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Questions and Answers
Which enzyme is used most frequently for tissue disaggregation?
What is primary culture in cell culture?
What is the purpose of using DNase in tissue culture?
How are primary cell cultures obtained after isolation?
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Why should fat and necrotic tissue be removed during dissection of the tissue?
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Which enzyme, although incomplete in disaggregation, is less harmful to the cells?
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What is the purpose of removing enzymes used for disaggregation by gentle centrifugation?
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Why should the concentration of cells in the primary culture be much higher than that used for subculture?
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Which technique is suitable for very small amounts of tissue in primary culture?
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What role do calcium-dependent cell adhesion molecules (CAMs) play in tissues?
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How can the purity of an enzyme affect its performance in disaggregation?
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What is the initial step when working with human or animal tissue?
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Which technique gives a better yield when more tissue is available for primary culture?
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What is the purpose of storing tissue in a refrigerator at 4ºC before transferring it to a balanced salt solution or culture medium?
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What is the main purpose of using DNase in tissue culture?
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Why is it important to remove fat and necrotic tissue during tissue dissection?
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Which enzyme is often used in conjunction with collagenase to digest the intracellular matrix?
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What is the purpose of finely chopping the tissue during dissection?
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Why are collagenase and dispase preferred over trypsin for tissue disaggregation?
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What is the initial stage of primary culture before the first subculture?
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What role do calcium-dependent cell adhesion molecules (CAMs) play in tissues?
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Why should fat and necrotic tissue be removed during dissection of the tissue?
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What is the purpose of using DNase in tissue culture?
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Which technique gives a better yield when more tissue is available for primary culture?
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What is the purpose of storing tissue in a refrigerator at 4ºC before transferring it to a balanced salt solution or culture medium?
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Which enzyme is used most frequently for tissue disaggregation?
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What is the initial step when working with human or animal tissue?
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Why should the concentration of cells in the primary culture be much higher than that used for subculture?
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Which enzyme, although incomplete in disaggregation, is less harmful to the cells?
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What is the purpose of removing enzymes used for disaggregation by gentle centrifugation?
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Study Notes
Tissue Disaggregation and Cell Culture
- Most Common Enzyme: Collagenase is frequently used for tissue disaggregation due to its ability to digest collagen in the extracellular matrix.
- Primary Culture Definition: Refers to the culture of cells directly obtained from living tissue. This is the first stage of culturing cells.
- DNase Purpose: Used to degrade DNA that may protect cells from disaggregation or provide a substrate that inhibits cell attachment.
- Obtaining Primary Cell Cultures: Primary cultures are established after enzymatically isolating cells from tissue, followed by plating into culture dishes.
- Importance of Removing Fat and Necrotic Tissue: These materials can hinder the culture's purity and health, obstructing nutrient exchange and leading to contamination.
- Less Harmful Enzyme: Trypsin is known to be less effective in complete disaggregation but poses less damage to cells compared to stronger alternatives.
- Gentle Centrifugation Purpose: This step is key to removing disaggregation enzymes, which, if left in the culture, can adversely affect cell viability.
- Higher Cell Concentration in Primary Culture: A greater initial concentration of cells enhances the likelihood of successful attachment and proliferation in the primary culture.
- Techniques for Small Tissue Amounts: Microdissection techniques are suitable for obtaining primary cultures from very small tissue samples.
- Calcium-Dependent Cell Adhesion Molecules (CAMs): CAMs facilitate cell-cell adhesion in tissues, crucial for maintaining structural integrity and signaling.
- Purity of Enzyme Effect: The effectiveness of disaggregation relies on the purity of the enzyme; impurities can inhibit enzyme action and lead to inconsistent outcomes.
- Initial Step with Human/Animal Tissue: The primary step involves the careful dissection of the tissue to isolate cells without introducing contaminants.
- Yield from Larger Tissue: For larger tissues, mechanical dissociation techniques, like mincing and macerating, yield better results in primary cultures.
- Refrigerator Storage Purpose: Storing tissue at 4ºC slows metabolic processes, preserving cell viability before transferring to culture media.
- Main Purpose of DNase: Apart from degrading DNA, it helps in preventing the clumping of cells during culture preparation.
- Necrotic Tissue Removal Importance: This removal is essential for maintaining healthy cell cultures; necrotic materials can create a hostile environment.
- Collagenase Synergy: Collagenase is often used alongside dispase to effectively digest tissues while preserving cell surface proteins.
- Finely Chopping Tissue Purpose: Chopping increases the surface area for enzymes, improving penetration and effectiveness during disaggregation.
- Preference for Collagenase and Dispase: These enzymes are gentler on cells than trypsin, allowing for better maintenance of cell morphology and function.
- Initial Stage of Primary Culture: This includes the attachment of isolated cells to the culture dish, which is critical for successful growth.
- Reiteration on CAMs' Role: CAMs are integral for tissue architecture, providing mechanical strength and facilitating communication between cells.
- Duplicate Importance of Fat and Necrotic Tissue Removal: Reinforcing the need to maintain the purity and viability of the cell culture environment.
- Enzyme Frequency in Tissue Disaggregation: Collagenase remains the most used enzyme, underscoring its significance in the disaggregation process.
- Human/Animal Tissue Initial Step: Ensuring proper handling during the initial isolation phase to safeguard cell integrity is fundamental.
- Comparison of Cell Concentrations: Primary cultures need denser cell populations compared to subcultures to ensure effective growth dynamics.
- Gentle Centrifugation Justification: This technique preserves cell integrity while eliminating residual disaggregating enzymes from the cell culture environment.
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Description
This quiz covers the stages involved in primary cell culture, from the acquisition of the sample to the culture after seeding into the vessel. It includes the process of isolation, dissection, and obtaining a primary cell culture.
