1.) CELL CULTURE - TECHNIQUES FOR PRIMARY CELL ISOLATION AND TISSUE CULTURE

Question 1

Define the term “Tissue/Cell Culture”?

Answer 1

Artificial growth under controlled conditions, of tissues and/or cells outside a living organism.

Question 2

Define the term “Cell Culture”?

Answer 2

Isolated primary cells or established cell lines.

Question 3

Define the term “Organ Culture”?

Answer 3

Whole organs can be maintained four hours/days by perfusion with oxygenated blood/serum. Use for metabolism and drug studies.

Question 4

Define the term “Explant Culture”?

Answer 4

Removes tissue and culture growth to produce primary cell culture.

Question 5

Define the Human Tissue Act 2004?

Answer 5

• Regulates activity regarding removal, storage, use and disposal of human tissue samples.
• Patient consent is needed for human tissue samples and ownership must be defined.
• Transfer of cell lines from labs must require a Material-Transfer Agreement

Question 6

Define the term Primary Cell Culture?

Answer 6

• Culture started from culture directly from tissues/organ.
• Cells are heterogenous, representing parent cell types and express tissue-specific properties.
• Culture is primary until subcultured from the first time then it’s a cell line.

Question 7

Define the term Continuous Cell Line?

Answer 7

Culture capable of an unlimited number of population doublings (i.e immortal). A cell line shouldn’t be considered continuous unless grown in culture for 6 months

Question 8

As cells continue to divide in culture, they generally grow to fill the available area/volume, what can cause cells to die?

Answer 8

• nutrient depletion
• accumulation of apoptotic/necrotic cells
• contact inhibition - cell-to-cell contact can stimulate cell cycle arrest, causing cells to stop dividing
• cell-to-cell contact can stimulate unwanted cell differentiation

Question 9

Define the term Splitting/passaging cells:

Answer 9

• Transferring a small number of cells into a new vessel.
• Suspension cells are diluted in fresh media.
• Adherent cells are detached from the substrate with enzymes, usually trypsin or trypsin/EDTA and seeded in fresh media.
• With microcarriers, it’s needed to only dilute fresh beads, enzymes are not needed.
• Different cell lines need different splitting rations - (1:3-10) but some cultures won’t grow unless a min cell concentration is added.

Question 10

Cells should not be passaged/sub-cultured continuously, why is that?

Answer 10

• The Number of passages depends on the cell line.

- Passaging should be minimal to reduce the possibility of phenotypic/genetic drift and contamination.

Question 11

Cell-Lines with a high passage number can have alterations in

Answer 11

• Cell Morphology
• Response to stimuli
• Growth Rates
• Protein Expression
• Transfection efficiencies
• Signalling

Question 12

What is the conditions for cell culture

Answer 12

• Gas Phase
• Temperature
• High Humidity

Question 13

Explain the Gas Phase in terms of the conditions of for cell culture?

Answer 13

-21% 02 Normal Atmospheric
-5% CO2 Good Buffering Capacity
Helps maintain culture pH (7-7.6) when used with biocarbonate buffers (need loose/gas permeable lids on culture flasks).

Question 14

Explain the ideal temperature with regards to ideal Cell Culture?

Answer 14

-37 degrees Celcius

Question 15

Explain the high humidity with regards to ideal cell culture?

Answer 15

Reduce evaporation which causes cells to be hypertonic.

Question 16

Explain Growth Media, what is the standard’s that it’s based upon?

Answer 16

• Based on standard salt mixes such as Earle’s salts - Eagles minimum essential medium.
• Many contain AA, glucose, vitamins, minerals, electrolytes, serum, GF’s, anti-oxidants, cell stablisers.
• Usually contain bio carbonate, which is buffered w/ 5% CO2 in the gas phase, helping to maintain the pH at 7.4.

Question 17

In growth Media, if a CO2 incubator isn’t available what is used instead?

Answer 17

• Experiment is carried out in the open, and the medium is supplemented with HEPES buffer to maintain the pH.

Question 18

Sometimes in Growth Media FCS is added, what is the significance of this?

Answer 18

It’s added to the 5-20% final concentration, containing Growth Factors and hormones. It contains PDGF, which stimulates fibroblast production. However, it’s still unknown and it’s should be batch tested.
Defined media doesn’t use FCS, replace with EGF and hydrocortisone.

Question 19

Why should media be regularly changed?

Answer 19

• Replenish nutrients
• Avoid the build-up of harmful metabolites by-products and dead cells.
• Suspension cells - (pellet cells and resuspend in fresh media)
• Adherent cells - remove media and replace

Question 20

Explain the significance of the Extracellular Matrix?

Answer 20

• It’s an important variable when trying to establish a cell line.
• It’s composed of proteins, glycoproteins, glycolipids and lipids.
• It’s digested by Proteolytic Enzymes.

Question 21

What are Speheroid 3D Cell Cultures?

Answer 21

• Sphere-shaped cell colonies grown in a viscous scaffold.

- It mimics the growth of naturally occurring human tumours.