Lab 7 - Mammalian Cell Culture and Transfection Flashcards
What are the tasks of this lab?
- Subculture and splitting for transfection
- Cell counting and Hemocytometer
- Lipid mediated transfection
- Mammalian cell lysis
When was tissue culture initially developed and why
Beginning of the 1900’s as a method for examining the behaviour of animal cells outside their host to allow researchers to test a specific hypothesis by treating cell lines under different conditions without the systemic variation that occur during animal testing
What are the 2 major advantages for utilizing mammalian cell culture?
- Control of the physiochemical environment of the cells which includes pH, temperature, osmotic pressure, O2, CO2, and surface tension
- Ability to modulate the intra and extracellular physiological conditions of cells
What do the majority of cell lines require?
The the media be supplemented with a combination of serum or other compounds
What must one be aware for supplements?
They can vary by batch and contain undefined elements such as hormones and other regulatory substances, all factors which can influence cellular activities
How must mammalian cell culture techniques be carried out?
Using strict aseptic techniques because animal cells grow much more slowly compared to bacteria, mold, and yeast, and are therefore susceptible to infection
What do mammalian cell cultures require a large amount of?
Skill and time to grow only a few milligrams of cells that can be used in an experiment
What is a major limitation of mammalian cell culture?
Altered cellular properties over time
- Every time the mammalian cell culture flask is confluent, i.e., the cells have no more room to grow, the mammalian cell culture is split by an enzymatic digestion, and an aliquot is placed into a new flask containing fresh media (Passage)
What happens with each passage?
Artificial selection causes small alterations in the phenotype of the cells of interest
What happens after numerous passages?
The cells which you are using in your research may not resemble the cells which were used at the beginning of the project
What must researchers be extremely diligent in?
Their record keeping and characterization of cell types to ensure that the cells remain true to their primordial line
How can the numerous types of cultures be separated?
1) Organ culture - derived from a specific organ in which the characteristics of the tissue in vivo are retained
2) Primary explant culture - a fragment of tissue is placed between a glass and liquid interface where it attaches
3) Mammalian cell culture - outgrowth of the primary explant culture and is dispersed into a cell suspension (as a monolayer or free floating in the media suspension)
What will we be utilizing in this lab?
A characterized human mammalian cell culture line that is derived from a primary explant of embryonic kidney epithelial tissue (HEK 293)
How can microorganisms be contaminated in tissue culture?
Bacteria, mycoplasma, yeast, and fungal spores, all of which are typically introduced by the researcher
To minimize contamination in the culture, what is it important to carry out?
1) Check the culture for the presence of bacteria, fungus, or any other “abnormal” substance by using an inverted microscope every time you handle a sample
2) The culture should contain an antibiotic to remove and cryptic contamination
3) The reagents are sterile
4) The bottles used should not be used for the maintenance of any other cell lines
5) Sterile techniques are used every time and at all times
What have biological containment devices been developed for?
To reduce the risks associated with performing cell culture, maintenance of sterile cell lines, and for the reduction of cross contamination
What is the use of proper microbiological procedures the primary method for?
Providing personal and environmental protection
What is the biological safety cabinet/laminar flow hood?
Filtered air moves at a steady velocity, is unidirectional, and moves in a parallel line to the worker
What does a laminar flow hood consist of?
A front opening, sash, exhaust high efficiency particulate air (HEPA) filter, rear plenum, supply HEPA filter, and a mechanical blower
What are various methods for transient transfection?
- Lipid-mediated
- Calcium-phosphate mediated
- DEAE-dextran mediated
- Electroporation
- Biolistics
What is lipofection?
A technique that introduces DNA into cultured mammalian cells
What are cationic lipids used for?
To create artificial membrane vesicles (liposomes) that have the ability to directly interact with the plasmid membrane of a cell or can be taken up by non-receptor mediated endocytosis
What liposomal transfection will we be focusing on for this lab?
Cationic not anionic liposomal transfection
How was cationic transfection developed?
When it was discovered that cationic lipids react spontaneously with DNA to form a unilamellar shell which can fuse to cell membranes
How have the types of cationic lipids evolved?
From a monocationic lipid, which was toxic to mammalian and insect cells, to polycationinc which are less toxic
What do the majority of lipofection reagents consist of?
A mixture of synthetic cationic lipid and a fusogenic lipid (phosphatidylethanolamine)
What will we be performing in this lab?
A transient cationic lipid-mediated DNA transfection into HEK 293 cultured cells
- The already isolated plasmid DNA forms a complex with unilamellar liposomes that are formed by cationic lipids in water
What are the main benefits of using the type of transfection that will be carried out in this lab?
- Higher efficiency
- Ability to transfect numerous different cell lines
- Lower cell toxicity
What is the largest drawback to using the type of transfection that will be carried out in this lab?
Cost associated with the transfection reagents
What is proper knowledge of total cell numbers required for?
Reproducible cell maintenance and reducing experimental variability
How can the concentration of cell suspension be determined?
By placing the cells in an optically flat chamber, called a hemocytometer, and viewing it under a microscope
- Cell number within a defined area of known depth is counted and the cell concentration is derived from the count
What is derived from the hemocytometer count?
Cell number within a defined area of known depth
