Cell culture technique(INCOMPLETE CBA)

Question 1

How are cells isolated?

Answer 1

Isolation through transfection

Question 2

What process is selection of cells through?

Answer 2

Selection through selection pressure

Question 3

Isolation of cells in blood vs tissue?

Answer 3

• In blood, cells are already in isolation Compared to tissue sample where they are closely packed

Question 4

How are blood samples centrifuged?

Answer 4

• Blood sample can be easily centrifuged
○ Used gradient from medium
○ Layer diluted blood on top of thicker blood

Question 5

What do different cells in blood form?

Answer 5

• Different cells in blood will form different layers
○ Based on cell density and cell size
○ These are not pure populations of cells

Question 6

What are the 2 ways to further purify cells using immune-purification methods?

Answer 6

1. Mix cells with antibody-coated magnetic beads

2. Can also use FACS

Question 7

Method of mixing cells with antibody coated magnetic beads to further purify cells using immune-purification methods

Answer 7

• Can further purify the cells using immune-purification methods
-Mix cells with antibody-coated magnetic beads
§ Antibody is a cell surface marker that the cell you want to isolate will express
§ Magnetic coated beads bind to cells with marker
§ Cells can be isolated with magnet

Question 8

Method of FACS to further purify cells using immune-purification methods

Answer 8

§ Mixture of cells can be mixed with antibody that has fluorescent strand
§ Antibody binds to cell with specific antigen (this is the cell you want to isolate)
§ Pass cell through fast-flowing stream of liquid that vibrates
§ Each cell is isolated on a single drop
§ Each drop that passes through laser detector has a single cell
§ The ones that fluoresce can be quantified so you know how many there are
§ They are then passed through series of electromagnets
§ Fluorescent cells have positive charge so are put into negatively charged container
-Cells you are interested in will go into this container whilst the others will not

Question 9

Steps involved in isolation of cells from solid tissues

Answer 9

• Isolate cells from placenta
• in order to get to fine layer of tissue: use scalpel, wash, enzymatic disruptions used, mechanical disruption
• Layer of cell clumps:
○ Use magnetic-coated beads
○ Cells in placenta CD31+
○ Use magnet to remove placental endothelial cells
○Cells can be placed in dish

Question 10

What are cells susceptible to for growth in culture?

Answer 10

Susceptible to contamination

Question 11

How are cells handled under for growth in culture?

Answer 11

Handled under aseptic condition

Question 12

Steps involved in cell growth in culture

Answer 12

• Cells are grown on treated plastic
• Maintained in a warm, humidified temperature
○ Put in incubator
○ Mimics conditions of human body i.e. 5% CO2, 37 degrees

Question 13

What do cells in growth culture produce and what has to be changed?

Answer 13

○ Cells produce toxic byproducts as they grow so the medium has to be changed constantly

Question 14

What are primary cells derived from?

Answer 14

Derived directly from tissue

Question 15

What are positive aspects of primary cells?

Answer 15

• Unmodified

-Represent origin (situation in body) as closely as they can

Question 16

What are negative aspects of primary cells?

Answer 16

• Negative aspects:
	○ Aberrant expression of some genes
	○ Variable contamination
	○ Poor growth characteristics (50-100 divisions)
	○ Inter patient variation
		§ Cannot generalize results to population
	○ Phenotypic instability
		§ Out of natural environement
Molecular manipulation difficult

Question 17

What are cell lines derived from?

Answer 17

Derived from cancerous tissues