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Cellular Pathology > Cell Culture Techniques > Flashcards

Cell Culture Techniques Flashcards

1
Q

1.What is density centrifugation?

A

-You isolate different cell populations depending on their density in a density gradient medium.

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2
Q
  1. In a density centrifugation, where would granulocytes/erythrocytes isolate?
A

They are dense so isolate at the bottom through the density gradient medium

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3
Q

3.Where would lymphocytes isolate in a density centrifugation?

A

In the white buffer coat

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4
Q

4.How does the process of immuno-purification work?

A

Magnetic beads are coated with antibodies
Antibodies bind to specific cell surface antigens
Antigens (of interest) are extracted by applying magnetic field

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5
Q

5.How does Fluorescence activated cell sorter (FACS) isolate cells of interest?

A

Using antibodies
Or
Physical Properties eg size

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6
Q

6.List three mechanisms to isolate cells from blood?

A
  1. Density Centrifugation
  2. Immuno-purifiation
  3. Fluorescence activated cell sorter
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7
Q

7.How would isolate cells from solid tissues?

A

First the tissue is either disrupted :
mechanically(scalpels/needles) or
by enzymatic digestion.
The cells of interest can then be isolated by magnetic immuno-purification

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8
Q

8.Give an example of a cell where isolation would be easy?

A

chondrocytes migrating away from a cartilage explant mean they will separate by themselves

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9
Q

9.What is meant by “primary cells”

A

Cells derived directly from tissue

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10
Q

10.What is a cell line

A

Cell line is a permanently established cell culture that will proliferate indefinitely given appropriate fresh medium and space.

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11
Q

11.What are two advantages of using primary cells for treatment?

A

They are unmodified

-Good for personalised medicine

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12
Q

12.What are 7 disadvantages of using primary cells for treatment?

A
  • Aberrant(abnormal) expression of some genes
  • Variable contamination
  • Limited
  • Short life-span
  • Inter patient variation
  • Difficult molecular manipulation
  • Phenotypic instability
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13
Q

13.How can you make cells immortal?

A
  • Cells are isolated from cancerous tissues (HeLa cells) that are derived from primary cultures.
  • Healthy primary cells are transformed to make them immortal through genetic manipulation.
  • Cells from prolonged cultures can be transformed (phenotypically) spontaneously when they obtain multiple ill-defined mutations.
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14
Q

14.To generate immortal cell lines, we target processes that regulate cellular growth and ageing , give some examples of this?

A

. tumour suppressor genes (p53, pRb)
or
the telomerase enzyme

Some cells require both

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15
Q

15.What does the telomerase enzyme do?

A

elongates telomeres e.g., in cancerous cells, stem cells, gametes etc

oAs cells divide over time, telomeres shorten, and eventually cell division stops → Apoptosis (p53,pRb).

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16
Q

16.How can we inhibit the function of tumour suppressor proteins or introduce telomerase to make them immortal?

A

By taking advantage of viral oncoproteins.

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17
Q

17.How does Simian Virus-40 (SV40) affect P53 and pRb?

A

• SV40’s T-antigen interacts with the binding domains of p53 and pRb to cause increased growth (can’t bind to DNA binding regions) without losing the function of these proteins.

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18
Q

18.What viral oncoproteins does the Human papilloma Virus (HPV) produce?

A

E6 and E7

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19
Q

19.How does the viral oncoproteins E6 and E7 affect P53 and pRb?

A

E6 targets p53 for degradation, and E7 binds to pRb inactivating it- these cells maintain a differentiated phenotype.

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20
Q

20 . How can the telomerase gene can be introduced into a target primary cell ?

A

-Using transfection methods
o This is done by designing a plasmid vector that contains the gene for selection and a growth promoting gene (telomerase).
o This is transfected into the primary cells which are allowed to grow.
o A selection pressure e.g. antibiotic is added, colonies are formed from bacteria that have taken up the genes. These are selected for culturing.

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21
Q
  1. What are 6 advantages of cell lines?
A
  • Good growth characteristics. Standard media
  • Phenotypic stability
  • Defined population
  • Molecular manipulation readily achieved
  • Good reproducibility
  • Good model for basic science
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22
Q
  1. .What are 5 disadvantages of cell lines?
A
  • Often lose differentiated function
  • Cell-substrate interactions dominate
  • Does not mimic real tumour conditions
  • Lacks cells heterogeneity
  • Phenotype needs to be validated
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23
Q

23.. To grow cells in a culture there are conditions and requirements, list these?

A

o Handled under aseptic conditions (ethanol sanitised surface, gloves).
o Grown on tissue culture treated plastic flasks/dishes.
o Maintained in a warm (37°C) humidified atmosphere (5% CO2).
o In ideal supplemented medium (growth medium RPM 11640 or DMEM) that needs to be replaced by a fresh one every 2/3 days due to nutrient depletion and the collection of debris/waste.

24
Q
  1. What are adherent cells?
A

Cells that grow attached to a solid surface

25
Q
  1. What are suspension cells?
A

Cells that grow suspended (floating) in a liquid medium

26
Q
  1. For the following features/characteristic, explain how ADHERENT cells would act in that situation:
    - Anchorage dependency
    - Agitation
    - Trypsinization
    - Tissue culture treated vessels
    - Yield
    - Growth limited
    - Types of cells
A

Anchorage dependency =Anchorage-dependent

Agitation=Not required

Trypsinization=Required

Tissue culture treated vessels=Required

Yield=Low

Growth limited=By the surface area

Types of cells=Most types of cell lines and primary cultures

27
Q

27 . For the following features/characteristic, explain how SUSPENSION cells would act in that situation:

  • Anchorage dependency
  • Agitation
  • Trypsinization
  • Tissue culture treated vessels
  • Yield
  • Growth limited
  • Types of cells
A

Anchorage dependency = Anchorage independent

Agitation= Continous agitation required

Trypsinization= Not required

Tissue culture treated vessels = Not required

Yield= High

Growth limited= By the conc of cells in the medium

Types of cells= Some non-adhesive cell lines such as hematopoietic

28
Q
  1. Do E6/ E7 and telomerase transformations result in cell lines with a differentiated phenotype??????
A

yeaaaaahhh

29
Q
  1. When cells are grown in a culture , there may be culture contamination.How would you notice contamination by Bacteria?
A

pH change
Cloudiness/Turbidity
Precipitation
Stink

30
Q

30.When cells are grown in a culture , there may be culture contamination.How would you notice contamination by Yeast?

A

Cloudiness

pH change

31
Q

31.When cells are grown in a culture , there may be culture contamination.How would you notice contamination by Fungus?

A

Spores furry growths

pH Change

32
Q

32.When cells are grown in a culture , there may be culture contamination.How would you notice contamination by Mycoplasma?

A

Often Covert
Poor Cell adherent
Reduced Cell Growth

33
Q
  1. What are Mycoplasma?
A

Parasitic bacteria lacking cell walls

34
Q
  1. When cells are grown in a culture , there may be culture contamination.How would you notice contamination by Virus?
A

Sometimes Cytopathic (structural changes in host cells that are caused by viral invasion)

35
Q
  1. One type of cell culture contamination , is when you have cell line cross-contamination. Why is this most likely to occur?
A
  • Poor tissue culture technique
  • Culture of multiple cell lines at one time
  • Accidental mixing of cell lines
36
Q
  1. List four new in vitro models?
A
  • 2D cell culture
  • 3D cell culture
  • 3D Spheroid Culture
  • 3D organic Culture
37
Q
  1. What is a medium pH indicator?
A

Phenol Red

38
Q
  1. When using Phenol red.What would the colours be for a basic, neutral and acidic medium and the correlating pH’s?
A

pH of 7.4 - 7.6 is BASIC , red/purple

pH of 7 - NEUTRAL , Tomato Red

pH of 6.8 = ACIDIC , Yellow

39
Q

39 . Please compare Organoid (O) vs Spheroid 3D Culture (S) :
- Where are Each derived from?

A 
O= Derived from stem cells
S= Derived from cell line monoculture
40
Q
  1. please compare Organoid (O) vs Spheroid 3D Culture (S) :
    - How many Cell lineages?
A 
O = Multiple Cell lineages
S= Represent Single/Partial Tissue components
41
Q
  1. please compare Organoid (O) vs Spheroid 3D Culture (S)

- How do they resemble actual physiology?

A 
O = Recapitulate organ physiological Parameters
S= Transiently (briefly) resemble cell organisation
42
Q

42 . Please compare Organoid (O) vs Spheroid 3D Culture (S) :
-Is the culture long term?

A 
O = Long term Culture
S= Difficult to maintain long term
43
Q
  1. How can we use patient derived Organoids to study cancer drug resistance?
A
  • Organoids are taken from tumour biopsies are taken and studies before and after treatment.
  • Study new drug targets, drug resistance (mutational analysis and survival analysis), histology and optical metabolic imaging.
44
Q
  1. What are four ADVANTAGES of Organoids?
A

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

45
Q
  1. What are four DISADVANTAGES of Organoids?
A

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

46
Q
  1. What is transfection?
A

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.

47
Q
  1. What are 6 chemical methods to transfection?
A 
lipofection
calcium phosphate
cationic polymer
DEAE-Dextran
Magnet-mediated transfection 
Activated dendrimers.
48
Q
  1. What is the viral method of transfection?
A

Infection

49
Q
  1. What are 5 physical methods of transfection?
A

Electroporation
Nucleofection
Microinjection
Biolistic Particle Delivery Laser-faction/Optoinjection

50
Q
  1. What are three benefits of using a cationic lipid transfection system?
A
  • Fast
  • Simple
  • Reproducible
51
Q
  1. What is the method of lipofection?
A

o The positively charged lipoplexes (synthetic nuclear acid carriers with cationic phospholipids) interacts with the negatively charged cell membrane and is taken up by endocytosis.
o It’s then released from the endosome and transported to the nucleus (entry to the nucleus is inefficient – may need mitosis).
o Liposomes as potential drug carriers for drug delivery.

52
Q
  1. What is the method of electroporation?
A

High electric field forms pores in the cell membrane which then reseal (rate of pore sealing is dependent on temperature).

53
Q
  1. What is the method of nucleofaction?
A

o Combination of electroporation and lipofection
o Increased efficiency particularly of non-dividing cells
o Technology is protected under patent
o Different solution and protocols are used for each cell type

54
Q
  1. What is the method of viral infection/transduction?
A

o Exploits the mechanism of viral infection: uses Retrovirus, Adenovirus, but most commonly Lentivirus.
o High transfection efficiency.
o Target cells need to express the viral receptor to work.
o There are safety aspects to consider.

55
Q
  1. Summarise this lecture?
A

o To overcome the short comings of primary cultures, cell lines have been produced
o Cell lines can be spontaneously induced or produced by transfection with viral oncogenes that inhibit tumour suppressor genes or by introducing/over-expressing telomerase.
o Cell transfection can be carry out using chemical, physical or viral methods.
o Rapidly dividing cells can lose differentiated functions → need for different models- Organoids

Cellular Pathology (13 decks)

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