Untitled Quiz

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