Cell Culture Techniques Quiz

Cell Culture Characteristics

• Where to Obtain Cells: A choice must be made whether to obtain cells directly from tissue or from a culture collection (cell banks). The choice depends on the experiment's objective and nature.

• Primary Cell Culture: A primary culture is established when cells from animal tissue are added to growth medium. Embryonic tissue is often used because the cells are easily dispersed and have high growth potential. The goal is selecting a single cell type from the tissue. Isolation involves tissue fragmentation (using forceps and scissors), enzymatic treatment (trypsin or collagenase), and low-speed centrifugation. The time enzymes are in contact with the cells should be minimized to avoid membrane damage.

• Culture Collections: These are banks where cells are extensively characterized in terms of growth, origin, and genetic traits.

• Tissue Culture Methods: Original tissue culture methods involved tissue fragments (explants) on a solid surface with nutrients. These are more useful if individual cells are separated before culture.

• Contamination: A major difficulty in cell culture is contamination with bacteria or fungi. Maintaining aseptic techniques throughout the procedure is crucial.

• Sterility: Dissection instruments and working surfaces need to be sterile and should be swabbed with 70% alcohol. The procedure should occur in a laminar flow cabinet under sterile conditions.

Cell Types and Morphology

• Animal Cells: Defined by tissue origin and characteristic shapes observable via light microscopy. Types commonly used in culture include fibroblasts, epithelial cells, muscle cells, neurons, and lymphocytes.

• Epithelial Tissue: Forms a layer covering organs and lining cavities (e.g., skin, alimentary canal). Epithelial cells in culture grow as a single cell monolayer and have a characteristic cobble-stone appearance; epithelial-like cells are polygonal and grow attached in discrete patches.

• Connective Tissue: Major structural component of animals (e.g., bone, cartilage). Fibroblasts (frequently used in lab cultures) have a spherical shape when dissociated but elongate into a spindle-shape on attachment to a surface.

• Muscle Tissue: Consists of contractile tubules formed from myoblast precursor cells, which fuse to subsequently form myotubes. Alignment of myoblasts during this muscle differentiation process is observable in muscle tissue cultures.

• Nervous Tissue: Composed of neurons (spindle-shaped cells) responsible for transmitting electrical impulses and glial cells. Neuronal cells may form cytoplasmic outgrowths known as neurites when nerve growth factor is added.

• Blood and Lymph: Part of connective tissues; involve suspended cells. Cells like lymphoblasts (white blood cells) are actively used in culture due to their ability to secrete immune-regulating compounds (cytokines).

Selecting a Specific Cell Type

• Primary Culture Diversity: A primary culture will usually contain diverse cell types with varying growth capacities.

• Specific Cell Isolation: Techniques for isolating a single cell type:

• Allowing Natural Growth: Fast-growing cell types (like fibroblasts) may dominate, potentially out-competing other cells.

• Growth Medium Control: Adding specific growth factors or known inhibitors can allow for the selective growth of desired cell types.

• Density Gradient Centrifugation: Separating cells based on their densities, this usually uses a nontoxic, high-molecular-weight material such as silica.

• Fluorescence-Activated Cell Sorting (FACS): Isolating cell mixtures based on their light scattering and fluorescent characteristics.

Normal Animal Cells

• Characteristics:
  • Diploid chromosome number (no gross damage)
  • Anchorage dependence (need surface for attachment and growth)
  • Finite lifespan (intrinsic growth potential)
  • Nonmalignant (cannot form a tumor in immuno-compromised mice)

Anchorage Dependence

• Definition: The requirement of cells to bind to a solid surface for attachment and growth.

• Laboratory Setup: This attachment occurs on solid surfaces, such as Petri dishes, T-flasks, or Roux bottles (often treated for a negative charge on glass or plastic).

• Interaction Mechanisms: The cell membrane's interaction with the growth surface is critical and involves a combination of electrostatic attraction and van der Waals forces. Proteins (e.g., fibronectin) help forming a layer at the growth surface prior to cell attachment. Conditioning factors released by cells into the medium also play a role in bond formation. The solid surface's charge density is important to maximize attachment. (e.g., alkali treatment on glass surfaces gives a negative charge; tissue culture-grade plastic ware contains sulfonated polystyrene and a surface charge).

Stem Cells

• Embryonic Stem Cells: Capable of unlimited growth and differentiation into any cell type. Isolated in 1998; cultured from inner cell mass and used for potential for differentiation.

  • Properties: Pluripotent (potential to become any cell type), capable of indefinite propagation (self-renewal), and directed differentiation (ability to follow specific pathways under chemical or cellular cues). Possessing certain markers (e.g., stage-specific embryonic antigen (SSEA), Octa-4 TF). Normal karyotypes, and high telomerase activity.
  • Applications: Studying development, cell therapy, and drug screening.

• Adult Stem Cells: Undifferentiated cells found among differentiated cells in tissues or organs. They have limited differentiation potential, replacing and repairing cells/tissues.

  • Applications: Replacing cells in damaged tissue, and creating cells for therapy, cell growth, and screening for drug activity, etc

• Transdifferentiation: A process by which adult stem cells can differentiate into cell types different from those in the original tissue, this is an active research area.

Transformed Cells

• Definition: Transformation in cell biology refers to the conversion of normal cells to cells capable of infinite growth. This is different from expressing foreign genes in bacteria or animal cells.

• Characteristics of Transformed Cells:

• Infinite Growth: The most noticeable characteristic.

• Anchorage-Independence: Can grow in the absence of a solid surface.

• Chromosomal Abnormalities: Often have chromosomal fragmentation and can exist in a non-diploid stage (aneuploidy).

Cells from Culture Collections

• Convenience: A primary source for cell lines, providing a ready selection of well-characterized cells.
• Safety and Stability: Cells are often already in a good state for use in research and are safely stored (e.g., in liquid nitrogen) so that specific cell lines can be isolated and studies in sufficient quantity for analysis.
• Cell Lines for Sale: Many cell samples are offered and shipped.
• Examples: There are many international establishments (e.g., ATCC and ECACC) that house and distribute cell lines.

Other Considerations

• Embryoid Body Formation: Embryonic stem cells can form embryoid bodies if allowed to clump. They then differentiate spontaneously.

• Directed Differentiation: Introducing specific growth factors to guide differentiated pathways of cell change. The capacity to direct such change is widely used for studying development and in cell therapy (regenerative medicine), and drug screening.

• Tumor Cell Examples: Examples of differentiated tumor cells (e.g., neuroblastomas) that are useful for study, often for their good growth and differentiated capacities, although they are cancerous.

• Culture Services: These establishments provide services that are valuable, from maintaining and preserving master stocks and testing for contamination to performing cell line characterization (e.g., using isoenzyme analysis, karyotyping, DNA fingerprinting, flow cytometry).