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
