Cell Biology: Cell Culture

Cell Biology

• The study of cell structure and function, typically focusing on Eukaryotic cells.
• Course topics include cell membrane transport, cellular energetics, protein trafficking/targeting, signal transduction, cell organization/movement, cell cycle, growth/development, the immune system and cancer.

Cell Culture: Primary Cells

• The genome contains instructions that differentiate a fertilized egg into diverse human body cell types.

Cell Culture Environment

• Key factors are temperature, pH, ionic strength, nutrients, special surfaces, and antibiotics.
• Cells placed in incubators maintain consistent temperature, humidity, and gas composition.
• Most mammalian cells are incubated at 37°C.
• 5% CO2 maintains appropriate media pH.

Cell Culture: Cell Lines

• Cultured cells are either primary cells or cell lines.
• Primary cells are directly generated from multicellular organisms.
• Tissue samples are exposed to enzymes that break down cell-cell adhesion
• Tissue is agitated by drawing cells into and out of a pipette.
• Primary cells undergo a limited number of divisions; some do not divide.
• Cell lines originate as primary cells but acquire mutations enabling indefinite growth and survival.
• Transformed cells exhibit cancer cell characteristics.
• Transformed cells grow faster than primary cells and eventually dominate the culture.
• These cells can be extracted and transferred to expand the culture (passaging).
• Cell lines can be frozen for later use (cryopreservation).

Assessing Cell Structure and Function

• Cell structure and function can be experimentally investigated in many ways.
• Course focuses include: Microscopy, Immunocytochemistry, Flow Cytometry, and Western Blotting.

Microscopy

• Enables magnification to observe objects in detail.
• Average cell length is in the tens of microns measure (millionth of a meter)
• Magnification is achieved using glass lenses.
• The objective lens is placed close to the specimen for greatest magnification.
• The projection lens is placed in the eyepiece or near camera.
• Total magnification is calculated by multiplying the magnifications of all lenses.

Microscopy Image Quality

• Affected by contrast, which is the ability to distinguish the sample from the background.
• Quality of magnified image is also affected by resolution, that is the ability to distinguish 2 closely apposed objects.
• Both contrast and resolution is a property of the microscope and cell/tissue

Light Microscopy

• Directed at a specimen to view.
• Brightfield microscopes use white light, containing all wavelengths, to illuminate a specimen.
• Fluorescence microscopes use a single wavelength or a narrow range of wavelengths.

Light Microscopy Orientations

• Light microscopes have upright and inverted orientations
• Upright microscopes have an objective lens above the specimen and shine light from below.
• Inverted microscopes shine light from above, with the objective lens below the specimenand are ideal for viewing cells in culture

Fluorescence Microscopy

• Used to detect fluorescent molecules, known as fluorophores.
• A fluorophore's electron is excited by light of a narrow range of wavelengths and known as excitation wavelengths.
• The excited electron returns to its ground state, emitting another photon.
• The emitted photon has a longer wavelength than the original. and known as emitted light
• A special dichroic mirror in fluorescence microscopes reflects the excitation wavelength towards the specimen and passes the emission wavelength to the eyepiece or camera.

Fluorophores

• Not innately present in cells, so must be experimentally introduced.
• Introduction strategy depends on the experiment's purpose such as using live vs. preserved cells, to determine the location of a protein, identifying cells expressing a specific protein, or observing expression changes over time.

Membrane Permeable Fluorophores

• Can be used in live cells.
• Once inside, the fluorophore is enzymatically cleaved so it can no longer cross the membrane
• Fluorescence changes in response to changes in ion concentration and voltage.

Fluorescently Tagged Recombinant Proteins

• The gene encoding a specific protein is modified to produce the normal protein fused to a fluorescent protein, for example the Green Fluorescent Protein (GFP).
• Can be used on live or preserved cells
• Determines which cells are expressing a given proteins, when it is expressed and where it is located in the cell