BIOL 4426: CELLULAR PHYSIOLOGY - ELECTRONIC LECTURE NOTES - PART II. METHODS FOR THE STUDY OF EUKARYOTIC CELLS
A - Observation of Eukaryotic Cells (Microscopy)
1. Brightfield Microscopy - Compound Brightfield Microscope
Visible Light: Light passes through the object, creating a light field with a darker sample.
Magnification: Achieved by two lens sets: Ocular and Objective.
Resolution: Approximately 0.2 \mu m (the smallest distance between two points that can be distinguished). This is limited by the physics of optics.
Maximum effective magnification is typically 1000-2000X.
Contrast Problem:
Optical Enhancement: Utilizes a condenser and an iris diaphragm.
Chemical Enhancement (Staining Techniques):
Fixation: Preserves cellular structure by killing the cells.
Dyes: Used for staining specific cellular structures.
Example: Hematoxylin-Eosin (H&E) is the most commonly used histological staining procedure.
Histochemical Staining: Detects specific enzymes using chromogenic substrates.
Immunohistochemical Staining (IHC): Detects specific proteins via antibodies conjugated with an enzyme.
Thickness Problem:
Sectioning: Achieved using a microtome.
This involves embedding the sample in resin and then cutting thin sections with a knife.
2. Fluorescence Microscopy - Fluorescent Staining
Fluorescent Dyes (Fluorophores):
Absorb light at one wavelength (color 1).
Emit light at a different, longer wavelength (color 2).
Examples: Fluorescein (emits green light), Rhodamine (emits red light).
Immunofluorescence Microscopy (IF):
Detects a target antigen using a labeled fluorescent antibody.
Offers high sensitivity and good resolution.
Examples: IF Glut2, Triple IF for Tubulin/Actin/Intermediate-Filaments.
Confocal Microscopy and Deconvolution Fluorescence Microscopy:
Both provide sharper images, even with relatively thick samples.
Confocal Microscopy: A laser beam is focused on a very thin plane within the sample.
Example: Alpha tubulin in a sea urchin egg.
Deconvolution Microscopy: Several images are captured at different focal planes and then mathematically analyzed to improve sharpness.
Example: Microtubules, microfilaments, and nuclei in macrophages.
Dynamic Fluorescence Microscopy of Living Cells (Live-Cell Imaging):
Enables the detection of molecules within living cells.
Examples:
Fluorescent detection of Ca^{2+} ions using Fura-2.
Observation of leukocytes with chemoattractants.
Fluorescent detection of GFP/CFP/YFP fusion proteins (chimeras), such as a Purkinje neuron with GFP.
Visualization of protein-protein interactions using FRET (e.g., rac + partner in fibroblast).
Observation of transgenic embryos of mice.
3. Phase Contrast & Nomarsky Interference (Differential Interference Contrast - DIC) Microscopy
These microscopes provide high contrast for living, unstained samples.
They utilize refraction and diffraction light phenomena (optics) to enhance contrast.
Example: Macrophages observed with Brightfield (BF), Phase Contrast (PC), and DIC microscopy offer differing levels of contrast for the same sample.
4. Transmission Electron Microscopy (TEM)
Provides very high resolution images of internal cellular structures.
Principle: Uses an electron beam (instead of visible light) to achieve significantly higher resolutions.
Resolution can be less than 0.1nm (>1,000,000X magnifications are possible).
Sample Preparation: Requires very thin sections, which are prepared using an ultramicrotome.
Staining: Metals are used to enhance contrast.
Examples of Staining Agents: Osmium tetroxide, lead citrate, uranyl acetate, gold, or platinum coating.
Example: TEM of a human plasma cell.
Immunogold Staining: Utilizes an antibody attached to a gold bead for specific protein detection.
Examples: Detection of HIV virion particles, detection of catalase in peroxisomes.
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5. Scanning Electron Microscopy (SEM)
Provides very high resolution images of the surface of samples.
Principle: A narrow electron beam scans the surface, causing secondary electrons to bounce off, which are then detected to create an image.
Sample Preparation: Can accommodate thick samples, which are usually coated with metal to facilitate imaging.
Examples: Red Blood Cells (RBC), blood vessels, intestinal epithelium.
Produces very high-resolution images of the sample's surface.
B - The Purification of Animal Eukaryotic Cells
1. Tissue Disaggregation - Methodology
The primary tool for disaggregating cells within a tissue involves enzymes that remove proteins crucial for tissue structure and cell adhesion.
These proteins include extracellular matrix (ECM) proteins and cell adhesion molecules.
Proteases: Can degrade both extracellular matrix components and cell adhesion molecules on the cell surface.
Example: Trypsin is the most widely used protease for separating cells.
Note: Many other proteases exist for specific cases, such as collagenase, elastase, papain, and dispase.
Combination Methods: Mechanical and chemical procedures can be combined with proteases to enhance effectiveness.
Cutting and Mincing: Helps generate smaller tissue pieces for enzymes to work on, requiring careful execution to avoid cell damage.
EDTA: A Ca^{2+} ion chelator often used with proteases.
Many cell adhesion molecules require calcium ions for proper function; removing these ions weakens cell-to-cell or cell-to-ECM adhesion.
2. Cell Separation Based on Physical Properties - Methodology
Centrifugation:
Separates cells based on differences in physical properties like size or density.
Only effective for a few cell types because most cells have similar sizes and densities.
In specific cases where cells have very different densities:
Example: Density gradient centrifugation works exceptionally well with blood to separate red blood cells and various types of white blood cells.
3. Flow Cytometry and Cell Sorting - Methodology
The instrument used for this method is the Fluorescence Activated Cell Sorter (FACS).
Principle: Membrane markers on the surface of desired cells are detected using fluorescent antibodies.
Cells in suspension are channeled through a capillary tube.
Lasers of specific colors hit each individual cell as it passes through the capillary.
The laser excites the fluorescent molecules, generating electrical charges.
An electric field can then deflect and separate cells based on the amount of marker (level of fluorescence/charge).
FACS can also detect cell size and shape, aiding in separation.
Significance: This is considered the most advanced method for cell separation.
Example: Separation of T-cells (T-Lymphocytes) from blood by detecting both CD3 and Thy1.2 markers using fluorescent antibodies (emitting red and green light, respectively).
Many diverse cell types, including hard-to-separate low-abundance cells like neural stem cells, have been isolated with FACS instruments.
C - Culture of Animal Cells
1. Importance and Materials Needed for Culturing Mammalian Cells
Importance:
The development of animal cell culture methods has significantly advanced knowledge of animal cells.
Beyond scientific knowledge, it holds great value for biotechnology and medicine.
Culturing animal cells is considerably more difficult than culturing bacterial cells; significant progress has been made since the 1950s.
Media, vessels, and environmental requirements are far more complex than for prokaryotic cells.
Media:
Unlike E. coli which can grow in a simple aqueous solution of glucose and ions, mammalian cells require more complex mixtures.
Mammalian Media Components: Glucose, various ions, several amino acids, and multiple vitamins.
Note: Nine amino acids are essential: Histidine (H), Isoleucine (I), Leucine (L), Lysine (K), Methionine (M), Phenylalanine (F), Threonine (T), Tryptophan (W), Valine (V). Three additional amino acids, Cysteine (C), Glutamine (Q), and Tyrosine (Y), are essential for most cell lines.
Growth Factors are Key: Mammalian cells will cease growing and undergo apoptosis unless they receive signals to grow, reflecting the complex regulation of cell division in eukaryotes.
Serum: Early success came from adding serum (typically around 10% or more) to culture media.
Serum contains multiple growth factors and hormones that promote cell survival and division.
Examples of Sera: Bovine newborn calf serum, horse serum, or fetal bovine serum (FBS).
FBS is generally the best for providing high levels of growth factors, though it is also more expensive.
Defined Media: Nowadays, fully synthetic media of known composition can be used by substituting animal sera with a mixture of lipids, trace elements, growth factors, and hormones.
These are more expensive but necessary when animal sera is unacceptable (e.g., for human medical procedures).
Examples of Growth Factors/Components: Insulin, Transferrin, Fibroblast Growth Factor (FGF), Epidermal Growth Factor (EGF), Platelet-Derived Growth Factor (PDGF).
Culture Plates:
Most mammalian cells (except blood cells) require attachment to a proper substratum, mimicking the extracellular matrix (ECM) or other cell surfaces in their natural environment.
Regular plastic dishes for bacterial culture have a hydrophobic surface unsuitable for mammalian cell adhesion.
ECM Production: A few cell types, like fibroblasts, can produce their own ECM proteins (e.g., collagen, fibronectin, laminin), which are often positively charged.
Treated Dishes: Mammalian cell culture dishes are treated to make the plastic surface hydrophilic and negatively charged, allowing ECM proteins to bind to the plastic, and cells to bind to the ECM.
External ECM/Substitutes: For cells that cannot produce their own ECM, ECM proteins can be added (extra work and expense) or a polymer like polylysine can be used as a cheaper alternative.
Environment Control:
Mammalian cells require high humidity, tight temperature regulation, and controlled CO_2 gas levels to replicate their natural environment.
Specialized cell culture incubators provide these conditions.
2. Primary Cell Cultures - Origin, Methodology, and Terminology
Origin: Primary cell cultures are derived directly from tissues.
A tissue piece is disaggregated enzymatically, and the cells are placed in a tissue culture plate with culture medium within a cell culture incubator.
Initial Survival: Many cells often die relatively quickly due to missing essential components in the medium or substratum.
Primary Cells: Some cells can survive for a significant period (days or weeks) if the medium and substratum are adequate.
Successful Expansion: If the medium and substratum provide all necessary signaling and conditions, some cells can multiply, leading to a successful expansion of the primary cell culture.
3. Cell Types and Primary Cell Culture Success
Ease of Culture: Not all cell types are equally easy to grow as primary cell cultures.
Easiest Type: Fibroblasts: They produce their own ECM and are not particularly delicate regarding growth factor requirements. In the body, they readily multiply to fill gaps and repair tissues.
Moderately Challenging: Macrophages, keratinocytes, myoblasts, among others, have been successfully grown. They often need more complex media formulations, artificial ECM, or both.
Very Challenging: Neurons, various stem cells, etc. Highly specialized and often expensive media formulations have been developed for some, but not all, of these cell types.
4. Advantages and Disadvantages of Primary Cell Cultures
Advantages:
Normal Cells: Primary cells are genetically normal and exhibit characteristics very similar to the cells in the original tissue.
Models: They serve as excellent models for studying cell function.
Disadvantages:
Purity/Homogeneity: Achieving a homogeneous (pure) culture of a single cell type can be challenging and may require stringent cell purification.
Example: Contamination by fibroblasts can be a significant issue, as they tend to multiply rapidly and outcompete other, harder-to-culture cells.
Limited Cell Division: The growth rate curve of primary cultures shows a characteristic pattern that eventually reaches a