Cell Culture Techniques

Question 1

What is a cell/tissue culture?

Answer 1

• A laboratory method (in vitro) by which cells are grown under controlled conditions outside their natural environment
• in vitro = lab method
• in vivo= natural organsim

Question 2

State the advantages of using cell cultures (8)?

Answer 2

• Control of environment: pH, temperature, osmolarity, levels of hormones, nutrients
• Control of the micro-environment of the cells
• Techniques: Cells can be stored in liquid nitrogen for long periods
• Cells can easily be quantified
• Cells can be easily characterised by cytological or immune-staining techniques and visualised using imaging techniques
• Reduces use of animals in scientific experiments
• Cheaper to maintain

Question 3

State the two types of cells found in cell cultures? VD

Answer 3

• Primary tissue cells
• Immortalised cell lines

Question 4

State characteristics of primary tissue cells? (4)

Answer 4

• Cells derived directly from tissues/patients (unmodified), good for personalised medicine
• Finite lifespan (~6-7 divisions)
• Cells can divide and/or differentiate
• Cells carry out normal functions - that they would normally in their tissue

Question 5

State the 2 main methods for isolation of primary tissue cells?

Answer 5

1. Cells are allowed to migrate out of an explant - natural method of isolation
   a. Explant = cells/tissue removed from body and placed in culture medium
2. Mechanical (mincing, sieving, pipetting) or/and enzymatic dissociation (trypsin, collagenase, hyaluronidase, protease, DNase) of tissue + method of detection of specific cell via immuno-purification

Question 6

State the exception to the standard methods of isolation of primary tissue

Answer 6

• Haemopoietic cells - Do not need to be disaggregated - They already are as individual cells circulating in blood
• HCs are SCs which give rise to other blood cells - any type of cell found in blood

Question 7

State the methods of isolation used here instead?

Answer 7

1. Density centrifugation - separation of components of blood into layers dependent on density using density gradient medium
2. Immuno-purification
3. Fluorescence activated cell sorter (FACS)

Question 8

List examples of primary cells
Non haematopoietic with Haematopoietic

Answer 8

• Liver - Stem, progenitor cells
• Endothelial cells - T and B cells
• Muscle - Monocytes
• Skin - Osteoblasts
• Nerves - Dendritic cells
• Fibroblasts - Neutrophils
• Prostate - Erythrocytes / Platelets

Question 9

State disadvantages of primary cells (6)?

Answer 9

• Inter-patient variation: Different effects due to cells specific to each person
• Limited number (small amount at high cost)
• Finite lifespan and hard to maintain
• Difficult molecular manipulation
• Phenotypic instability
• Variable contamination

Question 10

What are immortal cell lines?

Answer 10

• Cell lines are cultures of animal cells that can be propagated repeatedly and sometimes indefinitely
• They arise from primary cell cultures

Question 11

State characteristics of immortalised cell lines? (7)

Answer 11

• 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 12

State the 2 methods of production of cell lines?

Answer 12

1. Isolated from cancerous tissues (e.g. HeLa cells)
   - Cancerous tissues used as these can grow very quickly or forever
   - If this isn’t possible then
2. Immortalisation of healthy primary cultures (usually through genetic manipulation)

Question 13

How are cells lines produced via genetic manipulation? (Part 1)

Answer 13

• To generate cell lines we target processes that regulate cellular growth and ageing
• Telomerase, P53 and pRb
• As cells divide over time, telomeres shorten, and eventually cell division stops -> Apoptosis (p53, pRb)
• Telomeres shorten after each cell divison due to DNA end replication problem

Question 14

How are cells lines produced via genetic manipulation? (PART 2)

Answer 14

• p53 and pRB are the mediators of apoptosis
• Therefore p53 and pRB are inhibited so telomere stabilisation occurs and ‘immortality’ of cell lines

Question 15

State the two methods of genetic manipulation for cell line
production?

Answer 15

• Inhibition of tumour suppressor proteins
• Introduction of telomerase via transfection

Question 16

Describe the Inhibition of tumour suppressor proteins method of genetic manipulation for cell line production

Answer 16

• Take advantage of viral ‘oncoproteins’
• 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

Question 17

Describe the Introduction of telomerase via transfection method of genetic manipulation for cell line production

Answer 17

• 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 18

Describe the process of introduction of telomerase to form cell lines?

Comparison of primary tissue cell and cell lines

Answer 18

VD

Question 19

State the Key difference between 2D and 3D cell cultures?

Answer 19

• 2D = cells are grown on a flat dish
• 3D = artificially created environment in which cells are permitted to grow or interact with their surroundings in all three dimensions.
• Two types = spheroid and organoid

Question 20

State advantages and disadvantages of 2D cultures?

Answer 20

• Simple, well established
• Affordable |
• Forced apical basal polarity
• High stiffness
• Limited communication with other cells
• No diffusion of gradients
• Results not relevant to human physiology

Question 21

State advantages and disadvantages of 3D cultures?

Answer 21

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

Question 22

State the difference between spheroid and organoid 3D cultures and what they are generated from?

Answer 22

• Spheroid
• Generated from immortalised cell lines
• Composed of 1 cell type which grows and proliferate
• But doesn’t undergo differentiation or self-organisation (non-stem cells)
• Can exhibit enhanced physiological response

Question 23

State the difference between spheroid and organoid 3D cultures and what they are generated from?

Answer 23

• Organoids
• Generated from primary tissue cells
• Composed of stem cells which can differentiate

Question 24

State a usage of patient-derived organoids?

Answer 24

Allows for the study of cancer drug resistance

Question 25

Methods of cell transfection
What is 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 labs e.g. a plasmid, a CRISPR/Cas9 complex.
• Always occurs in 2D

Question 26

State the chemical and physical methods of transfection in the lab?

Answer 26

• Chemical: Lipofection, Calcium Phosphate, Cationic polymer, DEAE-Dextran, Magnetic mediated transfection, Activated dendrimers
• Physical: Electroporation, Nucleofection, Microinjection, Biolistic Particle Delivery, Laserfection/ optoinjection
• Viral: infection

Question 27

State key methods of cell transfection?

Answer 27

• Chemical: Lipofection
• Physical: Electroporation, Nucleofaction
• Viral: Infection

Question 28

State the components of lipofection?

Answer 28

• Liposomes: positive cationic head, linker and hydrophobic tail
• DNA: negatively charged
• When liposomes introduced to plasmid DNA via cationic lipid transfection systems, forms a lipoplexes with a net positive charge

Question 29

State the process of lipofection?

Answer 29

• 1. Interaction with the cell memorane
• 2. Taken up by endocytosis
• 3. Release from the endosome
• 4. Transport to the nucleus
• 5. Entry to the nucleus is inefficient and may lead to mitosis
• Express protein of interest

Question 30

What is the use of liposomes in drug delivery?

Answer 30

Used as potential drug carriers

Question 31

Describe the method of electroporation?

Answer 31

• Plates of capacitor charged
• High electrical field temporarily forms pores on cell membrane of cell and increases permeability of cell. After electroporation the pores will reseal.
• Allows DNA to go through
• Rate of pore resealing is dependent on temperature
• decrease temp, decrease rate of pore resealing

Question 32

What is nucleofaction and what is unique about it?

Answer 32

• Combination of electroporation and lipofection
• Used when the individual methods have been unsuccessful
• Increased efficiency particularly of non-dividing cells
• Technology is protected under patent
• Different solution and protocols are used for each cell type

Question 33

What is viral infection/transduction?

Answer 33

• Exploits the mechanism of viral infection.
• High transfection efficiency.
• Usage: Retrovirus, Adenovirus, but most commonly Lentivirus are used.
• Target cells need to express the viral receptor to work.
• There are safety aspects to consider - as dealing with live virus