BIOL3402 Cell Biology Quiz

Course Information

• Course title: BIOL3402 Cell Biology and Cell Technology
• Instructor: Dr. W. Y. Lui
• Office: Rm4N09 KBSB
• Email: [email protected]
• Lectures: 6

Learning Outcomes

• Describe and explain media formulation
• Explain cell dissociation and separation techniques
• Distinguish different types of cell cultures (primary vs. cell lines)
• Describe the characteristics of transformed cells
• Explain methods for modifying cells in culture and selection methods
• Explain how cell modifications in culture help answer scientific questions
• Describe essential laboratory facilities for cell culture
• Describe cryopreservation methods

Why Tissue/Cell Culture?

• Studying cell physiology
• Synthesizing valuable cell products (e.g., recombinant proteins via gene cloning and cell culture)

Metabolism

• Food (polymeric molecules) → Cellular molecules (small) → Energy → Cellular macromolecules → Cellular structure → Cellular functions
• Energy enables cellular physiology, biosynthesis, and biomolecule activation. Intermediate compounds are cellular molecules in energy pathways.

Obtaining Energy

• Stage 1: Breakdown of large macromolecules (proteins, polysaccharides, fats) into simple subunits (amino acids, simple sugars, fatty acids and glycerol)
• Stage 2: Breakdown of simple subunits to acetyl CoA, accompanied by production of limited ATP and NADH
• Stage 3: Complete oxidation of acetyl CoA to H2O and CO2, accompanied by production of large amounts of NADH and ATP, producing reducing power as NADH and ATP.

Intermediate Products in Cellular Respiration

• Intermediate products from cellular respiration are precursors for essential molecules synthesis

Culture Medium (Eagle's Minimal Essential Medium (EMEM, MEM))

• Used for various cell lines, such as HeLa cells
• Components include: glucose, 13 essential amino acids, vitamins, salts, and antibiotics. Additional components such as serum (e.g., FBS) might be needed.

Culture Medium (Ham's F12)

• Satisfies fastidious requirements of some cell lines
• Basic components of MEM plus: pyruvate, 7 non-essential amino acids, trace elements (e.g., Fe++, Cu++), and organic compounds (e.g., adenine, thymidine, hypoxanthine).

Culture Media Disadvantages of Serum-Supplemented Media

• Chemically undefined, inconsistent results between batches
• Expensive
• Frequently contaminated with viruses

Culture Media From Serum-Supplemented Media to Serum-Free Media

• Supplements of growth factors replace serum
• Not all cell lines can grow in serum-free conditions

Growth Factors-Supplemented Serum-Free Media

• Used to study endocrine regulation and simplify product purification
• Growth factors often increase cell growth of specific cell types
• Growth factors are used typically at low concentrations

Common Supplements for Most Cell Types

• Insulin for glucose uptake and amino acid metabolism
• Transferrin for iron transport
• Epidermal growth factor (EGF) for epithelial and fibroblastic cell growth promotion/mitogenesis
• Other growth factors (e.g., NGF, TGF) for specific cell types

Attachment Factors/Extracellular Matrix

• Required for some cells to adhere and attach to substrata (plastic culture dishes) in serum-free conditions
• Mimic in vivo cell attachment situations
• Common attachment factors include collagen, fibronectin, laminin, and poly-lysine.

Cell Dissociation and Separation Techniques

• Perfusion (e.g., for liver and kidney) for blood contaminant removal
• Mechanical dissociation (e.g., for spleen and thymus)
• Enzymatic dissociation (e.g., trypsin, collagenase, or hyaluronidase) is often used for tissue culture

Enzymatic Digestion

• Trypsin: Non-specific protease; cleaves peptide bonds on the carboxyl side of lysine and arginine residues
• Collagenase and hyaluronidase: Digest extracellular matrix glycoproteins like collagen
• DNase: Disperses DNA from lysed cells

Isolation of Cells (testicular cells and germ cells)

• Different isolation methods exist for different cell types. Details of these procedures have been outlined in the notes.

Cell Counting Methods

• Coulter Counter
• Hemocytometer

Five Cell Types

• Epithelial cells (polygonal/rectangular, layer cells of organs)
• Fibroblasts (spindle-shaped, attach to surfaces)
• Muscle cells (myoblasts can differentiate, and form myotubes that further differentiate into muscle fiber)
• Nerve cells (highly differentiated, do not divide)
• Blood cells (culture in suspension, e.g., lymphocytes)

From Primary Cell Culture to Cell Lines

• Primary cell culture: very few cells
• Continuous cell lines are established from cells taken directly from animal tissue; freshly isolated in cell culture.
• Many cells die in days/weeks

Transformation

• Cells lose sensitivity to growth control stimuli

Characteristics of Transformed Cells

• Loss of anchorage dependence (can grow without being attached to a surface)
• Lack of contact inhibition (overcrowded cells continue to grow)
• Chromosome fragmentation
• Alteration in chromosomal state (e.g., increase in chromosome number)
• High growth capacity

Obtaining Cell Lines

• ATCC (American Type Culture Collection)
• ECACC (European Collection of Animal Cell Culture)

Growth Parameter

• Cell line obtained from the culture collection
• Cells inoculated in growth medium; Cells stop growing if nutrient is limited, toxic metabolite accumulated, or lacking growth surface
• Sub-culturing/passaging: trypsinization/diluting, and transferring cells to fresh medium

Four Phases of a Culture

• Lag phase: no apparent increase in cells (synthesis of growth factors, duration varies)
• Log phase: exponential increase in cell amount
• Stationary phase: no further increase in cells (cell death = cell growth)
• Decline phase: exceeds cell growth; death associated with toxic products (apoptosis/necrosis)

Modifications of Cells in Culture

• Cell fusion: production of somatic cell hybrid; combine characteristics of different cells; fusion techniques e.g., PEG; electric currents (fusion of antibody-producing B lymphocytes with myeloma cells)
• Expression of cloned genes: DNA transfer through methods like calcium phosphate precipitation, DEAE-dextran, liposomes, microinjection, or electroporation; or infection of cells with viruses.

Selection System

• Select successful hybridomas or clones from a population; selection markers
• Selectable markers for hybridomas include Thymidine kinase (TK), Hypoxanthine-guanine phosphoribosyltransferase (HGPRT), and neomycin resistance genes

Two Pathways in Nucleotide Biosynthesis

• De novo biosynthesis (folic acid, dihydrofolate, x-aminopterin, tetrahydrofolate)
• Salvage pathway (hypoxanthine and thymine)

Selection System for Hybridomas

• Preselection of B-lymphocytes with bromodeoxyuridine and myelomas with 8-azaguanine
• Fusion of cells to form a mixed population (lymphocytes, myeloma, and hybridoma)
• HAT selection for specific hybridomas

Neomycin Resistance Gene (NeoR)

• Confer resistance to aminoglycoside antibiotics (e.g., G418)
• Cells carrying NeoR gene can replicate in G418 medium
• G418 prevents the replication of mammalian cells

Stable Transfection

• Cloning gene of interest into a vector containing NeoR genes
• Generating the "kill curve" to determine the G418 dosage for selection
• Transfection, selection, subculture, and checking for gene overexpression with PCR/western blot

Laboratory Facilities

• Water source, glassware/plasticware, laminar flow hoods, CO2 incubators, microscope, sterilization methods, chemicals, and contamination sources

Cryopreservation and Freezing and Thawing of Cells

• Storage at low temperatures (e.g., liquid nitrogen, -196 °C), moderate cooling, rapid warming
• Physical changes during freezing (dehydration and intracellular ice formation)
• Cryoprotectants (e.g., glycerol, DMSO)
• Cryo-injuries from low thawing rates
• Procedure for freezing and thawing cells (freezing, thawing)