Cell Culture

Question 1

Organ culture

Answer 1

The entire embryo or organ is excised from the body and culture

Question 2

Advantages and Disadvantages of organ culture

Answer 2

Advantages
- Normal physiological functions are maintained
-Cells remain fully differentiated
Disadvantages
-Scale-up is not recommeneded
-Difficult maintanence
-Fresh explantation is required for every experiment

Question 3

Tissue culture

Answer 3

Fragments of excised tissue are grown in culture media

Question 4

Ad and Dis of Tissue Culture

Answer 4

Advantages
-Some normal functions may be maintained
-Better than organ culture for scale-up but still not ideal.
-Can prolong tissues indefinitely by submerging them in liquid Nitrogen
Disadvantages
-Original organization of tissue is lost.

Question 5

Cell culture

Answer 5

Tissue from an explant is dispersed, mostly enzymatically, into a cell suspension which may then be cultured as a monolayer or suspension culture.

Question 6

What is celll culture used for

Answer 6

-model systems for studying basic cell biology and interactions between disease- causing agents and cells
-Toxicity testing: new drugs
-Cancer research-How cells turn cancerous
-Virology: vaccine
-Genetic engineering: Large scale production of proteins or viruses
-Gene Therapy: Replace non-functional genes in cell

Question 7

Ad and Dis of cell culture

Answer 7

advantages
-absolute control of physical environment
-Homogeneity of sample
-Less compounds needed than in animal models
-Large upscaling possible
Disadvantages
-Hard to maintain
-Not cheap
-Dedifferentiation (loss of specialized function)
-Instability and aneuploidy after accumulating many mutations after many replications

Question 8

Major developments in cell culture technology

Answer 8

-antibiotics inhibit the growth of contaminants ( penicillin and streptomycin)
-Trypsin removes adherent cells to subculture further from the culture vessel
-Culture medium which uses complex chemicals to provide nutrition to cells

Question 9

Primary culture

Answer 9

these are cells directly taken from tissue but their disadvanatge is they can only replicate a set number of times

Question 10

contact inhibition

Answer 10

It is a growth mechanism which functions to keep cells growing into a o layer one cell thcik

Question 11

Secondary cultures

Answer 11

They are derived from primary cultures and exhibit contact inhibiton and anchorage dependance

Question 12

Immortalised cell line

Answer 12

Cells will keep replicating but are not cancerous.
To prevent mutations, you must stop replicating cells after a certain point

Question 13

Transferd cell line

Answer 13

This occurs when a cell turns cancerous

Question 14

Qualities of cancer cells

Answer 14

• Growth factor independency
  -No response to growth factors
  -Evasion of apoptosis
  -Can promote angiogenesis
  -Unlimted proliferation
  -Invasive

Question 15

What are the 4 cell types

Answer 15

-Fibroblasts
-Epithelial
-Lymphoblastic
-Neuronal

Question 16

Epithelial cells

Answer 16

They are attached to substrate and appear flat and polygonal

Question 17

Lymphoblast cells

Answer 17

Cells remain in suspension with a spherical shape

Question 18

Fibroblast cells

Answer 18

Cells are attached to substrate and appear elongated and bipolar

Question 19

Anchorage dependance

Answer 19

cells that will grow only on suitable substrates. Most cells are like this except for suspension cells

Question 20

What is Mr Frosty

Answer 20

It is a piece of equipment used to freeze cells to eventually reach -178 degrees when cooled in liquid nitrogen. However this device cools the cell down progressively to prevent formation of water crystals

Question 21

Confluency

Answer 21

It is how much of the available space is taken up by cells.

Question 22

What is normal confluency

Answer 22

It is anything between 70%-80% as if they are at 100%, then the cells are in high competition and will be continuously stressed.

Question 23

Adherent cells

Answer 23

These are cells that are anchorage dependant

Question 24

What does EDTA do

Answer 24

It binds to Mg and CA in a cell dish and removes them from the medium. It is added in addition to Trypsin in order to deattach cells

Question 25

Human breast adenocarcinoma

Answer 25

They are cancer cells that do not have hormone receptors and so is hard to develop drugs for.
-mainly affects women of colour

Question 26

Human embryonic kidney cells (HEK)

Answer 26

Epithelial morphology
-used to determine if drug kills healthy cells

Question 27

suspension cells

Answer 27

are not anchorage dependent
-They are easier to culture but harder to assay

Question 28

Why sub culture

Answer 28

In order for cells to be healthy and in a growing stage they must be sub cultured
this usually occurs at 90% confluency
Therefore sub culturing reduces confluency

Question 29

What do cells need to grow?

Answer 29

Substrate or liquid
-For cells to bind to or float in
Nutrients
Environment:
-CO2, temperature at 37, humidity so that they don’t dry out and that the osmolarity of the plate remains constant
Sterility: aseptic technique and antibiotics

Question 30

Why is pH control important

Answer 30

Physiological pH is at 7
pH can affect:
-Cell metabolism
- Growth rate
-Protein synthesis
-Availability of nutrients
CO2 acts as a buffering agent to maintain the same pH.

Question 31

Types of contaminants of cell cultures

Answer 31

Chemical- difficult to detect but could be caused by metal ions or disinfectants
Biological- Cause visible effects on culture and could be yeast, bacteria or fungus

Question 32

MTT assay

Answer 32

-It is used to determine the percentage cell survival in response to a drug, antibody, etc.
-MTT is a yellow salt that becomes converted into purple formazan crystals in the mitochondria of viable cells.
-The absorbance of formazan is directly proportional to cell viability and it is read at 570nm.
-Since formazan is only produced in viable cells, the higher the absorbance of formazan ( the darker the color), the higher the number of viable cells

Question 33

enzyme in MTT

Answer 33

Mitochondrial Succinate Dehydrogenase converts MTT into Formazan

Question 34

How to passage cells

Answer 34

Question 35

Why passage cells

Answer 35

-To maintain cell culture, so that they don’t overgrow and become stressed
-to increase cell number for experiments/storage

Question 36

Factors affecting cell behaviour in Vivo

Answer 36