CELL CULTURE TECHNIQUES

Question 1

Name some characteristics of Primary Cell Cultures

Answer 1

Characteristics of primary cell cultures include: Cells derived directly from tissues (unmodified) Finite lifespan (~6-7 divisions) They can be grown in 2D or 3D Cells divide and/or differentiate Cells carry out normal functions Good for personalised medicine

Question 2

What are examples of methods of isolation of cells?

Answer 2

1. Cells allowed to migrate out of an explant 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 In which case, we can use eg density centrifugation and we can use eg Immuno-purification (uses antibodies) or Fluorescence activated cell sorter (FACS) (uses fluorescent markers) to isolate specific cells

Question 3

What are some examples of non-haematopoietic and haematopoietic primary cells?

Answer 3

Non-haematopoietic: Liver Endothelial cells Muscle Skin Nerves Fibroblasts Haematopoietic: Stem, progenitor cells T and B cells Monocytes, macrophages Osteoblasts Dendritic cells Neutrophils Dendritic cells Erythrocytes Platelets

Question 4

What are some disadvantages of primary cells?

Answer 4

Inter-patient variation Limited Finite lifespan (ie dont survive for longer than a week) Difficult molecular manipulation Phenotypic instability Aberrant expression of some genes Variable contamination

Question 5

What are some characteristics of Cell line cultures?

Answer 5

Immortalised cells Less limited number of cell divisions (~30) or unlimited (whereas much more limited with primary cells) They can be grown in 2D or 3D Phenotypically stable, defined population Limitless availability Easy to grow Good reproducibility Good model for basic science

Question 6

What are some methods of production for cell line cultures?

Answer 6

. Isolated from cancerous tissues (e.g. HeLa cells) 2. Immortalisation of primary cultures: a) Spontaneously from prolonged culture: multiple ill-defined mutations transformed phenotype b) Through genetic manipulation: artificial transformation of healthy primary cells Production through genetic manipulation To generate cell lines we target processes that regulate cellular growth and ageing - As cells divide over time, telomeres shorten, and eventually cell division stops → Apoptosis (p53, pRb)

Question 7

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 7

taking advantage of viral ‘oncoproteins Oncoproteins are proteins encoded by oncogenes which are involved in the regulation or synthesis of proteins linked to cancer tumour cell growth. Current oncoprotein research focuses on antibodies that can directly target the oncoproteins inside cancer cells and suppress aggressive cancer growth. eg virus: Human Papilloma Virus (HPV) viral oncoprotein: E6 E7 cellular targets: p53 pRb HPV’s 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 - 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”

basically nicola’s voicenote made sense but the above doesnt:

so looking at the second part of ‘finite’ so in a primary cell, there is a finite number that the cell can divide and if it divides more than that number, it will die-apoptosis and that number is 50(hayflicks constant!?) but that is what we want to prevent! we want to turn primary cells into cell lines so we can use as a culture add drugs etc becuase we can’t directly do that to primary cells as they will die (ie adding stuff to see how they respond etc),

we therefore need to do 2 things:

1. remove tumour supressors from primary cells
2. induce telomerase (you want to integrate telomerase into your primary cells) (which affects telomeres by adding a specific sequence to the end of the ) chromosome) but essentilaly telomerase is causing the cancer eg to be prominent- it allows the cancer to have that infinite number of divisions, and causes the tumour supressors to not supress the tumour.

the break and the accelerator analogy:

the break is the tumour suppressor- so you get rid of that (foot now on pedal) but you want the telomerase but essentially theres no stop to it- uncontrolled division

Question 8

What conditions and requirements are required for growth in cultures?

Answer 8

-Handled under aseptic conditions - Grown on tissue culture treated plastic flasks/dishes -Maintained in a warm (37°C) humidified atmosphere (5% CO2) -ideal supplemented medium that needs replacing every 2/3 days! if medium is yellow=acidic, if medium is tomato red= neutral, if medium is red to purple in colour= basic

Question 9

What are types of cell culture contamination that are a potential hazard?

Answer 9

• Bacteria (pH change, cloudiness/turbidity, precipitation, stink) - Yeast (cloudiness, pH change) - Fungus (spores furry growths, pH change) - Mycoplasma (often covert, poor cell adherent, reduced cell growth) - Virus (sometimes cytopathic)

Question 10

What can cause cell line cross contamination?

Answer 10

• Poor tissue culture technique - Culture of multiple cell lines at one time - Accidental mixing of cell lines

Question 11

What are some differences between organoid and spheroid 3D cultures?

Answer 11

cells are grown in suspension, which bunch together and form a bigger blob of cells (known as organoids or spheroids) organoids and spheroids are helpful for modelling specific organs in the body Organoid: derived from stem cells multiple cell lineages long term culture eg so you can take a biopsy/sample of the tumour and grow organoid samples so you can test different drugs etc before giving it to the patient! Spheroid: derived from cell line monoculture represent single/partial tissue components difficult to maintain long term

Question 12

What are some advantages of organoids?

Answer 12

Advantages: - Gene expression as in vivo (87% phenotype and genotype similarity) - Cells-cell communication re-established - Cells are orientated in same ways as tissue - Ideal platform for individualized therapeutic screening

Question 13

What are some limitations of organoids?

Answer 13

Limitations: - Limited amount of tissue in some cases (e.g. prostate) - Organoids in the same culture are heterogeneous - Absence of immune cells in culture system - Unable to mimic in vivo growth factor/signalling gradients

Question 14

What is transfection?

Answer 14

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 15

What does lipofection involve? (as a method of transfection)

Answer 15

Using cationic lipid transfection systems Lipofection- introduces DNA into cells through liposomes(vesicles which can easily merge with the cell membrane of the eukaryotic cell because liposomes are made with phospholipid bilayers) liposomes have a net positive charge 1. Interaction with the cell membrane 2. Taken up by endocytosis 3. Release from the endosome 4. Transport to the nucleus 5. Entry to the nucleus inefficient and may need mitosis so cell expresses gene/material we have introduced better used for when material is small so liposomes are used as potential drug delivery carriers

Question 16

What does electroporation involve? (as a method of transfection)

Answer 16

where an electric field is applied to cells to increase their permeability to allow chemicals/drugs etc to be introduced into cells etc

Question 17

What is nucleofection? (as a method of transfection)

Question 18

What does viral infection/transduction involve? *not a method of transfection

Question 19

Summary of the lecture

Question 20

Comparison summary table of primary cells and cell lines

Question 21

What is density centrifugation of blood?

Answer 21

1. Density centrifugation
   o Gradient formed by centrifugation

o Cells/sample placed on top of gradient and centrifuged
o Each subcellular component will move up or down when centrifuged until it reaches a position where its density matches its surroundings and then will move no further  distinct bands formed  most dense at bottom
o If you use blood can isolate different blood cell population using density medium using density centrifugation method. Advantage of diff density of diff cell pop. e.g Ficoll (density of 1.077 g/ml).
o Mix sample with Ficoll after centrifugation we can see the different layers.
- Granulocytes / RBCs are denser than mononuclear cells, the sediment with the density gradient medium so we can isolate them from the bottom layer of the differential layers.
- Less dense mononuclear usually remain in the top layer of the plasma.
- For example lymphocytes can try to isolate the white layer (buffing coat), all lymphocytes usually stay there.

Question 22

What is FACS?

Answer 22

o Label surface of cells with specific marker for particular cell type
o Laser used to detect which cells are labelled

o Labelled cells diverted to particular tube (see flow cytometry)
o Can isolate cells based on size and markers

Question 23

What is immuno-purification?

Answer 23

o Antibodies attracted to magnetic beads
o Antibody binds to antigen on cell surface of protein

o Magnet used to attract proteins with magnetic beads  purified (other unwanted proteins filtered out)

Question 24

How do we isolate from solid tissues?

Answer 24

o Mechanical and enzymatic disruption (e.g. collagenase, dispase, trypsin)
o Can also isolate cells using explant culture – tissue harvested in aseptic manner, often minced, and pieces placed in cell culture dish containing growth media. Over time progenitor cells migrate out of the tissue and onto the surface of the dish. (these cells can then be further expanded and transferred into fresh dishes)

o e.g. placenta, the haemopoietic stem cell can diff into any blood cell lineage.

Question 25

How do we maintain good cell cultures?

Answer 25

o Isolated and maintained under aseptic conditions – reducing possibility of contamination, protecting cells and individual researching (in case cells from humans are infected)
o Cells grown on treated plastic – plates positively charged (as cells negative), also contains ECF proteins, growth media

o Maintained in warm humidified atmosphere
o Antibiotics also added to prevent contamination
o Glutamine added for eliminating ammonia
o Factors important for growth: temperature, pH, nutrients, GFs (via serum or recombinant proteins)

Question 26

What are the pros and cons of primary cells?

Answer 26

o Derived directly from tissue
o Positive aspects:

+ Good for personalised medicine
+ Unmodified
o Negative aspects:
- Aberrant expression of genes (genetic errors more likely if cultured for longer)
- Variable contamination
- Poor growth characteristics, esp. human primary cells
- Interpatient variation – variation in responses of cells likely
- Phenotypic instability – e.g. loss of receptors after hours of isolation, etc
- Molecular manipulation difficult

Question 27

What are the pros and cons of cell lines?

Answer 27

o Positive aspects:
+ Good growth characteristics. Standard media

\+ Phenotypic stability
\+ Defined population
\+ Molecular manipulation readily achieved
\+ Good reproducibility
\+ Good model for basic science
o Negative aspects:
- Often lose differentiated function
- Cell-substrate interactions dominate
- Does not mimic real tumour conditions
- Lacks cells heterogeneity
- Phenotype needs to be validated

Question 28

How do we produce cell lines?

Answer 28

Production of cell lines:
o Isolated from cancerous tissues (e.g., HeLa cells, can survive by them self without manipulation, however they can be manipulated to become immortal).

o Derive from primary cultures
o Can be spontaneous:
o From tumours or prolonged culture – cells tend to be poorly differentiated, since abnormal, something has triggered them to keep dividing
o Multiple ill-defined mutations – longer we work with culture, more likely to find out mutation
o Transformed phenotype
o From genetic manipulation:
o Transformation of healthy primary cells
o To generate cell lines, we target processes that regulate growth and ageing, examples:
o P53 – antioncogene/TSG
o Retinoblastoma gene (Rb) – antioncogene/TSG
o Telomerase
 Telomerase is an enzyme that elongates the chromosomes. In each cell division there is telomere shortening.
 Telomeres: short tandem nucleotide repeats at the end of each chromosomes they help maintain stability and stop fusion with other chromosomes.
 As cells divide over time, telomeres shorten, and eventually cell division stops → Apoptosis (p53,pRb)
 Hayflick limit- when number of division reaches critical.

Question 29

What cells have an active telomerase?

Answer 29

 Stem cells
 Gametes

 Cancer cells

Question 30

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 30

o Taking advantage of viral ‘oncoproteins’. Target p53 and pRB.
o Interacting with DNA bonding protein domains. By interacting with those domains hey stop p53/pRB from interacting with the domains. Still present in cells and functional.

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

Question 31

Why target p53 and Rb?

Answer 31

o They are tumour suppressor genes
o Regulate different parts of cell cycle

o P53 mutated or missing in many cancers
o Many have established that cell lines have lost a functional p53
o Rb inhibits DNA synthesis and arrests cells in G1 of the cycle

Question 32

How p53 works in a normal cell:

Answer 32

o Telomeres at ends of DNA get shorter every time replication happens
o There are telomere binding proteins at the end to protect telomere

o When telomeres get short, the binding protein is lost (when point of senescence is reached)
o P53 can bind to unprotected chromosomes
o Activated p53 triggers growth arrest or cell death

Question 33

How can using viral genes stimulate growth?

Answer 33

o Examples: t and T-antigen of Simian Virus-40, E6/7 protein of Human Papilloma virus (HPV)
o T-antigen interacts with normal p53 and Rb – increase growth without loss of function

o HPV – E6 targets p53 for degradation, E7 binds to Rb (HPV more functionally stable)
o Cells made using E6/7 are believed to maintain a differentiated phenotype

Question 34

How can using telomerase simulate growth?

Answer 34

o Most adult human cells don’t have telomerase that can increase length of telomeres
o Having telomerase will therefore extend lifespan of particular cell – binding protein will remain at ends and no place for p53 to bind

o Therefore: increase expression of telomerase  blockage of p53  generate cell line.
o - The telomerase gene can also be introduced into a target primary cell.
o - Some cells need both introduction of the telomerase gene and inactivation of the pRb/p53 for “immortalisation”
o  E6/ E7 and telomerase transformations are believed to result in cell lines with a differentiated phenotype

Question 35

How do we generate a cell line?

Answer 35

o TRANSFECTION (delivery of DNA or RNA into eukaryotic cells):
- Use plasmid with gene for selection (gene that allows us to select for cells that have successfully taken up GPG (growth promoting gene), e.g. enzyme that can break down toxic compound like antibiotics) and growth promoting gene (e.g. large T-antigen, E6/E7)

o SELECTION PRESSURE ADDED
- Toxic compound (e.g. antibiotic) added to culture
- If cell has taken up gene for selection, it will therefore successfully divide as it can break down toxic compound, unsuccessful cells will die
- We then know these cells have also successfully taken up GPG
- Hope is to incorporate growth promoting gene into host’s genome, so every time cell divides it will express these genes
o COLONIES SELECTED
- Colonies will form from cells that have successfully taken up GPG
- Colonies selected can be grown individually, can then be investigated

Question 36

What hurdles are there in generating a cell line?

Answer 36

o Getting DNA into the cells – DNA is toxic, negatively charged
o Getting cells to stably incorporate DNA once inside

Question 37

What are the conditions and requirements for a cell culture?

Answer 37

1. Handled under aseptic conditions
2. Grown on tissue culture treated plastic flasks/dishes
3. Maintained in a warm (37°C) humidified atmosphere (5% CO2)
   - 37°C and 5% CO2 replicate the same conditions inside the human body.
4. In ideal supplemented medium that needs to be replaced by fresh one every 2/3 days*
   - Neutral pH and space in flask and dishes. Added supplements and nutrients.
   - Growth Medium RPMI 1640 and Growth Medium DMEM
   - This needs to be replaced every 2/3 days the nutrients from the medium will have been used up and metabolised, also debris will contain waste.
   - Phenol red is a medium pH indicator. Indicates that growth medium must be changed.

Question 38

Define adherant cells and suspencion

Anchorage dependant

Agitation

Trypinisation

Tissue culture treated vessels

Yield

Growth limited

Types of cells

Question 39

How can cell culture be contaminated?

Answer 39

o Microbial contamination
 Bacteria (pH change, cloudiness/turbidity, precipitation, stink)

 Yeast (cloudiness, pH change)
 Fungus (spores furry growths, pH change)
 Mycoplasma (often covert, poor cell adherent, reduced cell growth)
 Virus (sometimes cytopathic)
o Cell lines cross-contamination
 Poor tissue culture technique
 Culture of multiple cell lines at one time
 Accidental mixing of cell lines

Question 40

What are the new in vitro models?

Answer 40

 New type of in vitro cell line model used to overcome the negative aspects of the cell line. 2 types of 3D models - organoid and spheroid.
 Polarisation is not biased. grown how they are in real life conditions.

 Spheroid – very reproducible can be grown in different laboratories and can observe same results.
 Organoid – very useful in drug resistant studies. As if we extract primary cells from tumour and grown in 3D. Here we can test different drugs and see how drugs respond we can then extrapolate those results to the patient.
 Patient-derived organoids allow the study cancer drug resistance

Question 41

What are organoids and spheroids and what are the pros and cons?

Question 42

What is Transfection?

What is calcium phosphate precipitation?

What is lipofection?

What is endocytosis?

What is electroporation?

What is nucleofection?

What is viral transfection?

Answer 42

Methods of transfection
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.
CaPO4 co-precipitation
o Forms molecule with Ca2+ and DNA  irritates cell membrane and reduces negative interaction of 2 negative entities (DNA and membrane)
1. Lipofection
o Method of injecting DNA into cell via a liposome (artificially prepared vesicle composed of lipid bilayer)
o Uses positively charged lipids (cationic head group) and combined with negatively charge genetic material  net positive charge (so can ‘mask’ negative charge of DNA – more effective transfection)
o Current discussion on whether liposome gets endocytosed or fuses with membrane (or both)
o Can be used in the transfection of drugs.
o Liposomes as potential drug carriers for drug delivery

o Endocytosis:

• Positive liposome interacts with cell membrane and is taken up by endocytosis
• DNA released from endosome (as liposome breaks down)
• DNA transported to the nucleus
• Entry to nucleus inefficient (small amount taken up, and even smaller amount stably incorporated)

2. Electroporation
   o Plates of capacitor charged
   o The rate of pore sealing is dependent on temperature
   o High electrical field temporarily forms pores on cell membrane of cell  increases permeability
   o Allows DNA to go through
   o Rate of pore resealing is dependent on temperature – maintaining lower temperature after electroporation reduces rate of pore resealing
   o High electric field forms pores which then reseal
3. Nucleofection
   o Combination of electroporation and lipofection
   o Punch holes cell via electroporation  DNA enters via liposome (lipofection)  and then enters nucleus
   o Increased efficiency (but less than viral transfection, however less hazardous)
   o Different solution and protocols used for each cell type
4. Viral transfection
   o Most commonly used are lentiviruses, as can enter cells not undergoing cell division.
   o Exploits normal mechanisms of viral infection
   o Usually has high transfection efficiency
   o Three gene-targeting vectors commonly used based on three types of viruses: retrovirus, adenovirus and adeno-associated virus.
   o Retrovirus, Adenovirus, but most commonly Lentivirus are used.
   o Target cells need to express the viral receptor to work.
   o There are safety aspects to consider.
   o Disadvantage: viruses used can be harmful to humans (biological scientist)