BIOL3402 Cell Biology Quiz

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Questions and Answers

What is one primary reason for performing tissue or cell culture?

To study cell physiology (correct)

To create artificial intelligence

To develop new tissues for organ transplants

To increase the size of cells

Which of the following is a key characteristic that differentiates primary cell culture from culture using cell lines?

Primary cells can multiply indefinitely.

Cell lines are derived from tissues while primary cultures are not.

Cell lines are less sensitive to growth factors than primary cells.

Primary cell cultures have a finite lifespan. (correct)

Which method is essential for preserving cell viability during long-term storage?

Cell dissociation

Cell transformation

Cryopreservation (correct)

Cell line expansion

What is the role of media formulation in cell culture?

To provide essential nutrients and support cellular processes Signup and view all the answers

Which process is NOT associated with the modification of cells in culture?

Increased cellular reproduction Signup and view all the answers

Which type of medium is commonly used for a wide variety of cell lines?

Eagle’s Minimal Essential Medium (EMEM) Signup and view all the answers

What is the main purpose of using transformed cells in research?

To facilitate easy manipulation and study of cellular functions Signup and view all the answers

Which component is crucial for energy production during cellular respiration?

Intermediate products Signup and view all the answers

What is the primary function of Sertoli cells in separation methods?

To provide support to germ cells Signup and view all the answers

Which cell type is characterized by a spindle-like shape when attached to a surface?

Fibroblasts Signup and view all the answers

What is the main characteristic of a continuous cell line compared to a primary cell culture?

Unlimited proliferation Signup and view all the answers

Which method can lead to the transformation of normal cells into continuous cell lines?

Spontaneous mutations Signup and view all the answers

Which of the following characteristics is NOT typically associated with transformed cells?

Contact inhibition features Signup and view all the answers

How does the transformation of cells affect their growth behavior?

They lose sensitivity to growth control stimuli Signup and view all the answers

Which cell type is known for not being able to divide in culture?

Nerve cells Signup and view all the answers

What is a feature of primary cell cultures after a few days?

Majority of cells die after some time Signup and view all the answers

Which vitamin is included at a concentration of 10 mM for HeLa cells?

Biotin Signup and view all the answers

What is the concentration of Glutamine provided in mM for HeLa cells?

2 Signup and view all the answers

Which of the following amino acids is provided at a concentration of 0.2 mM?

Valine Signup and view all the answers

What is the role of NaCl in the nutritional medium for HeLa cells?

Osmotic balance Signup and view all the answers

Which essential amino acid has the lowest concentration in HeLa cell culture medium?

Tryptophan Signup and view all the answers

Which of the following is NOT listed as a component in the miscellaneous section for HeLa cells?

Gentamicin Signup and view all the answers

What is the concentration of KCl provided in the HeLa cell culture medium?

5 mM Signup and view all the answers

Which antibiotic is included in the HeLa cell culture medium at a percentage?

Penicillin Signup and view all the answers

What is the main purpose of using perfusion in cell dissociation techniques?

To prevent blood contamination Signup and view all the answers

Which enzyme is a non-specific protease used in the dissociation of cells?

Trypsin Signup and view all the answers

Which of the following is NOT a common attachment factor used in cell culture?

Gelatin Signup and view all the answers

What is the role of DNase in the cell dissociation process?

To disperse DNA released from lysed cells Signup and view all the answers

What are Sertoli cells primarily cultured from after undergoing enzymatic digestion?

Rat testes Signup and view all the answers

What method is used to filter and purify germ cells after mechanical preparation?

Filtering through nylon mesh Signup and view all the answers

In the enzymatic procedure for preparing Sertoli cells, what is the temperature during the incubation step with trypsin and DNase?

37 °C Signup and view all the answers

Which method is primarily used to dissociate spleen and thymus tissue?

A combination of mechanical and enzymatic methods Signup and view all the answers

Which of the following is used to prevent the action of trypsin in the Sertoli cell culture preparation?

Soybean trypsin inhibitor Signup and view all the answers

What type of cells are primarily isolated through the mechanical method from rat testes?

Germ cells Signup and view all the answers

At what rate of cooling is considered moderate during the freezing process?

1‐2 oC/min Signup and view all the answers

What are the critical temperatures for cooling during the freezing process?

‐5 oC to ‐40 oC Signup and view all the answers

Which cryoprotectant is known to be permeable to cells?

Glycerol Signup and view all the answers

What effect can slow warming have during the thawing process?

Recrystallization of intracellular ice Signup and view all the answers

What is the purpose of using a freezing medium containing DMSO?

To prevent dehydration Signup and view all the answers

What should be done with the DMSO containing medium after thawing cells?

Discard it Signup and view all the answers

During the freezing process, what is an important step after resuspending cells in freezing medium?

Aliquoting into freezing vials Signup and view all the answers

What is the recommended temperature for prewarming the medium before thawing cells?

37 oC Signup and view all the answers

What occurs during the Lag Phase in cell culture?

No apparent increase in cell concentration Signup and view all the answers

What is the correct formula to calculate the final cell concentration after a certain number of generations?

N = N0 * 2^x Signup and view all the answers

Which statement describes the Stationary/Plateau Phase in culture?

Cell growth is limited due to nutrient depletion Signup and view all the answers

What happens during the Decline Phase of cell culture?

Cell death exceeds cell growth Signup and view all the answers

Which method utilizes an electric field to fuse cells?

Electrofusion Signup and view all the answers

What is a key application of hybridoma technology?

Antibody production Signup and view all the answers

What does the NeoR gene confer to transfected cells?

Resistance to a specific antibiotic Signup and view all the answers

Which method would be least effective for introducing DNA into cells?

Filtration Signup and view all the answers

What is the main purpose of autoclaving in laboratory settings?

To sterilize and disinfect Signup and view all the answers

Which characteristic is essential for cells produced through cell fusion techniques?

They can produce specific antibodies Signup and view all the answers

What is the typical temperature for a water jacketed CO2 incubator?

37°C Signup and view all the answers

Which method is NOT a form of sterilization or disinfection?

Microwaving Signup and view all the answers

What type of contamination can arise from the source itself, such as the medium or serum?

Biological contamination Signup and view all the answers

Which of the following does NOT play a role in cryopreservation of cells?

Use of G418 Signup and view all the answers

Study Notes

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)

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Description

Test your knowledge on cell biology and technology with this quiz covering media formulation, cell culture techniques, and the characteristics of transformed cells. Designed for students in BIOL3402, this quiz challenges your understanding of key concepts essential for laboratory practices in cell biology.