Lecture 1a: Cell and Tissue

Question 1

Define cell culture

Answer 1

the maintenance and growth of the cells of multicellular organisms outside the body under precise conditions of temperature, humidity, nutrition, and freedom from contamination

Question 2

Define tissue culture

Answer 2

fragments of tissue from an animal or plant are transferred to an artificial environment where they can continue to survive and function. The cultured tissue may consist of a single cell, a pop of cells, or a whole part of an organ

Question 3

Primary cell culture

Answer 3

first line of cells taken directly from the target

Question 4

Secondary cell culture. What do they tend to be?

Answer 4

Primary cell cultures that have been sub-cultured
For example, they have been split and transferred into fresh media. They tend homogenous

Question 5

What is a cell line?

Answer 5

a cell culture capable of continued growth and passage

Question 6

What are the two types of cells lines?

Answer 6

Finite cells lines
Continuous cell lines

Question 7

Finite cell lines (4). Give two examples

Answer 7

1. Limited replicative potential
2. 20-100 generations before senescence (cell cycle arrest - stop dividing)
3. Contact inhibition and anchorage dependence (need a solid surface)
4. Grow slowly - double in 24-96hours
   Ex? WI-38, HUVEC

Question 8

Continuous cell lines 4

Answer 8

1. Indef replication
2. No contact inhibition nor anchorage dep
3. Growth rate is rapid - 12-24 hrs
4. Also know as “transformed”
   Ex/ HeLa cells

Question 9

Transformed cells have undergone a ______ change in _____ through a _______ ____

Answer 9

permanent, phenotype, genetic transformation

Question 10

What is the most common transformation?

Answer 10

Viral

Question 11

How does the virus SV40 transform cells?

Answer 11

SV40 T antigen distrupts the host cell tumor suppressor proteins RB and p53, by binding to them and deregulating cell prolif and cell cycle progression. Cells are not immortal

Question 12

Papillomavirus produces ___ and ___ which bind the the host cells p53 and RB proteins

Answer 12

E6 and E7

Question 13

E6 protein binding leads to the _______ of ______

Answer 13

ubiquination of p53

Question 14

E7 binding to ___ prevents its action as a brake on cell prolid

Answer 14

Rb

Question 15

What can be used instead of or in addition to viral oncogenes?

Answer 15

hTERT human telomerase reverse transcriptase

Question 16

Why is hTERT useful?

Answer 16

It is useful because it can yield cells that behave like primary cultures but propagate like immortalized cell lines. It can establish non-cancer, normal cell line

Question 17

HeLa cells

Answer 17

Human cervical cells transformed by HPV18

Question 18

Jurkat cells

Answer 18

Derived from human T-cell leukemia and grown in suspension culture

Question 19

HEK293 cells

Answer 19

from human embryonic kidney and transformed by adenovirus

Question 20

What are advantages of HEK293 cells?

Answer 20

They are easy to grow, transfect
They are used lots in transfection-based experiments, protein expression, and vaccine production

Question 21

HUVEC cells

Answer 21

primary cell line
Human umbilical vein endothelial cells
Used in CVD and solid tumor progression

Question 22

Transfection

Answer 22

Overexpress a protein of interest
Knock-down expression of a protein of interest (siRNA)
Transient vs stable

Question 23

What are 4 methods of transfection

Answer 23

Calcium phosphate
Lipid based (lipofectionO)
Electroporation
Viral systems

Question 24

What can be used to increase the surface area and increase the number of cells?

Answer 24

Polystyrene beads

Question 25

How do HEK293 grow?

Answer 25

In a monolayer on a coated surface (polystyrene beads)

Question 26

Why do tissue culture plates/flasks not accurately mimic in vivo conditions?

Answer 26

Cells do not normally grow in a monolayer in an organism

Question 27

Matrix environment mimics..

Answer 27

native cell-cell contact and signaling

Question 28

What are some considerations for stem cell cultures? 5

Answer 28

Theoretical infinite lifespan
Specialized media
Prone to contamination
“feeder cells” need food
Induced differentiation

Question 29

List some pros and cons of tissue cultures

Answer 29

Pros
Low cost
minimal time involved
easily manipulated
cell type specificity

Cons
contamination
physiological relevance??
Not every cells type is culturable

Question 30

List some pros and cons of animal models

Answer 30

Pros
True in vivo model
physiologically relevant
knock in and knock out models
Tissue, organ, and whole organism anaylses

Cons
$$$
Very time consuming
ethics

Question 31

Transformed cells have ____ gene expression

Answer 31

altered

Question 32

Do transformed cells have contact inhibition?

Answer 32

no

Question 33

What type of lifespan do transformed cells have?

Answer 33

infinite

Question 34

What type of lifespan do primary cells have?

Answer 34

Finite

Question 35

What type of gene expression occurs in primary cells?

Answer 35

normal

Question 36

Quiescence

Answer 36

lack of nutriton and grown factors and the cell goes into a quiet state where it is not in the cell cycle but it can divide

Question 37

What are the 3 main issues with cell lines?

Answer 37

1. All cell lines exhibit altered expression of p16
   which is a CDk inhibitor and is responsible for cell cycle control
2. Cell culture does not mimic native environment
3. Altered function of p53 and Rb tumour suppressors

Question 38

What affects p53?

Answer 38

E1A, SV40 T-antigen

Question 39

What affects Rb?

Answer 39

E7

Question 40

Normal cells have competent ____ whereas transformed cells do not

Answer 40

growth arrest, growth arrest