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Cellular Pathology > Cell Culture Techniques > Flashcards

Cell Culture Techniques Flashcards

1
Q

What are the methods of cell isolation from blood?

A

Density centrifugation
- entire cell populations

Immunopurification / FACS
- specific cell types

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2
Q

How does density centrifugation work?

A

Density centrifugation takes advantage of the varying densities of the blood cell populations and the density gradient medium (DGM) that we use

Depending on the medium we use, we can isolate different types of cells

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3
Q

Name an example of a density gradient medium used in density centrifugation

A

We can mix our blood sample with Ficoll (DGM) that has a density of 1.077 g/mL
After centrifugation we can observe different layers

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4
Q

Which blood cell type sediments as the bottom layer?

A

Granulocytes and erythrocytes are denser than the other cell layers and so sediment through the DGM to the bottom

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5
Q

Which cells are found towards the top of the blood centrifugation?

A

Less dense mononuclear cells usually remain towards the top in the plasma interface - can isolate and centrifuge to separate the mononuclear cells

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6
Q

What is the buffy coat?

A

Can also isolate the buffy coat white layer (lymphocytes) containing copies of the germline info

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7
Q

What techniques can be used to isolate more specific cell types?

A

In order to isolate more specific type of cells we can use other techniques e.g.

  • Immuno-purification
  • Fluorescence Activated Cell Sorter
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8
Q

Outline the process of immunopurification

A
  1. Magnetic beads coated with antibodies - bind to
    specific antigen on cell of interest surface
  2. Coated beads mixed with blood sample and only bind
    to cell type of interest
  3. By application of magnetic fields, we can instruct beads
    bound to cells of interest
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9
Q

How does FACS isolate specific cell types?

A

uses cell size and antibodies to separate cells of interest

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10
Q

How are cell types differentiated in FACS?

A

Uses cell surface markers and size to differentiate between cell types

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11
Q

Give an example where cells are isolated from tissues

A

E.g. Isolation of hematopoietic placental stem cells that can differentiate into any cell lineage

Or placental endothelial cells

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12
Q

What is the difference between cell isolaton from blood and from tissue?

A

In tissues cells require isolation from solid tissue so need to carry out a mechanical enzymatic disruption

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13
Q

How do we carry out a mechanical enzymatic disruption of cells?

A
  • Scalpels used to disrupt tissue

- Pass tissue through needles to extract individual cells

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14
Q

What other technique is used to disrupt cells alongside mechanical disruption?

A

Mechanical disruption is usually combined with digestive enzymatic disruption
e.g.
Dispase, trypsin, collagenase

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15
Q

How are specific cell types extracted from tissue after disruption?

A

We can now apply magnetic immunopurification techniques to extract the cell type of interest

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16
Q

Outline how mechanical enzymatic disruption is used in explant cultures

A

Easily carried out in chondrocytes as they migrate away from a cartilage explant - doesn’t require any additional techniques, can occur spontaneously

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17
Q

What are the advantages of using primary cell cultures?

A

Unmodified; carry all genetic info belonging to patients tumour / tissue they’re isolated from

Makes them good for personalised medicine e.g. therapeutic drug assays: assess response of patient to certain drugs based on their genetic profile

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18
Q

Why are primary cell cultures not good for basic science?

A

However, for in vitro contours for basic science, primary cells aren’t ideal models as they carry other non-functional genes - contribute in contamination (e.g. bacteria from organism extracted form)

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19
Q

What are some disadvantages of primary cell cultures?

A

Short life span - can’t be handled after a certain no. of times

Can’t carry out reproducible in vitro analysis using other cell lines - primary cell population will have different characteristics to others

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20
Q

Where are cell lines isolated from?

A

Can be isolated from healthy or cancerous tissues (e.g., HeLa cells)

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21
Q

Where are cell lines derived from?

A

Spontaneously
from prolonged culture, multiple ill-defined mutations transformed phenotype

Through genetic manipulation
Transformation of healthy primary cells

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22
Q

How are cell lines generated?

A

To generate cell lines we target processes that regulate cellular growth and ageing

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23
Q

What 3 genes are manipulated to form immortal cells?

A

P53
pRB
Telomerase enzyme

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24
Q

What are the roles of p53 and pRB?

A

P53 and pRB are encoded by tumour suppressor genes and maintain cell cycle checkpoints and regulate genomic instability

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25
Q

What are telomeres?

A

Telomeres are short tandem nucleotide repetitions found at the ends of each chromosome.

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26
Q

What is the function of telomeres?

A

To maintain stability of chromosomes and prevent fusion with other chromosomes

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27
Q

What is telomerase?

A

Telomerase is an enzyme that elongates chromosomes

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28
Q

Why does telomerase elongate chromosomes?

A

Every time DNA is replicated in a cell, DNA pol. is unable to elongate telomere sequences completely ∴ in each cell division there is telomere shortening

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29
Q

How does certain no. of cell divisions lead to apoptosis?

A

When cell division no. reaches its limit, telomeres are very short in length causing chromosomal damage
⇒ activates p53 and pRB; lead cell to apoptosis due to damage signal

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30
Q

Which cells contain active telomerase?

A

Other types of cells also contain active telomerase e.g.

  • Gametes
  • Stem cells
  • Cancer cells
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31
Q

What is the advantage of activated telomerase?

A

By activating TERT (telomerase enzyme) we can create immortal cells

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32
Q

How can we produce immortal cells?

A

We can inhibit the function of tumour suppressor proteins, or introduce telomerase in order to alter a cell’s capability for its finite number of divisions by taking advantage of viral ‘oncoproteins

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33
Q

Which viruses do we use to produce immortal cells?

A

can use SV40 and HPV to manipulate telomerase enzymes in their viral oncoproteins.

34
Q

How is SV40 used to produce immortal cells?

A

SV40’s large T-antigen DNA binding domains interact with p53 and pRb preventing them from binding to E6 and E7 domains. This can cause increased growth without loss of function of these proteins

35
Q

How are HPV manipulated to produce immortal cells?

A

E6 in HPV targets p53 for degradation, and E7 binds to pRb inactivating it

36
Q

Describe the phenotype of viral oncoprotein manipulated cell lines

A

E6/ E7 and telomerase transformations are believed to result in cell lines with a differentiated phenotype

37
Q

Outline how the telomerase gene is introduced to immortalise primary cells

A
  1. Plasmid vector designed carrying gene for selection
    marker e.g. antibiotic resistance marker
  2. Insert into plasmid, the telomerase gene sequence we
    want to transfect into cells
  3. Completed plasmid vector transfected
  4. Positively transfected cells identified via antibiotic
    resistance marker
  5. Only positively transfected cells show antibiotic
    resistance as they have antibiotic resistance gene
38
Q

Why are cell lines a good model for basic science?

A

Cell lines show good reproducibility as different labs using the same cell line can obtain the same results from an assay

> makes cell lines a very good model for basic science

39
Q

Why are cell lines an unreliable source?

A

Cell lines usually lose differentiated function and revert to become stem cell like without a differentiated phenotype

40
Q

What are the disadvantages of cell line use?

A

Show bias to cell substrates in their actions; acquire polarity due to attachments to cell surfaces

Phenotype needs to be validated by an external source - identify cross contaminations, acquired mutations etc.

41
Q

Outline the conditions and requirements of growth in culture

A

a) Handled under aseptic conditions
b) Grown on tissue culture treated plastic flasks/dishes

c) Maintained in warm (37°C) humidified atmosphere (5%
CO2) - mimics internal human conditions

d) In ideal supplemented medium that is replaced by fresh
one every 2/3 days*

42
Q

What conditions need to be maintained for cell culture growth?

A

Need to ensure cells are grown at optimum pH (7.0) and temperature (37.0c)

Ensure there is enough space to grow in the dish

We need to add the corresponding growth factors and supplements to the medium

43
Q

What is the consequence of a lack of any of the conditions required for cell culture growth?

A

If the cells lack any of the aforementioned requirements they will arrest in the cell cycle ⇒ quiescent cells

44
Q

What are quiescent cells?

A

Quiescent cells - still alive, but won’t divide and proliferate

45
Q

Give examples of 2 common growth culture mediums

A

Growth medium RPMI 1640

Growth Medium DMCM

46
Q

Why does the growth medium need to be replaced so often?

A

Medium needs to be replaced every 2-3 days to replenish nutrients and metabolite excretion

47
Q

What is phenol red?

A

Phenol red is a medium pH indicator

48
Q

What causes a colour change in phenol red?

A

Colour change based on medium pH dependent on metabolite presence

Ideal colour - red (neutral)

49
Q

What are the two ways culture cells can grow?

A

Cells can either grow in suspension or attached to a surface

50
Q

Outline the features of adherent cells

A
  • Grow attached to solid surface
  • Anchorage-dependent
  • Trypsinization required
  • Tissue culture treated vessels
  • Low yield
  • Growth limited by SA
  • Most cell lines and cultures
51
Q

Outline features of suspension cells

A
  • Grow suspended in a liquid medium
  • Anchorage-independent
  • Continuous agitation needed
  • High yield
  • Growth limited by [cells] in medium
  • Some non-adhesive cell lines
52
Q

What are some of the microbial contaminations found in cell cultures?

A

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)

53
Q

What are the types of cross contaminations that can occur in cell cultures?

A
  • Poor tissue culture technique
  • Culture of multiple cell lines at one time
  • Accidental mixing of cell lines
54
Q

What is the purpose of new in vitro models?

A

Used to overcome cell line disadvantages

55
Q

What are the 2 types of 3D cell culture models?

A
  • Organoid

- Spheroid

56
Q

How are spheroid cells made?

A

Spheroids are made from established immortalised cell lines that normally grow in 2D but are grown in 3D

57
Q

What are organoid cells?

A

Organoids are 3D cultures derived from stem cells normally present in normal tissues

58
Q

Why are organoids and spheroids better than 2D models?

A

Organoids and spheroids are better models than the 2D cell cultures, as they replicate 3D growth as in humans - no bias to cell attachments

59
Q

What areas in cell culturing are spheroids and organoids respectively used for?

A

Spheroids good for in vitro model = reproducible

Organoids good for drug resistance studies

60
Q

Outline the features of organoid cells

A
  • Derived from stem cells
  • Multiple cell lineages
  • Recapitulate organ physiological parameters
  • long term culture
61
Q

Outline the features of spheroid cells

A
  • Derived from cell line monoculture
  • Represent single / partial tissue components
  • transiently resemble cell organisation
  • Difficult to maintain long term
62
Q

Explain how patient derived organoids are used to study cancer drug resistance

A
  1. Extraction of primary cells from a tumour
  2. Grown in 3D in the lab
  • Allows tumour to maintain its 3D structure and cell-cell
    contacts
  • can test different drugs and their efficacy and response -
    simulates body well
63
Q

What are the advantages of organoid use?

A
  • 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
64
Q

What are the limitations of organoid use?

A
  • 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
65
Q

What is transfection?

A

Process by which foreign DNA is deliberately introduced into a eukaryotic cell through non-viral methods (chemical and physical methods in the lab)

e.g. a plasmid, a CRISPR/Cas9 complex

66
Q

Give examples of chemical methods of transfection

A 
Lipofection
Calcium phosphate
Cationic polymer
DEAE-Dextran
Magnet-mediated transfection
Dendrimers
67
Q

Outline the physical transfection methods

A 
Electroporation
Nucleofection
Microinjection
Biolistic Particle Delivery
Laserfection/ opto injection
68
Q

Explain how lipofection occurs

A

Lipofection takes advantage of the cationic nature of liposomes

DNA by nature is -ve charged - can introduce DNA into those liposomes allowing it to fuse

69
Q

What are liposomes?

A

small vesicles surrounded with phospholipid bilayer with positive heads

70
Q

Outline the mechanism of lipofection

A

Lipoplexes positively charged
Membrane is negatively charged

  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
71
Q

When is lipofection commonly used?

A

Lipofection significantly used in transfection of drugs

72
Q

How can we make liposomes tissue specific?

A

Can make liposomes tissue specific by attaching antigens to their surface

73
Q

What is the disadvantage of lipofection?

A

Lipofection however can be quite toxic for the cells

74
Q

What is the process of electroporation?

A

Application of electric fields enables us to open pores in the cell membranes making them permeable to the plasmids

Once the plasmid is in the cell, the membrane pores reseal

75
Q

What needs to occur in electroporation for transcription to occur?

A

In order to activate the transcription machinery, the cell needs to incorporate the plasmid in its nuclear DNA

76
Q

What is nucleofection?

A

Combination of electroporation and lipofection

77
Q

Outline the features of nucleofection?

A

Increased efficiency particularly of non-dividing cells

Technology is protected under patent

Different solution and protocols are used for each cell type

78
Q

How effective is viral infection as a transfection method?

A

High transfection efficiency

79
Q

How is viral infection manipulated for transfection?

A

Package viral components into plasmid and infect other cells

Material release containing both viral components in the supernatant

80
Q

Name examples of viruses used to transfect cells

A

Retrovirus, Adenovirus, but most commonly Lentivirus are used

81
Q

What are the limitations of viral infection as a transfection method?

A

Target cells need to express the viral receptor to work

There are safety aspects to consider

Cellular Pathology (13 decks)

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