Cell Culture Techniques

Question 1

What are the characteristics of primary cells culture?

Answer 1

1. cells derived directlly from tissues (unmodified)
2. finite lifespan (~6-7 divisions)
3. can be grown in 2D or 3D
4. cells divide and/or differentiate
5. cells carry out normal functions
6. good for personalised medicine (from pt)

Question 2

Describe some methods of isolating cells from a biopsy to form a primary cell culture

Answer 2

1. cells allowed to migrate out of explant
2. Mechanical (mincing, sieving, pipetting) or/and enzymatic dissociation (trypsin, collagenase, hyaluronidase, protease, DNAase)
   - - exception is haematopoietic cells – Do not need to be disaggregated – They already are
3. density centrifugation (less dense at top/most dense at bottom)
4. immuno-purification (using antibodies attracted to antigen-expressing cells)
5. fluorescence activated cell sorter (FACS) - (using different fluorescent markers)

Question 3

State some examples of non-haematopoietic primary cells

Answer 3

any kind of cell you can extract from tissue organs other than blood
liver, endothelial cells, muscle, skin, nerves, fibroblasts

Question 4

State some examples of haematopoietic primary cells

Answer 4

stem cells/progenitor cells
T and B cells
monocyte, macrophages
osteoblasts
dendritic cells
neutrophils
erythrocytes
megakaryocytes, platelets

Question 5

What are some disadvantages of primary cells culture?

Answer 5

• Interpatient variation (no reproducibility as genetics different)
• Limited growth - only grow small amount at high cost (expensive medium, need a lot of care)
• Finite lifespan
• Difficult molecular manipulation
• Phenotypic instability
• Abnormal expression of some genes
• Variable contamination (e.g. bring bacteria from body)

Question 6

What are the characteristics of cell lines culture?

Answer 6

• immortalised cells (one type of primary cell)
• less limited number of cell divisions (~30) or unlimited (inexpensive medium)
• can be grown in 2D or 3D
• phenotypically stable (keep morphology and behaviour thru divisions), defined population (same type of cell)
• limitless availability
• easy to grow
• good reproducibility (e.g. so can validate results)
• good model for basic science

Question 7

Describe some methods of production for cell lines culture

Answer 7

1. isolated from cancerous cells (e.g. HeLa cells) (immortal by nature)
2. immortalisation of primary cultures (from non-cancerous tissues):
   a) spontaneously from prolonged culture: multiple ill-defined mutations transformed phenotype
   b) thru genetic manipulation: artificial transformation of healthy primary cells

Question 8

How do we produce cell lines through genetic manipulation?

Answer 8

• we need to make the cells immortal by targetting processes that regulate cellular growth and ageing
  – for example; thru p53 or retinoblastoma that are tumour suppressor genes. They can slow/stop at a specific point in cell cycle.
  Or activate or introduce the telomerase (enzyme normally present in germ cells or adult stem cells or cancer cells but not somatic cells)

Question 9

What is the role of telomerase?

Answer 9

To elongate the telomeres.

Question 10

What is the role of the telomeres?

Answer 10

end caps that protect the chromosome

Question 11

What normally happens to telomeres which results in apoptosis?

Answer 11

• Normally, as cells divide over time, telomeres shorten and eventually cell division stops as two key genes are affected that flank the telomere which initiates apoptosis. This happens thru p53 and pRb.

Question 12

How can cells be genetically manipulated in order to immortalise them?

Answer 12

• RB and/or p53 are inactivated to inhibit cell death mechanisms and stabilisation of telomeres is also inhibitted via telomerase.

Question 13

How can we inhibit the function of tumour suppressor proteins, or introduce telomerase in order to alter a cell’s capability for its finite number of divisions?

Answer 13

By taking advantage of viral ‘oncoproteins’ and transfecting the cell with telomere gene.