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2 - Cellular Pathology > cell culture techniques > Flashcards

cell culture techniques Flashcards

1
Q

isolating cells depends on what?

A

the tissue they come from

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2
Q

what does density centrifugation take advantage of?

A

the different densities of different cell population and the density gradient medium we use

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3
Q

what can we observe after centrifugation?

A

different layers

  • granulocytes and erythrocytes are denser than the mononuclear cells, and they sediment through the density gradient medium, so we can isolate them from the bottom layer
  • mononuclear cells usually remain in the top layer, in the plasma interface
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4
Q

what is the buffy coat and its importance?

A

for lymphocytes we can try and isolate the white intermediate layer, known as the buffy coat – this intermediate layer is isolated when you want to look at germ line mutations

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5
Q

how does Immunopurification work?

A
  • magnetic beads with an antibody that binds to one cell surface receptor (antigen) present on the cells we want to isolate
  • mix coated beads with cells, beads will only bind to the cells of interest
  • then extract the cells of interest using a magnet
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6
Q

how does FACS work?

A

Fluorescence activated cell sorter

using antibodies to isolate the cells of interest but it can also isolate cells based on their physical properties, eg. their size

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7
Q

if we need to isolate cells from solid tissue (eg. placenta), how would we go about doing this?

A
  • mechanical disruption (eg. passing the tissue through different needles)
  • usually combined with enzymatic disruption too, eg. collagenase
  • after disruption, then apply the magnetic immunopurification and isolate cells of interest
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8
Q

what doesn’t require mechanical disruption?

A

Explant culture - don’t need to do any mechanical disruption because the cells are migrating

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9
Q

positives of primary cells (derived directly from tissues)?

A

positives

  • unmodified
  • carry genetic info
  • very good for personalised medicine = we can do therapeutic drug assays to see if the cells of the patients respond well to the drugs depending on their genetic profile
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10
Q

negatives of primary cells (derived directly from tissues)?

A
  • Aberrant expression of some genes
  • Variable contamination
  • Short life-span
  • Phenotypic instability
  • Difficult molecular manipulation
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11
Q

where can cell lines come from?

A

healthy or cancerous tissue

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12
Q

where do HeLa cells lines come from?

A

cervical carcinoma – they manage to survive spontaneously without manipulation, but sometimes they needed to be genetically manipulated to transform them and make them immortal and work with them in the lab

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13
Q

What need to be targeted in order to make the cells immortal?

A

3 different proteins that can be genetically manipulated in order to produce immortal cells

-p53, Rb and telomerase enzyme

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14
Q

role of p53 and Rb?

A

p53 and Rb are both encoded by tumour suppressor genes, in charge of maintaining cell cycle checkpoints, and they also look after genomic stability

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15
Q

what are telomeres?

A

short tandem nucleotide repetitions that are at the end of each chromosomes

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16
Q

function of telomeres?

A

maintain chromosome stability and prevent them from fusing with other chromosomes

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17
Q

why is there telomere shortening?

A

Every time the cell replicates its DNA, the DNA polymerase can’t replicate the telomere sequence completely, so in each cell division there is telomere shortening

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18
Q

Highflick limit?

A

when the number of cell divisions reaches a Highflick limit (threshold), the telomeres reach a very short length, so much so that the chromosomes start getting damaged and p53 becomes activated, leading to cell apoptosis

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19
Q

function of telomerase?

A

an enzyme that elongates the telomeres on chromosomes

– elongation only happens in cell where telomerase is activated

20
Q

which cell types have active telomerase?

A

stem cells, gametes and many types of cancer cells

21
Q

when will immortal cells be generated?

A

if p53 and Rb is inhibited, and telomerase is expressed

-introduce telomerase into the cell via transfection methods

22
Q

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?

A

taking advantage of viral ‘oncoproteins’

-Viruses contain viral oncoproteins that can target p53 and pRb

23
Q

name some viruses and viral oncoproteins they contain?

A

Simian Virus-40
(SV40)
-large T antigen
-small t antigen

Human Papilloma Virus (HPV)
-E6 and E7

24
Q

SV40’s T-antigen function?

A

interacts with p53 and pRb. This can cause increased growth without loss of function of these proteins

-don’t interact directly with the proteins but interact with the DNA binding domains, so the activity of the 2 proteins aren’t carried out but they are still present in the cell

25
Q

HPV E6 and E7 function?

A

E6 targets p53 for degradation

E7 binds to pRb inactivating it

26
Q

Cell lines Production

A

eg. add neomycin, only the cells that contain the plasmid of interest will be able to survive as they will be resistant to that antibiotic

27
Q

Cell lines

-positives and negatives

A

positives

  • Good growth characteristics. Standard media
  • Phenotypic stability
  • Defined population
  • Good reproducibility

negatives

  • Often lose differentiated function
  • Cell-substrate interactions dominate
  • Does not mimic real tumour conditions
  • Lack cells heterogeneity
  • Phenotype needs to be validated
28
Q

Growth in Culture

-conditions and requirements?

A

a) Handled under aseptic conditions
- spray all the reagents with ethanol

b) Grown on tissue culture treated plastic flasks/dishes

c) Maintained in a warm (37°C) humidified atmosphere (5% CO2)
- same conditions that the cells grow in the human body

d) ideal supplemented medium
-needs to be replaced by fresh one every 2/3 days
-Neutral pH (ph marker too)
Enough space
Have enough nutrients
-any of these conditions aren’t right, the cell cycle will arrest and they will stop growing, they will be alive but they won’t divide or proliferate

29
Q

why does the medium need to be replaced every 2-3 days?

A

the nutrients will be depleted and used by the cells

also the cells release metabolites they don’t want and debris, so the medium will start becoming old and the balance between metabolites and waste and nutrients is broken – build up of waste products could be toxic for the cells – they can die or go to a quiscent state

30
Q

what do most mediums contain?

A

phenol red marker – colour changes depending on the pH of the medium, and the pH is affected by metabolites
red-purple is basic
red is neutral
yellow is acidic medium

31
Q

Adherent vs Suspension cells

A

anchorage dependency
AD - dependent
SUS - independent

agitation
AD - not required
SUS - continuous agitation required

yield
AD - low
SUS - high

growth limited
AD - by the surface area
SUS - by the concentration of cells in the medium

types of cells
AD - most types of cell lines and primary cultures
SUS - some non-adhesive cell lines such as haematopoietic

32
Q

what is meant by ‘suspension cells’

A

Cells that grow suspended (floating) in a liquid medium

33
Q

cell culture contamination?

A

Microbial contamination

a) Bacteria (pH change, cloudiness/turbidity, precipitation, stink)
b) Yeast (cloudiness, pH change)
c) Fungus (spores furry growths, pH change)
d) Mycoplasma (often covert, poor cell adherent, reduced cell growth)
e) Virus (sometimes cytopathic)

Cell lines cross-contamination

  • Poor tissue culture technique
  • Culture of multiple cell lines at one time
  • Accidental mixing of cell lines
34
Q

what are the 2 types of 3D models?

A

esperoids and organoids

35
Q

what are the 2 types of 3D models?

A

esperoids and organoids

-both models are better than the 2D models

36
Q

what are organoids?

A

Organoids come from primary cells extracted from an individual. V good in drug resistance studies

-we can test different drugs and see how the cells respond, and depending on results we can extrapolate the results to the patients.

37
Q

spheroids?

A

Spheroid is established from an immortalised cell line. V reproducible, can be grown in different laboratories.

38
Q

adv and disadvantage of organoids?

A

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

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 invivo growth factor/signalling gradients
39
Q

what is meant by the term transfection?

A

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

40
Q

how is infection different to transfection?

A

a transfection carried out via viral methods is infection

41
Q

name some types of transfection

A
  1. Lipofection
  2. Electroporation
  3. Nucleofection
  4. Viral infection/transduction
42
Q

what is lipofection?

A

using liposomes

  • using cationic lipid transfection systems
  • cationic head, linker, hydrophobic tail
  • net positive charge
  • DNA has negative charge

lipoplexes are positively charged, membrane is negtively charged - lipoplex interacts with cell membrane and taken up by endocytosis. release from the endoscope and transported to the nucleus

43
Q

potential function of liposomes?

A

Liposomes as potential drug carriers for drug delivery

-make them tissue specific by attaching different tissue specific antigens to the surface of the liposome

44
Q

electroporation?

A

Application of electric fields

  • High electric field forms pores which then reseal
  • Rate of pore sealing is dependent on temperature
45
Q

nucleofection?

A
  • 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
  • quite toxic for the cells, transfect DNA inside the nucleus, so gene expression will be rapidly carried out
46
Q

viral infection/transduction?

A

v. efficient

  • 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.

2 - Cellular Pathology (24 decks)

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