- Log/Growth Phase: Exponential increase in cells, used to calculate doubling time.
- Stationary/Plateau Phase: No further increase, cell growth equal to cell death.
- Decline Phase: Viable cell concentration decreases.
Cryopreservation and Freezing and Thawing of Cells
- Storage at very low temperatures.
- Methods for successful cell storage and thawing.
Laboratory Facilities
- Water source (distilled/purified)
- Glassware/Plasticware (sterilization/recycling)
- Laminar Flow Hood (horizontal/vertical)
- CO2 (water-jacketed) Incubators
- Microscopes (standard/inverted)
- Other equipment like sterilizers, filtration systems, chemicals and methods of contamination avoidance
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