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What is needed for preparation of primary cell culture?
- Primary culture for explants
What are the advantages and disadvantages of cell culture?
- Advantage- Development of cells line over generations
- Disadvantage- may lose differentiated characteristics
What are the most commonly used enzymes for disaggregating cells?
Trypsin and Pronase
What are the 2 types of continuous culture?
- Cell lines
- Continuous Cell lines
What is transformation?
Spontaneous or induced permanent phenotypic changes resulting from change in DNA and gene expression
What is transfection?
Introduction of DNA into a cells (like viral DNA)
What is the difference between histotypic and organotypic cultures?
- Histo- single cell line
- Organo- multiple cell lineages
What are the cell cycle phases and what happens in each?
- G0/G1- pre-DNA synthesis
- S- DNA synthesis
- G2- period between DNA synthesis and mitosis
- M- mitosis (actual cell division)
How do we measure cell growth?
- Population doubling level (generation #)
- Multiplication rate (r)
- Population doubling time (PDT)
What is the equation for cell growth?
- n= 3.32 (logNH- logN1)
- N1- initial cells
- NH- harvested cells
What is a cell line?
Cell population derived from a primary culture at the first passage
What is SOP (Standard Operating Procedure)?
- Detailed instructions for technique used
- All aspects of tasks involved
What are the 2 sub-zero temperatures used to cryopreserve cells?
Why freeze cells?
- Reduce risk of microbial contamination, cross contamination, genetic drift and morphological changes
- Reduce cost
What are the properties of Cryoprotectant?
- Lowering freezing temperature
- Forming Viscosity
- Diffuse through membrane
What are conventional cryoprotectants?
What are the main factors the influence cell viability?
Cryoprotectant, cooling rate, storage, temperature, and warming rate
What are the advantages and disadvantages of a -135 freezer?
- Pros- Easy to maintain
- Steady Temp
- Low running cost
- Cons- Requires liquid nitrogen (LN) back up
- Mechanically complex
- Temperature is high compared to LN
What are the advantages and disadvantages of Liquid Phase Nitrogen?
- Pros- Steady Ultra Low Temp
- Simple and Mechanically reliable
- Cons- Requires steady supply of LN
- High Cost
- Risk of cross-contamination
What are the pros and cons of Vapor Phase Nitrogen?
- Pros- No risk of cross contamination
- Low temp
- Simplicity and reliability
- Cons- Temperature fluctuation
- Requires steady supply of liquid nitrogen