Cell Culture Techniques

Question 1

History of Cell Culture

Answer 1

1882: Sidney Ringer develops solutions of salt to maintain frog heart

1885: Wilhelm Roux cultures embryonic chick tissue

1940-50: Development of cell culture techniques for growing viruses

1951: Jonas Salk and his team grow polio virus in monkey kidney cells

1951: George Otto Gey propagates HeLa cells from Henrietta Lacks – ‘The Immortal Life of Henrietta Lacks’ – they have been used in ~75,000 studies

1954: Enders, Weller and Robbins receive Nobel prize

Question 2

Define Cell/Tissue culture

Answer 2

Laboratory method (in vitro) by which cells are grown under controlled conditions outside their natural environment

Question 3

List some advantage of cell culture

Answer 3

• 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 4

What are the types of cells in culture

Answer 4

• Primary tissue cell
• Immortalised cell lines

Question 5

What are the differences between primary tissue cells and immortalised cell lines

Answer 5

Primary Tissue cells:
- Limited lifespan retains cell identity
- Taken from the tissue
- Pre-characterised and ready to use
- study cells with varied donor characteristics

Immortalised cell lines:
- Infinite lifespan, loses cell specificity
- from a vial with high mutations and clonal selections
- authentication required before use
- study single donor repeatedly

Question 6

What are the characteristics of primary tissue cells

Answer 6

• Cells derived directly from tissues/patients (unmodified), good for personalised medicine
• Finite lifespan (~6-7 divisions)
• Cells divide and/or differentiate
• Cells carry out normal functions

Question 7

What are the methods of isolation for primary tissue cells

Answer 7

• Cells allowed to migrate out of an explant
• Mechanical and/or 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 8

What are the methods of isolation for hematopoietic cells

Answer 8

• Density centrifugation
• Immune-purification
• Fluorescence activated cell sorter (FACS)

Question 9

Give some examples of Non-haematopoetic primary cells

Answer 9

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

Question 10

Give some examples of haematopoetic primary cells

Answer 10

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

Question 11

What are some disadvantages of primary cells

Answer 11

• Inter-patient variation
• Limited number (small amount at high cost)
• Finite lifespan and hard-to-maintain
• Difficult molecular manipulation
• Phenotypic instability
• Variable contamination

Question 12

What are some characteristics of Cell lines

Answer 12

• 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 13

What are some methods of production of cell lines

Answer 13

• Isolated from cancerous tissues (e.g. HeLa cells)
• Immortalisation of healthy primary cultures (usually through genetic manipulation)

Question 14

Describe the production of cell lines through genetic manipulation

Answer 14

• To generate cell lines we target processes that regulate cellular growth and ageing (p53, pRb and Telomerase)
• As cells divide over time, telomeres shorten, and eventually cell division stops (hayflick’s limit)→ Apoptosis (p53, pRb)
• Inactivation of p53 and RB can cause immortality

Question 15

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 15

• Take advantage of viral oncoproteins which are able to target p53 and RB

Simian Virus-40
Human Papiloma virus

Question 16

How does the viral oncoprotein work

Answer 16

• SV40’s T-antigen interacts with p53 and pRb. This can cause increased growth without loss of function of these proteins
• E6 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 17

How does telomerase play a role in cell immortalisation

Answer 17

• The telomerase gene can also be introduced into a target primary cell.
• Some cells need both introductions 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 18

How is TERT transfected into cells

Answer 18

• Used plasmid which contains genes for neomycin resistance and TERT
• Plasmid is circularised and transfected into cells
• Cells grown in neomycin solution to only produce colonies which have been positively transfected

Question 19

What is the difference between 2D and 3D cell cultures

Answer 19

2D is grown on a flat surface whereas 3D is grown in a matrix either in a spheroid or organoid culture

Question 20

What are 3D cultures

Answer 20

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

Question 21

Compare 2D and 3D cultures

Answer 21

2D Cultures:
- Forced apical-basal polarity
- High stiffness
- Limited communication with other cells
- No diffusion of gradients
- Results not relevant to human physiology
- Simple, well established
- Affordable

3D Cultures:
- Adhesion in all three dimensions
- No forced polarity
- Variable stiffness
- Diffusion gradients of nutrients and waste products
- More relevant to human physiology
- More complex
- Added expense

Question 22

Describe what is a Spheroid culture

Answer 22

Generated from cell lines

A 3d cellular aggregate is composed of one or more cell types that grow and proliferate.
May exhibit enhanced physiological responses but do not undergo differentiation or self-organisation

Question 23

Describe what is a Organoid culture

Answer 23

Generated from primary tissue

3d structure derived from either PSCs, neonatal tissue stem cells or adult progenitors in which they can self-organisation and differentiate. Can resemble their in vivo counterparts and recapitulate some functions of organs

Question 24

How are patient derived organdies good for cancer drug resistance studies

Answer 24

• biopsy cell straight from the patient
• can create organoids from the tumour biopsy cells
• test which drugs on the organoid model
• use the results to better treat the tumour in the patient

Question 25

Define transfection

Answer 25

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

Question 26

Explain Lipofection

Answer 26

• Introduced DNA into cells using lysosomes
• can easily merge with cell membrane as they share the same membrane structure
• Liposomes as potential drug carriers for drug delivery

1. Positively charged lipoplexes interaction with cell membrane
2. Taken up by endocytosis
3. Release from the endosome
4. Transport to the nucleus
5. Entry into nucleus inefficient and may need mitosis

Question 27

Explain Electroporation

Answer 27

• An electric field is applied to cells which increases their permeability
• allows entry of DNA into the cells from the pores created

Question 28

Explain Nucleofection

Answer 28

• Combination of electroporation and lipofection
• Increased efficiency particularly of non-dividing cells
• Technology is protected under patent
• Different solution and protocols are used for each cell type

Question 29

Explain Viral infection/transduction

Answer 29

• 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.