Cell Culture Quiz: Master Techniques and Methods

Cell culture is the process of growing prokaryotic, eukaryotic, or plant cells under controlled conditions.
Tissue culture is the removal of cells, tissues, or organs from an animal and placing them into an artificial environment.

Primary culture cells are obtained by surgically or enzymatically removing cells from an organism and placing them into a suitable culture environment.
Stages of obtaining primary culture: acquisition of the sample, isolation of the tissue, dissection and/or disaggregation, and culture after seeding into the culture vessel.

A cell line is obtained after the first subculture or passage of primary culture cells.
Finite cell lines have a limited lifespan and may die out or acquire a stable, heritable mutation that gives rise to a continuous cell line.

Subculturing involves replacing the old culture medium with fresh medium and transferring some of the cells to a new vessel to promote further growth.
Enzymes such as trypsin in combination with EDTA break the cellular "glue" that attaches cells to the surface, allowing them to be subcultured.

The growth cycle consists of three phases: lag phase, log phase, and plateau phase.
The lag phase is a period of adaptation during which the cell replaces elements lost during trypsinization and attaches to the surface.
The log phase is the period of increase in cell number, and the plateau phase is when the culture becomes confluent.

Cell counting is done using a Burker chamber or a hemocytometer.
The cell suspension is mixed and a sample is placed in the counting chamber, and the cells are counted under a microscope.

Cells should be frozen slowly to allow water to leave the cell and to minimize ice crystal growth.
Cryoprotectants such as DMSO are used to protect biological tissue from freezing damage.
Cells should be thawed rapidly to minimize intracellular ice crystal growth during the warming process.

A certified biological safety cabinet to protect both the cells and the worker from biological contaminants.
A centrifuge to centrifuge the cells.
A microscope for examination of cell cultures and for counting cells.
A humidified incubator set at 37°C with 5% CO2 in air.

Fetal Bovine Serum (FBS) is commonly used to promote cellular multiplication.
L-Glutamine is an essential amino acid required by most mammalian cells grown in culture.
Antibiotics may be added to prevent contamination by microorganisms.

Different types of cells have different growth requirements, and a number of chemically-defined formulations have been developed.
The various nutrients present in medium include glucose, fats and fatty acids, lipids, phospholipids, and sulpholipids, ATP and amino acids, vitamins, and minerals.

Prepared medium should be stored at 4°C and warmed up to 37°C only for the time necessary to perform a given experiment.

Disposable polystyrene vessels are commonly used for tissue culture work.
Some cell lines require further treatment of the growth surface before they will attach and proliferate, such as coating the surface with poly-L-lysine.### Cell Culture Basics
Mycoplasmas are the smallest free-living organisms and can contaminate cell lines, which can be screened using the fluorochrome DAPI and detected using a fluorescent microscope.
Cross-contamination with an existing continuous cell line or mislabeling can occur, and HeLa cells have contaminated 10-20% of all cell lines currently in use.
Cell authentication is essential to ensure that the cells being worked on are not cross-contaminated.

Cell culture provides an excellent model system for studying:
- Normal physiology, cell biology, and biochemistry of cells
- The effect of drugs, radiation, and toxic compounds on cells
- Carcinogenesis and mutagenesis
Cell culture is used in drug screening and development, and large-scale manufacturing of biological compounds (e.g., blood clotting factors, tissue plasminogen activator, interferons).

Advantages:
- Controlled physico-chemical environment (e.g., pH, temperature)
- Homogeneous genetic population
- Reagent saving: reduced volumes, direct access to cells
- Reduction of animal use (e.g., for cytotoxicity and screening of pharmaceutics, cosmetics)
Limitations:
- Sterile handling
- Risk of chemical, microbial, and cross-contamination
- May not represent in vivo phenotype/genotype
- Continuously growing cells often show genetic instability and may lose differentiated characteristics

Fibroblasts:
- Largest group of connective tissue cells
- Spindle-shaped cells of mesenchymal origin
- Synthesize proteins of extracellular matrix (e.g., collagen, fibronectin, laminin)
- Produce matrix metalloproteinases and growth factors
3T3L1 mouse fibroblasts:
- Isolated from the embryo of a mouse
- Can be used to study cellular mechanisms associated with diabetes, obesity, and related disorders
A375 human melanoma cells:
- Adherent melanoma cell line derived from the primary tumor
- Cells demonstrate epithelial morphology

Qualitative techniques:
- Immunocytochemistry
- Detection of apoptosis and necrosis
Quantitative techniques:
- Metabolic activity (e.g., cytotoxicity tests like XTT)
- Western blot from cells lysates
- Flow cytometric methods (e.g., determining the phase distribution of the cell cycle)

Cytotoxicity is a complex event in vivo, associated with direct cellular damage, physiological effects, and systemic effects
In vitro assays determine effects at the cellular level, called cytotoxicity
Cytotoxicity assay: XTT test measures the number of viable cells present compared to positive (toxin) and negative (vehicle) control treatments

The cell cycle is divided into four phases: G1, S, G2, and M
Progression around the cycle is driven by cyclins interacting with CDC kinases
Flow cytometric analysis of cell cycle: DNA content of cells can be stained by DNA binding dyes, which bind proportionally to the amount of DNA present in the cell

Immunofluorescent staining makes use of antibodies to locate and identify patterns of protein expression in cells
Primary antibody binds to antigen, and a secondary antibody conjugated to a fluorochrome binds to the primary antibody
Fluorochrome emits light at its own characteristic wavelength (fluorescence) and allows detection of antigen-antibody complexes

Cell fixation: using formaldehyde or methanol
Cell permeabilization: using Triton X-100
Immunofluorescent cell staining: incubating cells with a primary antibody, secondary antibody, and fluorochrome

Filamentous actin: an element of cell cytoskeleton, visualized using phalloidin
Nuclei: stained using Hoechst, a blue fluorescent dye that binds to DNA
Cortactin: a protein that promotes the polymerization and rearrangement of the actin cytoskeleton