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Questions and Answers
What is the primary risk of cross contamination in cell culture?
Which method is recommended for confirming the identity of cell lines?
What is a limitation of human STR analysis?
Which of the following techniques is NOT recommended for cell culture to avoid contamination?
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What is the best way to minimize contamination while working with cell cultures?
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Why are 'false' cell lines particularly costly for researchers?
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Which of the following cell types is most prone to misidentification due to morphology similarities?
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What type of analysis can help detect contamination from animal species in cell cultures?
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What is a key consideration when using enzymatic dissociation to isolate single cells from solid tumors?
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How does the concentration of FBS in culture media affect the cell culture process?
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Why is it important to examine a histological section of the tumor by a pathologist?
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Which enzyme is NOT commonly used for dissociating tumor cells from solid tissue?
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What happens to the cell viability if the temperature during enzymatic dissociation is incorrectly set?
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Which statement about cell culture media is true?
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What is a potential complicating factor in the establishment of tumor cell lines?
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In cell line establishment, what should researchers extensively review before beginning the process?
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What is a critical consideration when characterizing tumor cells in culture?
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What assay is used to assess the tumor potential of cells?
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Which characteristic is typically checked to confirm tumor cell identity during culture?
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How many passages in culture are fibroblasts capable of persisting?
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Which technique could help detect mesenchymal origin in cultured cells?
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What does chromosome instability indicate about tumor cells?
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What is a common challenge in differentiating between fibroblasts and tumor cells?
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What is an essential procedure to ensure the quality of tumor cell lines over time?
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Study Notes
Tumor Cell Line Establishment
- Mechanical Dissociation: A common method to separate tumor cells. Tissue is cut into small 1-2 mm³ pieces and can be further homogenized by filtration through a nylon filter, vortexing, and repeating these steps to remove dead cells, debris, and other cell types.
- Enzymatic Dissociation: Uses enzymes like collagenase, trypsin, papain, and elastase to digest tissue into single cells. Factors like enzyme concentration, temperature, and incubation time are critical to maintain cell viability and integrity.
- Cell Culture Media: A variety of basic media are available and can be customized with additives to meet the specific needs of the cells. It's important to maintain proper osmolality, especially when adjusting FBS concentration. Human blood has an osmotic value of 300 mOsmolkg-1, while most basic media range from 280–340 mOsmolkg−1.
Cell Contamination
- Viral Contamination: Difficult to detect and can be a significant issue. Unless cytopathic, viral contamination may have a minimal effect on host cells.
- Cell-Cell Contamination ("False" Cell Lines): A widespread problem, especially with primary cells. Can occur due to human errors, such as mislabeling, unsterile working conditions, cross-contamination, and improper technique (e.g., using feeder layers or xenografting).
- DNA Profiling: STR (short tandem repeat) DNA profiling is used for routine identification and authentication of human cell lines, stem cells, and tissues. It examines specific DNA regions to distinguish between individuals. However, it cannot identify contamination from other species, like mice or rats. Isoenzyme analysis can be used for species detection.
Ten Golden Rules for Successful Tumor Cell Line Establishment
- Fibroblast Contamination: Fibroblasts can often outgrow tumor cells and are not always easily recognizable by their morphology. It's important to test for tumor potential as soon as possible.
- Tumor Potential Characterization: Early characterization is crucial as few tumor cells may be available initially. Morphological observation, colony-forming unit assays, and flow cytometry for cell cycle analysis can be used to evaluate tumor potential.
- Tumor Marker and Genetic Profile Consistency: Tumor markers and genetic profiles should remain constant during cultivation, although some markers may be lost.
- DNA Profiling for Identification: STR analysis should be used to confirm the identity of the tumor cell line and confirm its origin.
- Regular Cryopreservation: Regularly cryopreserve tumor cell lines to ensure their preservation and viability.
- Extended Culture Duration: Tumor cell lines should be passaged more than 100 times and maintained in culture for more than 12 months.
- Genetic Phenotype and Morphology Maintenance: Tumor cells should maintain their genetic phenotype and morphology during cultivation.
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Description
This quiz covers the essential methods for tumor cell line establishment, including mechanical and enzymatic dissociation techniques. It also explores the importance of culture media and the risks of contamination. Understanding these concepts is vital for successful cell culture practices in research and clinical applications.
