Test 1: Cell Culture

Question 1

How do you isolate and purify cells for culture? 3 general steps

Answer 1

1. Remove tissue or the explant
2. Dissociate all cells from each other and extracellular matrix
3. Purify cells of interest

Question 2

How do you dissociate cells from each other and extracellular matrix?

Answer 2

Protease and EDTA

Question 3

Collagenase and Trypsin are used to…

Answer 3

digest proteins

Question 4

EDTA is used to…

Answer 4

bind to Ca2+, which is required for cell-cell binding.

Question 5

What are the 4 ways to purify cells of interest? Explain briefly.

Answer 5

1. Centrifuge: separate by size, shape, density
2. Adhereability: to glass or plastic
3. Cell Surface Proteins: Antibodies for specific cell surface proteins attached to heavy beads to separate cells of interest by weight.
4. Fluorescence Activated Cell Sorting (FACS): Antibodies attached to flourescent molecule.

Question 6

Explain how FACS work.

Answer 6

1. Flourescence-tagged and untagged cell is passed one cell per droplet.
2. Laser shines onto the cell
3. If fluorescence is detected, detector attaches to the cell a (-) charge . (No fluorescence is (+)).
4. Droplets then pass electron magnet to shunt cells one way or another. (so + side gets - fluorescent cells, - side gets + non-fluorescent cells)

Question 7

Primary culture is…

Answer 7

culture directly from explant

Question 8

Cells grow in a distinctive characteristic. Describe the three stages and how it looks on graph.

Answer 8

1. 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.
2. Log Growth: Attached cells actively growing.
3. Plateau/Saturations: Cells stop dividing due to contact inhibition.

Question 9

What is anchorage dependent growth?

Answer 9

cell must e anchored to culture dish to live

Question 10

What is Generation Time?

Answer 10

Time taken to double cell number for a specific population

Question 11

What is Double Time?

Answer 11

Time taken to double cell number as an AVERAGE of the whole culture.

Question 12

What is Saturation density, contact inhibition, and confluence?

Answer 12

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

Question 13

What is Secondary Culture? Why is it necessary?

Answer 13

Culture plate from 1’ Culture. Necessary because plateau usually doesn’t have enough n for experiment.

Question 14

The process by which 1’ Culture is turned into 2’ Culture is called ____. Give the 3 steps of this process.

Answer 14

Passaging cells.

1. Take cells from 1’ Culture
2. Dilute
3. Replate onto cells with lower density. (This is due to contact inhibition)

Question 15

What is the downside of 2’ Cultures?

Answer 15

Cells multiply for limited time (20-25) before dying due to shortening telomere.

Question 16

What is a Continuous Line? What kind of cell is it? It’s also called…

Answer 16

Cell that infinitely replicates. This is a cancerous cell or transformed cell.

Question 17

What gives Continous Line its characteristics? How?

Answer 17

Infinite replication because its telomere length stays the same. This is caused by DNA damage at oncogene.

Question 18

T/F: Transformed cells are exactly as it was before its mutation. Why?

Answer 18

F. It loses contact inhibition, anchorage dependent growth, and forms tumors.

Question 19

Characteristics of tumors are… (2)

Answer 19

1. . Angiogenic (recruits blood vessel to feed self)

2. Invasive

Question 20

Continuous Lines: Pros and Cons

Answer 20

Pro: + n
Cons: Atypical, so must be replicated in healthy 1’ Culture.

Question 21

Stem Cell Lines are divided into 3 categories, which are

Answer 21

1. Adult SC
2. Embryonic SC
3. iPSC

Question 22

Adult SC lines are (multipotent/pluripotent). It can be used for… (3)

Answer 22

Multipotent.

1. Leukemia Treatment (good stem cell, kill rest, reinsert)
2. Bone marrow SC -> Cardiac Cells
3. Tracheae Reconstruction (new from cartilage)

Question 23

Embryonic SC lines are (multipotent/pluripotent). It is from… The 2 conditions that cause differentiation are…

Answer 23

Pluripotent. From inner cell mass of blastocyst.

1. Retinoic Acid -> Neuron
2. Retinoic Acid + Dibuteral cAMP -> Smooth Muscle

Question 24

Why is ESC not used to fix degenerative diseases without Therapeutic Cloning?

Answer 24

Rejection.

Question 25

The solution to rejection of ESC is…

Answer 25

Therapeutic Cloning

Question 26

Therapeutic Cloning procedures?

Answer 26

1. Nuclei from patient extracted. Unfertilized Cell DNA extracted
2. Egg tricked into thinking fertilized after patient nuclei insertion
3. Blastocyst forms.

Question 27

Therapeutic Cloning Pro and Con?

Answer 27

Pro: no rejection
Con: needs lots of eggs

Question 28

iPSC lines are derived from…

Answer 28

Fibroblast from skin.

Question 29

Fibroblast turns into iPSC due to expression of these 3 genes by… (name genes)

Answer 29

virus infection to express Oct3/4, Sox2, Kif4 proteins

Question 30

iPSC pro and con

Answer 30

pro: no rejection
con: inefficient and introducing virus-infected cell into patient problematic.

Question 31

What are 2 phases of SC growth? What are the major differences?

Answer 31

1st Phase: Self renewal, reamins undifferentiated, and conserves telmere length
2nd phase: differentiation is terminal.