Test 1: Cell Culture Flashcards
How do you isolate and purify cells for culture? 3 general steps
- Remove tissue or the explant
- Dissociate all cells from each other and extracellular matrix
- Purify cells of interest
How do you dissociate cells from each other and extracellular matrix?
Protease and EDTA
Collagenase and Trypsin are used to…
digest proteins
EDTA is used to…
bind to Ca2+, which is required for cell-cell binding.
What are the 4 ways to purify cells of interest? Explain briefly.
- Centrifuge: separate by size, shape, density
- Adhereability: to glass or plastic
- Cell Surface Proteins: Antibodies for specific cell surface proteins attached to heavy beads to separate cells of interest by weight.
- Fluorescence Activated Cell Sorting (FACS): Antibodies attached to flourescent molecule.
Explain how FACS work.
- Flourescence-tagged and untagged cell is passed one cell per droplet.
- Laser shines onto the cell
- If fluorescence is detected, detector attaches to the cell a (-) charge . (No fluorescence is (+)).
- Droplets then pass electron magnet to shunt cells one way or another. (so + side gets - fluorescent cells, - side gets + non-fluorescent cells)
Primary culture is…
culture directly from explant
Cells grow in a distinctive characteristic. Describe the three stages and how it looks on graph.
- Plating Efficiency (PE): small “v” shape; decline caused by cells killed during purification and cell death because it is unable to attach to tissue culture dish.
- Log Growth: Attached cells actively growing.
- Plateau/Saturations: Cells stop dividing due to contact inhibition.
What is anchorage dependent growth?
cell must e anchored to culture dish to live
What is Generation Time?
Time taken to double cell number for a specific population
What is Double Time?
Time taken to double cell number as an AVERAGE of the whole culture.
What is Saturation density, contact inhibition, and confluence?
Saturation density is when cells are spread out to the point that they are all touching. At this point, they stop diving (confluence) because of contact inhibition
What is Secondary Culture? Why is it necessary?
Culture plate from 1’ Culture. Necessary because plateau usually doesn’t have enough n for experiment.
The process by which 1’ Culture is turned into 2’ Culture is called ____. Give the 3 steps of this process.
Passaging cells.
- Take cells from 1’ Culture
- Dilute
- Replate onto cells with lower density. (This is due to contact inhibition)
What is the downside of 2’ Cultures?
Cells multiply for limited time (20-25) before dying due to shortening telomere.
What is a Continuous Line? What kind of cell is it? It’s also called…
Cell that infinitely replicates. This is a cancerous cell or transformed cell.
What gives Continous Line its characteristics? How?
Infinite replication because its telomere length stays the same. This is caused by DNA damage at oncogene.
T/F: Transformed cells are exactly as it was before its mutation. Why?
F. It loses contact inhibition, anchorage dependent growth, and forms tumors.
Characteristics of tumors are… (2)
- . Angiogenic (recruits blood vessel to feed self)
2. Invasive
Continuous Lines: Pros and Cons
Pro: + n
Cons: Atypical, so must be replicated in healthy 1’ Culture.
Stem Cell Lines are divided into 3 categories, which are
- Adult SC
- Embryonic SC
- iPSC
Adult SC lines are (multipotent/pluripotent). It can be used for… (3)
Multipotent.
- Leukemia Treatment (good stem cell, kill rest, reinsert)
- Bone marrow SC -> Cardiac Cells
- Tracheae Reconstruction (new from cartilage)
Embryonic SC lines are (multipotent/pluripotent). It is from… The 2 conditions that cause differentiation are…
Pluripotent. From inner cell mass of blastocyst.
- Retinoic Acid -> Neuron
- Retinoic Acid + Dibuteral cAMP -> Smooth Muscle
Why is ESC not used to fix degenerative diseases without Therapeutic Cloning?
Rejection.
The solution to rejection of ESC is…
Therapeutic Cloning
Therapeutic Cloning procedures?
- Nuclei from patient extracted. Unfertilized Cell DNA extracted
- Egg tricked into thinking fertilized after patient nuclei insertion
- Blastocyst forms.
Therapeutic Cloning Pro and Con?
Pro: no rejection
Con: needs lots of eggs
iPSC lines are derived from…
Fibroblast from skin.
Fibroblast turns into iPSC due to expression of these 3 genes by… (name genes)
virus infection to express Oct3/4, Sox2, Kif4 proteins
iPSC pro and con
pro: no rejection
con: inefficient and introducing virus-infected cell into patient problematic.
What are 2 phases of SC growth? What are the major differences?
1st Phase: Self renewal, reamins undifferentiated, and conserves telmere length
2nd phase: differentiation is terminal.
