Cell Culture Techniques

Question 1

What is meant by culturing cells?

Answer 1

Cell/tissue culture: laboratory method (in vitro) by which cells are grown under controlled conditions outside their natural environment.

Question 2

What are the advantages of cell cultures? (7)

Answer 2

Control of the physiochemical environment (pH, temperature, osmolarity..) and physiological conditions (levels of hormones and nutrients)
Control of the micro-environment of the cells (matrix, cell-cell interactions and cell substrates attachment)
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 (cryopreservation)
Cells can be easily quantified
Reduces use of animals in scientific experiments
Cheaper to maintain

Question 3

What are the two types of cell cultures?

Answer 3

Primary tissue cells are not immortalised
The cells are directly derived from living tissue
Able to simulate body environment
Immortalised cell lines are immortal, derived from primary tissue cells
Less heterogenous because they are only derived from one specific type of cell

Question 4

What are the characteristics of primary tissue cells?

Answer 4

Cells derived directly from tissues/patients (unmodified), good for personalised medicine
Finite lifespan (~6-7 divisions)
Cells divide and/or differentiate mainly due to the presence of stem cells in the tissue
Cells carry out normal functions

Question 5

What are the modes of isolation for primary tissue cells? What is the exception?

Answer 5

1. Cells allowed to migrate out of an explant as a way to naturally isolate a certain type of cell such as chondrocytes.
   Some cells do not do that
   
   2. Mechanical (mincing, sieving, pipetting) or/and enzymatic dissociation (trypsin, collagenase, hyaluronidase, protease, DNAase)
      Exception – Haemopoietic cells – Do not need to be disaggregated – They already are as individual cells circulating in blood

Question 6

What are the different methods of isolation for hematopoietic cells?

Answer 6

• Density centrifugation
  This allows us to isolate specific cell populations by taking advantage of the density differences between the leukocytes and the medium we choose to use
  
  • Different mediums of different densities capture different cell populations
  • Immuno- purification
  • Fluorescence activated cell sorter (FACS)
  • Using the last 2 you can isolate cells of interest

Question 7

Name some examples of non-haematopoietic primary cells?

Answer 7

• Liver
  
  • Endothelial cells
  • Muscle
  • Skin
  • Nerves
  • Fibroblasts
  • Prostate

Question 8

Name some examples of haematopietic primary cells?

Answer 8

• Stem, progenitor cells
  
  • T and B cells
  • Monocyte
  • Osteoblasts
  • Dendritic cells
  • Neutrophils
  • Erythrocytes
  • Megakaryocytes, platelets

Question 9

What are the disadvantages of primary cells?

Answer 9

Inter-patient variation makes it difficult to reproduce results
Limited number (small amount at high cost)
Finite lifespan and hard to maintain, require expensive medium
Difficult molecular manipulation
Phenotypic instability
Variable contamination- sensitive to being contaminated

Question 10

What are the characteristics of immortalised cell lines?

Answer 10

Developed through immortalisation of just one type of primary cell tissue
Characteristics:
- Immortalised cells
- Less limited number of cell divisions (~30) or unlimited
- Phenotypically stable, defined population
- Limitless availability
- Easy to grow
- Good reproducibility
- Good model for basic science

Question 11

What are the methods of production of immortalised cell lines?

Answer 11

1. Isolated from cancerous tissues (e.g. HeLa cells)

2. Immortalisation of healthy primary cultures (usually through genetic manipulation)

Question 12

How can we produce cell lines through genetic manipulation?

Answer 12

Production through genetic manipulation:

- To generate cell lines we target processes that regulate cellular growth and ageing 
- P53 is the mitotic checker
- Telomerase elongates the repetitive sequences at the end of the chromosomes, protecting them from degradation

As cells divide over time, telomeres shorten, and eventually cell division stops → Apoptosis (regulated by p53, pRb)
So you have to inhibit p53 and pRb
So p53 and RB inhibition and spontaneous telomere stabilisation through expression of TERT lead to cell immortalisation

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

Taking advantage of viral ‘oncoproteins’
Some oncoviruses inhibit p53 and pRB functions
- Simian virus-40
- Human Papilloma Virus (HPV)

Question 14

How do Simian virus-40 and HPV inhibit p53 and pRB functions?

Answer 14

SV40’s T-antigen interacts with p53 and pRb.
This can cause increased growth without loss of function of these proteins
E6 from HPV targets p53 for degradation, and E7 binds to pRb inactivating it
Cell lines made using E6/ E7 oncoproteins are believed to maintain a differentiated phenotype

Question 15

Why would you introduce a telomerase gene into a target primary cell?

Answer 15

The telomerase gene can also be introduced into a target primary cell.
Some cells need both introduction of the telomerase gene and inactivation of the pRb/p53 for “immortalisation”
→ E6/ E7 and telomerase transformations are believed to result in cell lines with a differentiated phenotype

Question 16

How can you introduce a telomerase gene into a primary target cell?

Answer 16

The part of the telomerase that is usually inactivated in somatic cells is TERT
You make a vector/plasmid containing a selection marker like a neomysin resistance and sequence and code in telomerase as a growth promoting gene through transfection
Then we treat those cells with neomycin and only the cells that have been positively transfected will survive

Question 17

What are 3D cell cultures like?

Answer 17

In 3D cultures we provide a matrix that works as a scaffolding along with other protocols that enables those cells to aggregate and form these types of balls/spheroids

Question 18

What are the advantages and disadvantages of 2D cell cultures?

Answer 18

Advantages:
- Simple and well established
- Affordable
Disadvantages:
- Forced apical-basal polarity
- High stiffness
- Limited communication with other cells
- No diffusion of gradients
- Results not relevant to human physiology

Question 19

What are the advantages and disadvantages of 3D cell cultures?

Answer 19

Advantages:
- Adhesion in all three dimensions
- No forced polarity
- Variable stiffness
- Diffusion gradients of nutrients and waste products
- More relevant to human physiology
Disadvantage:
- More complex
- Added expense

Question 20

What do cell lines generate?

Answer 20

Spheroids
Composed from usually one cell type, do not undergo differentiation or self-organisation
Very homogenous

Question 21

What is generated from primary cell tissue?

Answer 21

Organoids
Derived from stem cells so can self organise, differentiate and continuously top up the cell pool
They mimic in vivo functions so are known as ‘mini organs’
Require a fine tune environment
(remember the origins)

Question 22

How do patient-derived organoids allow the study of cancer drug resistance?

Answer 22

If we extract the biopsy, isolate the cells and culture them in 3D then we can treat the organoids with different drugs and see which are the most effective

Question 23

What is transfection and what are the different types of transfection?

Answer 23

Transfection is the process by which foreign DNA is deliberately introduced into a eukaryotic cell through non-viral methods including both chemical and physical methods in the lab.
e.g. a plasmid, a CRISPR/Cas9 complex
Always performed when cells are growing in 2D otherwise it might not reach all the cells in 3D
Chemical transfection most common example is lipofection
In physical transfection the most popular types are electroporation and nucleofection
Viral infection is the exception to the non-viral methods rule

Question 24

How does lipofection happen?

Answer 24

Liposomes have a net positive charge
The plasma membrane of the cell is negatively charged

1. Interaction with the cell membrane
2. Taken up by endocytosis
3. Material inside (e.g. plasmid) is released from the endosome
4. Transport to the nucleus so plasmids can release their gene
5. Entry to the nucleus inefficient and may need mitosis

Question 25

What could liposomes be used for?

Answer 25

Liposomes as potential drug carriers for drug delivery
Can be used to deliver drugs that are hydrophobic or hydrophilic
We can do this by attaching tissue-specific antigens to the surface of the liposomes to ensure that the drug is delivered into the tissue of interest

Question 26

What is electroporation?

Answer 26

An electric field is applied to the cells of interest in order to increase their permeability, allowing drugs, DNA or chemicals etc. to be introduced into the cells

Question 27

What is nucleofection?

Answer 27

Combination of electroporation and lipofection

• Increased efficiency particularly of non-dividing cells
• Technology is protected under patent so we don’t really know how it works
• Different solution and protocols are used for each cell type

Question 28

What is viral transduction?

Answer 28

Exploits the mechanism of viral infection.

• High transfection efficiency.
• Retrovirus, Adenovirus, but most commonly Lentivirus are used.
• Target cells need to express the viral receptor to work.
• There are safety aspects to consider.