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Questions and Answers
What is the primary purpose of using trypsin during cell isolation?
What is the primary purpose of using trypsin during cell isolation?
Why should the exposure time of cells to trypsin be minimized?
Why should the exposure time of cells to trypsin be minimized?
What additional agents can be used to aid tissue digestion besides collagenase?
What additional agents can be used to aid tissue digestion besides collagenase?
What is one of the major challenges encountered during the primary culture establishment?
What is one of the major challenges encountered during the primary culture establishment?
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What is a characteristic of lymphoblast-like cells?
What is a characteristic of lymphoblast-like cells?
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Which of the following methods can be used to isolate a specific cell type from a mixed culture?
Which of the following methods can be used to isolate a specific cell type from a mixed culture?
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What is the recommended practice for dissection instruments when preparing a primary culture?
What is the recommended practice for dissection instruments when preparing a primary culture?
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What is the term used for the process of transferring cells from one culture to another?
What is the term used for the process of transferring cells from one culture to another?
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What phenomenon occurs when fibroblasts outgrow other cells in a culture?
What phenomenon occurs when fibroblasts outgrow other cells in a culture?
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What is isopycnic sedimentation used for?
What is isopycnic sedimentation used for?
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When is a secondary culture established in relation to the primary culture?
When is a secondary culture established in relation to the primary culture?
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What is the purpose of using a serum-supplemented medium after trypsinization?
What is the purpose of using a serum-supplemented medium after trypsinization?
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What materials are typically used to form gradients for cell separation?
What materials are typically used to form gradients for cell separation?
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What should be done after centrifugation to minimize granulocyte contamination when isolating lymphocytes?
What should be done after centrifugation to minimize granulocyte contamination when isolating lymphocytes?
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What is the typical recovery rate of lymphocytes from the original blood sample using this isolation method?
What is the typical recovery rate of lymphocytes from the original blood sample using this isolation method?
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Which cell type is known to form the lower layer after centrifugation in this context?
Which cell type is known to form the lower layer after centrifugation in this context?
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What characteristic is primarily associated with undifferentiated tumor cells?
What characteristic is primarily associated with undifferentiated tumor cells?
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Which of the following can help maintain the differentiated state of specific cell types in culture?
Which of the following can help maintain the differentiated state of specific cell types in culture?
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Which factor plays a crucial role in the formation of gap junctions between cells?
Which factor plays a crucial role in the formation of gap junctions between cells?
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What role do chemical agents such as dimethyl sulfoxide (DMSO) have in maintaining the differentiated state of cells?
What role do chemical agents such as dimethyl sulfoxide (DMSO) have in maintaining the differentiated state of cells?
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What is the significance of collagen in relation to hepatocytes in culture?
What is the significance of collagen in relation to hepatocytes in culture?
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What occurs when a cell population covers an available growth surface?
What occurs when a cell population covers an available growth surface?
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What can be a limitation of culture systems used for investigating metabolic changes associated with differentiation?
What can be a limitation of culture systems used for investigating metabolic changes associated with differentiation?
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What is one characteristic of differentiated tumor cells like neuroblastomas?
What is one characteristic of differentiated tumor cells like neuroblastomas?
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What role do serum-derived glycoproteins play in cell culture?
What role do serum-derived glycoproteins play in cell culture?
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Which modification helps improve the negative charge on glass surface containers used in cell culture?
Which modification helps improve the negative charge on glass surface containers used in cell culture?
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What defines the process of differentiation in cells?
What defines the process of differentiation in cells?
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What is a key limitation of differentiated cells in culture?
What is a key limitation of differentiated cells in culture?
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Which of the following is NOT a reason for the loss of differentiated properties in cultured cells?
Which of the following is NOT a reason for the loss of differentiated properties in cultured cells?
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What factors influence cell attachment to solid substratum in culture systems?
What factors influence cell attachment to solid substratum in culture systems?
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In which context does differentiation primarily occur?
In which context does differentiation primarily occur?
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What is one of the key characteristics of transformed cells?
What is one of the key characteristics of transformed cells?
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What types of cells are typically observed in cultures derived from animal tissues?
What types of cells are typically observed in cultures derived from animal tissues?
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What process results in normal animal cells acquiring infinite growth capacity?
What process results in normal animal cells acquiring infinite growth capacity?
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Which of the following is an example of cells derived from a carcinoma?
Which of the following is an example of cells derived from a carcinoma?
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Aneuploidy refers to what condition in transformed cells?
Aneuploidy refers to what condition in transformed cells?
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Which gene type is associated with the formation of tumorigenic cells?
Which gene type is associated with the formation of tumorigenic cells?
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What is a common feature of transformed cells in culture?
What is a common feature of transformed cells in culture?
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Which method is a particularly effective way to immortalize cells?
Which method is a particularly effective way to immortalize cells?
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What term describes the ability of transformed cells to grow without sensitivity to environmental stimuli?
What term describes the ability of transformed cells to grow without sensitivity to environmental stimuli?
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What is the typical doubling time for fibroblast and epithelial cells in culture?
What is the typical doubling time for fibroblast and epithelial cells in culture?
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Which type of cells are responsible for the transmission of electrical impulses in nervous tissue?
Which type of cells are responsible for the transmission of electrical impulses in nervous tissue?
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What do myoblasts fuse to form during muscle tissue development?
What do myoblasts fuse to form during muscle tissue development?
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What is the role of nerve growth factor in neuron cultures?
What is the role of nerve growth factor in neuron cultures?
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Which type of cells are lymphoblasts, and what is their primary function in culture?
Which type of cells are lymphoblasts, and what is their primary function in culture?
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What characteristic shape do neurons typically exhibit?
What characteristic shape do neurons typically exhibit?
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Which cells in muscle tissue are capable of differentiation to form myotubes?
Which cells in muscle tissue are capable of differentiation to form myotubes?
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In culture, which type of cells have not been observed to divide?
In culture, which type of cells have not been observed to divide?
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Study Notes
Cell Culture Characteristics
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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.
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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.
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Culture Collections: These are banks where cells are extensively characterized in terms of growth, origin, and genetic traits.
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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.
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Contamination: A major difficulty in cell culture is contamination with bacteria or fungi. Maintaining aseptic techniques throughout the procedure is crucial.
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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
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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.
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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.
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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.
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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.
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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.
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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
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Primary Culture Diversity: A primary culture will usually contain diverse cell types with varying growth capacities.
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Specific Cell Isolation: Techniques for isolating a single cell type:
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Allowing Natural Growth: Fast-growing cell types (like fibroblasts) may dominate, potentially out-competing other cells.
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Growth Medium Control: Adding specific growth factors or known inhibitors can allow for the selective growth of desired cell types.
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Density Gradient Centrifugation: Separating cells based on their densities, this usually uses a nontoxic, high-molecular-weight material such as silica.
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Fluorescence-Activated Cell Sorting (FACS): Isolating cell mixtures based on their light scattering and fluorescent characteristics.
Normal Animal Cells
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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
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Definition: The requirement of cells to bind to a solid surface for attachment and growth.
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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).
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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
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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.
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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
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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
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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.
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Characteristics of Transformed Cells:
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Infinite Growth: The most noticeable characteristic.
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Anchorage-Independence: Can grow in the absence of a solid surface.
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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
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Embryoid Body Formation: Embryonic stem cells can form embryoid bodies if allowed to clump. They then differentiate spontaneously.
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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.
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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.
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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).
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Description
Test your knowledge on cell culture techniques including the use of trypsin for cell isolation, methods for isolating specific cell types, and challenges in primary culture establishment. This quiz covers essential practices and terminology related to cell culture and tissue digestion.
