Cell Culture Techniques

Question 1

What is a cell culture?

Answer 1

Laboratory method (IN VITRO) by which cells are grown under controlled conditions outside of they’re natural environment.

Question 2

What are the advantages of cell cultures?

Answer 2

• control of the physiochemical environment (pH, temperature) and physiological environment (levels of hormone and nutrients)
• control of micro-environment of cells (matrix, cell-cell interactions)
• cells can be easily characterised by cytological or immune staining techniques and visualised using imaging techniques
• cells can be stored in liquid nitrogen for long periods of time
• cells can be easily quantified
• reduces use of animals in scientific experiments
• cheaper to maintain

Question 3

What is the difference between primary tissue cells and immortalised cell lines?

Answer 3

Primary living cells:
- taking from living tissues
- limited lifespan retains cell identitiy
- pre-characterised and ready to use
- study cells with varied donor characteristics

Immortalised cell lines:
- derived from one cell
- infinite, lifespan, loses cell specifity
- from a vial with high mutations and colonal selections
- authentication required before use
- study single donor repeatedly

Question 4

What are primary tissue cells?

Answer 4

Cells derived directly from tissue/patients, good for personalised medicine

Finite lifespan (6-7 divisions)

Cells divide and/or differentiate

Cells carry out normal functions

Question 5

What methods of isolations are used for primary tissue cells?

Answer 5

1. Cells allowed to migrate out of explant
2. Mechanical (mincing, sieving, pipetting) or enzymatically (trypsin, protease, DNAase) isolate the tissue

ONLY EXCEPTION - haematopoetic cells do not need to be disaggregated - they already are as individual cells circulating blood

3. Haematopoetic cells are separated using:
   - density centrifugation
   - immuno-purification (mixed with antibody coated magnetic beads and antigen expressing cells are isolated)
   - fluorescent activated cell sorter (FACS)

Question 6

Give some examples of non-haematopoetic primary cells?

Answer 6

• liver
• skin
• endothelial cells
• muscle
• nerves
• fibroblasts
• prostate

Question 7

Give some examples of haematopoetix primary cells?

Answer 7

• T cells and B cells
• stem progenitor cells
• dendritic cells
• monocyte
• neutrophils
• erythrocytes
• megakaryotyes
• osteoblasts

Question 8

What are the disadvantages of primary tissue cells?

Answer 8

• inner patient variation
• limited number (small amount at high cost)
• finite lifespan and hard to maintain
• difficult molecular manipulation
• phenotypic instability (have different characteristics)
• variable contamination

Question 9

What are cell lines?

Answer 9

Immortalised cells

Less limited number of cell divisions (~30) or unlimited

Phenotypically stable, define population

Limitless availability

Easy to grow

Good reproducibility

Good model for basic science

Question 10

What are methods for producing cell lines?

Answer 10

1. Isolated from cancerous tissues
2. Immortalisation of healthy primary cultures (genetic manipulation)

Question 11

How can cell lines be produced from genetic manipulation?

Answer 11

Targeting processes that regulate cellular growth and ageing

• p53 inactivation
• telomerase activated
• pRb inactivation

As cells divide over time, telomeres shorten , and eventually cell division stops (APOPTOSIS)

Question 12

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

Answer 12

Taking advantage of viral “Oncoproteins”

SV40:
- SV40 T antigen interacts with p53 and pRb
- This can cause increased growth without loss of function of these proteins

E6+E7:
- targets p53 for degradation, and E7 binds to pRb inactivating it
USUAL METHOD

Telomerase gene can also be introduced into target primary cell - using plasmid cultures with antibiotics

SOME CELLS NEED BOTH INTRODUCTION OF TELOMERASE GENE AND INACTIVATION OF THE PRB/p53 for “immortalisation”

Question 13

What is a 3D cultures?

Answer 13

Artificially created environment in which cells are permitted to grow or interact with their surroundings in all three dimensions

Question 14

What are the advantages of using 3D cultures?

Answer 14

• adhesion in all three dimensions
• no forced polarity
• variable stiffness
• more relevant to human physiology
• diffusion gradients of nutrients and waste products

Question 15

What are the negatives of 3D cell cultures?

Answer 15

• more complex
• added expenses

Question 16

What are the advantages of using 2D cell cultures?

Answer 16

• Simple, well established
• affordable

Question 17

What are the disadvantages of the 2D cell culture?

Answer 17

• forced apical-based polarity
• high stiffness
• limited communication with other cells
• no diffusion of gradients
• results not relevant to human physiology

Question 18

What are the two main types of 3D cultures?

Answer 18

Spheroids
- generated from cell lines
- no cell differentiation or organisation
- usually one cell type or two max

Organoids
- derived from primary tissues
- derived from stem cells in the adult tissue
- can self organise into properly differentiated cell types and progenitors
- recapitulate come function of the organ “mini organs”

Question 19

How can organoiods allow the study of cancer drug treatment?

Answer 19

Patient derived organoids from tumour biopsy

We can treat the mini organ with different drugs to see which drugs are more effective with treating cancer cells

Can extrapolate results and make treatment decisions

Question 20

What is cell transfection?

Answer 20

The process by which foreign DNA is deliberately introduced into a eukaryotic cell through NON VIRAL METHODS including both chemical and physical methods

Always performed in 2D because in 3D the transfection might not meet all the cells

Question 21

What is lipofection?

Answer 21

A transfection technique that introduces DNA into the cell using liposomes

Liposomes are vesicles that can easily merge with cell membrane

Liposomes have a net posative charge

Liposomes can be used as potential drug carriers for drug delivery

Plasmid membrane of cells is negatively charged

Question 22

Describe what happens during lipofection?

Answer 22

1. Interaction with the cell membrane
2. Taken up by ENDO cytosol
3. Release from the ENDO some
4. Transport to the nucleus
5. Entry to the nucleus inefficient and may need mitosis

Question 23

What is electroporation?

Answer 23

A type of transfection where a electric field is applied to the cell to increase they’re permiability, allowing drugs,DNA, to enter the cell (in plasmid)

Question 24

What is nucleofection?

Answer 24

Combination of electroporation and lipofection

Increased efficiency particularly if non-dividing cells

Technology is protected under patient

Different solution and protocols are used for each cell type.

Question 25

What is viral infection/transduction ?

Answer 25

A type of transfection that exploits the mechanism of viral infection High transfection efficiency Retrovirus, Adenovirus, but most commonly, lentivirus Target cells need to express viral receptor to work Saftey aspects to consider