Cell Culture Techniques Flashcards
Explain the difference between in vivo vs in vitro
In vivo – research performed on living organism
In vitro – “in glass”, research performed under controlled conditions outside of organism
-Often involves cell/tissue culture
What is cell culture? What are some of its pros and cons
Growth and maintenance of cells outside of a living organism
Pros:
- Researchers can manipulate culture
environment/conditions
- Less expensive (most of the time)
- Faster than many in vivo studies
Cons
- Are immortalized cells a good model?
- Do cells in a dish behave similarly to cells in an
organism?
What are immortalized cells?
- Tumor cells/manipulated cells that replicate indefinitely
- Will eventually proliferate to cover surface of culture dish
- Must be maintained by passaging or splitting – transferring a portion of cells from a dish to new dishes to provide space for future proliferation
- Homogenous and genetically identical – provide consistent, reproducible results
List Cell culture equipment and reagents
Biosafety hood
Cell Incubator
Cell culture flasks/plates
Water Bath
Microscope
Centrifuge
Fridge/Freezer
What is cell culture medium?
Provides nutrients, glucose, and growth factors necessary to promote survival and proliferation
What is cell culture medium composed of?
- Basal media – contains amino acids, vitamins,
glucose, and buffering agents (sodium bicarbonate)
to maintain physiological pH (7.4) in a 5-10% CO2
environment
-Ex. Dulbecco’s Modified Eagle Media (DMEM) - Serum (often Fetal Bovine Serum or FBS) – provides
growth factors necessary for survival and
proliferation - Antibiotics- Prevent growth of bacteria, may also be
used for selection
Specific media/media components may be necessary for some cells
Give two examples of immortalized cell lines
- HEK-293T – immortalized cells generated by
infecting kidney cells with adenovirus
- Reliable growth
- High transfection efficiency - HeLa – cervical cancer cells isolated from a tumor
from Henrietta Lacks
- First immortal human cell line
- Remarkably easy to grow
- Have been found invading/contaminating other
cell culture lines
What is primary culture?
Primary culture involves isolating cells from live organisms and culturing them in vitro
Give three examples of primary culture
Slice Culture: Retain the characteristic anatomic organization of the tissue of origin.
Explant culture: A small piece of tissue is surgically removed from an organism and grown in a controlled environment outside the body,
Dissociated culture: A cell culture where tissue or cell culture is broken down into individual cells
What are the pros and cons of primary culture
Pros:
- Allows researchers to study differentiated cell types
from organism of interest
- Can compare how cells from WT/mutant organism
respond
Cons:
- Cells are not immortal
- Cells/tissue may be difficult to isolate and culture
- Cells will be more heterogenous than immortalized
cells
What is an explant culture?
Explant cultures – primary culture of intact fragments of tissue
- Maintain endogenous mixture of cell types
- Some connectivity maintained
- Often used in coculture systems
What is a disassociated cell culture?
Dissociated cell cultures – tissue is dissociated into individual cells that are then grown under primary culture conditions
- Enzymatic digestion separates cells from one
another - Can be maintained for days to weeks under ideal
conditions
What does Stem cell potency reflect?
The diversity of cell types they can differentiate in to
Totipotent > Pluripotent > Multipotent > Unipotent
How are stem cells classified?
by their source and the tissues they give rise to
What do stem cells require to maintain their undifferentiated state and promote self renewal?
specially formulated media
