Cell Culture Techniques

Question 1

How can you isolate cells from a blood sample using Density centrifugation?

Answer 1

Use density gradient medium

Mix the blood sample with the medium and then centrifuge the mixture.
We can then observe the different layers
More dense - sediment through the medium and therefore isolate them from the bottom later
Less dense - mononuclear - remain in the plasma interface
can then isolate the mononuclear cells

Intermediate layer - full of lymphocytes that have the copies of the germ-line

Question 2

What order the cells form into after a density centrifugation?

Answer 2

1. Plasma
2. PMBCs
   Density Gradient Medium
   Granulocytes and erythrocytes

Question 3

What other techniques can be used?

Answer 3

Immuno-purification:
Antibodies that bind to the cell surface receptor on the cells of interest are used - an antibody that is complimentary to the antigen of interest
The antibodies are coated with magnetic beads
They are then mixed with the sample
The coated antibodies will only bind to the cells of interest
By using a specific magnetic field - can isolate the cells that express the specific antigen

Fluorescence activated cell sorter - FACS
Based on physical properties
can isolate cells by cell surface markers and by size of the molecule but using fluorescence

Question 4

How can you isolate cells from solid tissues?

Answer 4

Interested in Haematopoietic cells
A the tissue is not liquid you need to use mechanical enzymatic disruption
- this is done by passing the tissue between different needles that then disrupts the cells
- then use enzyme that digest e.g trypsin

Question 5

What is an Explant tissue?

Answer 5

It is when you use a tissue that has cells that are spontaneously migrating away from the tissue to then isolate the cells of interest

Chondrocytes - migrate away from the cartilage explant
can pick up the chondrocytes and then isolate them

This doesn’t require the disruption techniques

Question 6

What are primary cells and what are the advantages of using them?

Answer 6

They are cells derived directly from the tissues

• they are unmodified and carry all the genetic information that belong to the specific patient tissue
• they are good for personalised medicine

Question 7

What are the negatives of using primary cells?

Answer 7

• They express other genes - unfunctional expression
• Variable contamination
• Limited
• Short life span
• Inter-patient variation
• Difficult moleuclar manipulation
• Phenotypic instability - cant carry out in other labs as the characteristics vary between the primary cells therefore not reproducible results

Question 8

How and why can cancerous tissue be used in primary cultures?

Answer 8

the cells can be isolated from the cancerous tissue - e.g HeLa cells

once derived from primary cell cultures they can be:

• spontaneously grow from the prolonged culture - as they have multiple mutation that cause them to proliferate excessively
• manipulate the cells genetic information - transform the healthy primary cells to make them immortal to use in the lab

Question 9

What 3 components of cellular growth and ageing are targeted when generating cell lines?

Answer 9

p53
pRb
Telomerase

They are all genetically manipulated to then produce immortal cells

Question 10

What are p53 and pRb?

Answer 10

They are tumour suppressor genes
They are check points in the cell cycle - they look for genomic stability

p53 checks for:

• unreplicated or damaged DNA after G2
• Chromosome misalignment - after M
• Damaged DNA - after G1
• Unreplicated or damaged DNA - during S

Rb binds to E2F and stops transcription whereas unbound E2F will cause transcription

Question 11

What is telomerase?

Answer 11

Prevents the fusion of chromosomes and elongates the chromosomes

Every time the cell divides there is shortening of the chromosomes - due to DNA polymerase unable to completely elongate the telomere
As cell division increases the shortening also increases and then there is damage to the chromosomes
This then activated RB causing apoptosis
THEREFORE
The telomerase is used to elongate the telomere in only the cells that need it (where the enzyme is active)

Cells that have activated telomerase:

• Stem cells
• Gametes
• Cancer cells - why they survive and grow infinitely

Question 12

How do you create immortal cells?

Answer 12

Limit the function of p53 and pRb
Activate telomerase

Some cells need both introduction of the telomerase gene and inactivation of p53 and pRb for immortalisation

Question 13

How can we inhibit the function of tumour suppressor proteins, or introduce telomerase into cells?

Answer 13

Using viral ‘oncoproteins’

Virus:

• Sv40 = has viral oncoprotein - Large/Small T antigen
• HPV = E6/7

They both target p53 and pRb

They also induce telomerase activity

Question 14

What do the T-antigens do?

Answer 14

SV40’s T-antigen
Interacts with p53 and pRb protein domain in the DNA - this is the region the p53 and RB usually bind
They stop the p53 and Rb from binding to the binding regions on the domain - there is no tumour suppression
Cause increased growth without the loss of function of the proteins

Question 15

What do E6/E7 do?

Answer 15

E6 targets p53 for degradation
E7 binds to pRb and inactivates it

Cell lines made by using these oncoprotein are believed to maintain a differentiated phenotype

Question 16

How is telomerase introduced into the target primary cell?

Answer 16

It is introduced into cells by the telomerase gene

Question 17

How are plasmids used in cell line production?

Answer 17

The plasmid with the gene for selection and the growth promoting gene is made - it is used to transfer the primary cells with them
- antibiotic resistance is also mixed into the plasmid

The plasmid is then transfixed onto the plate

Selection pressures are then added - can be a antibiotic

The plasmids should be antibiotic resistant therefore if they have transfected properly they will survive in the cell culture as they will be resistant

Question 18

What are the positive aspects of cell lines?

Answer 18

• Good growth characteristics - standard media
• Phenotypic stability
• Define population
• Molecular manipulation readily achieved
• Good reproducibility
• Good model for basic science

Question 19

What are the conditions and requirements for growth in culture?

Answer 19

Handled under ascetic conditions - ethanol is used to clean everything

Grown on tissue culture treated plastic flasks/dishes - the plates can be of different size

Maintained at 37 degrees with humidified atmosphere - 8% CO2
- need these conditions to represent the human conditions

An ideal supplemented medium

• needs to be replaced by fresh one every 2/3 days = metabolites and nutrients to reach the cell and to remove debris and waste
• should be the perfect pH, have the growth factors and nutrients

Question 20

What happens when the requirements and conditions do not occur?

Answer 20

Lack of any of the conditions leads to arresting of the cells - they will stop growing

They will be alive but they won’t proliferate and divide

Question 21

What is Phenol Red and what is it used for?

Answer 21

It is a medium pH indicator

The balance between metabolites and waste is represented by the colour

The colour of the medium changes based on the presence of metabolites

Yellow - acidic
Red/purple - basic
Red - neutral

Question 22

What is an adherent cell?

Answer 22

Cells that grow attached to a solid surface

It is anchorage dependent

Agitation is not required

Trypsinisation is required

Tissue culture treated vessels are required

Yield is low

Growth is limited by the surface area

Types of cells - most types of cell lines and primary cultures

Question 23

What is a suspension cell?

Answer 23

Cells that grow suspended in a liquid medium

They are anchorage-independent

Continuous agitation is required

Trypsinisation is not required

Tissue culture treated vessels are not required

Yield is high

Growth is limited by the concentration of cells in the medium

Types of cells - some non-adhesive cell lines such as hematopoietic

Question 24

How can cell cultures be contaminated?

Answer 24

1. Microbial contamination
   - Bacteria
   - Yeast
   - Fungus
   - Mycoplasma
   - Virus
2. Cell lines
   - Poor tissue culture technique
   - Culture of multiple cell lines at one time
   - Accidental mixing of cell lines

Question 25

What kind of changes do the microbial contaminations cause?

Answer 25

Bacteria

• pH change
• Cloudiness
• Precipitation
• Stink

Yeast

• Cloudiness
• pH change

Fungus

• Spores furry growth
• pH change

Mycoplasma

• they often convert
• Poor cell adherent
• Reduced cell growth

Virus
- Can sometimes be cytopathic

Question 26

What are the negatives of using cell lines?

Answer 26

• Often lose differentiated function
• Cell substrate interactions dominate
• Does not mimic real tumour conditions
• Lacks cells heterogeneity
• Phenotype needs to be validated

Question 27

What is an example of a new in vitro model?

Answer 27

3D models - the cell culture is now 3D

Can be a 3D spheroid culture or an 3D organdie culture

Question 28

What are the differences between organoid and spheroid cultures?

Answer 28

Organoids are derived from stem cells whereas spheroids are derived from cell line monoculture

• organoids are good for drug resistance studies
• spheroids are good to observe the same result

Organoids are from multiple cell lineages whereas spheroids only represent single/partial tissue components

Organoids also reacpitualte organ physiological parameters whereas spheroids only resemble cell organisation

Organoids also are used fro long term culture whereas spheroids are difficult to maintain long term

Question 29

How do organoids allow the study of cancer drug resistance?

Answer 29

Tumour biopsy is taken
Then turn into an organdie cell culture
- the biopsy can be genome profiled to see if there are any new drug targets

Then treat the organoids with the new drug/treatment
- if there is resistance you can do mutational analysis by undergoing genomic profiling

Then use the treated/passed organoids for survival analysis, histology or optical metabolic imaging

Question 30

What are the advantages of organoids?

Answer 30

• Gene expression is similar to in vivo methods
• Cell-cell communication re-established
• Cells are orientated in same ways as tissue
• Ideal platform for individualised therapeutic screening

Question 31

What are the limitations of organoids?

Answer 31

• Limited amount of tissue in some cases
• Organoids in the same culture as heterogenous
• Absence of immune cells in culture system
• Unable to mimic in vivo growth factors/signalling gradients - therefore cant replicate the exact conditions and get the accurate results

Question 32

What is transfection?

Answer 32

It is the process by which foreign DNA is deliberately introduced into a eukaryotic cell through non-viral methods
- includes chemical and physical methods in the lab

Using viral methods = infection

Question 33

What is lipofection?

Answer 33

Method uses the cationic nature of the head of liposomes
liposomes - small vesicles that are surrounded by bilayer of phospholipids

The lipid heads are positive and the DNA is negative - introduce the DNA into the liposomes and they are then taken up into the cells by endocytosis

1. Lipoplexes are positively charged and the membrane is negatively charged - interaction of the lipoplex with the cell membrane of cell
2. taken up by endocytosis
3. lipoplex is released from the endosome
4. transport to the nucleus
5. entry to the nucleus is inefficient and may need mitosis

can make it tissue specific by attaching antigens to the liposomes - make sure the liposomes only go to the cells of interest
- can be potential drug carriers for drug delivery

Question 34

What is electroporation?

Answer 34

The cells with the plasmid DNA are in a medium with plates of a capacitor

High electric field - forms pores which then reseal
- The plasmid DNA then enters the cell when the pores are opened

The rate of pore sealing is dependent on the temperature

Question 35

What is nucleofection?

Answer 35

Combination of electroporation and lipofection
- lipofection can be toxic to cells sometimes

increased efficiency particularly of non-dividing cells - the transfection is straight into the nucleus - faster

Technology is protected under patent

Different solution and portals are used for each cell type

Question 36

What is viral infection?

Answer 36

• 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.