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Techniques of Molecular Medicine > Cells and cell culture - basics > Flashcards

Cells and cell culture - basics Flashcards

1
Q

What is cell culture?

A

Techniques the involves the isolation and maintenance in vitro of cells

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2
Q

Where are the two sources were cells isolated from?

A

Tissues

Whole organs

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3
Q

Which species of cells are used in cell culture?

A

Animals

Microbes

Plants

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4
Q

Why is it important to grow cells under controlled conditions?

A

Every time you do the same experiment you obtain the same results

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5
Q

What are requirements of cells culture?

A

Requires a sterile pure culture of cells

Adopts aseptic techniques

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6
Q

What can cell cultures be used to study?

A

Basic cell biology

Protein synthesis

Signal transduction mechanisms

Cell-cell interactions

Celll-disease interactions

Aging and nutrition

Effects of drugs on cells

Gene therapy

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7
Q

What are techniques of good cell culture?

A

Characterization and maintenance of essential characteristics

Quality assurance

Recording

Reporting

Safety

Education and training

Ethics

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8
Q

What are the types of cells used in cell culture?

A

Primary cells

Cell lines

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9
Q

Primary cells retain the characteristics and reflect the true activity of the cell type in vivo

TRUE or FALSE

A

TRUE

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10
Q

Primary cell cultures retain a differentiated phenotype in culture

TRUE or FALSE

A

TRUE

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11
Q

All primary cells are anchorage dependent

TRUE or FALSE

A

FALSE

Primary cells are mainly anchorage dependent

Blood derived cells are anchorage independent

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12
Q

What is contact inhibition of primary cells?

A

When two cells come into contact they are signalled to stop proliferating

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13
Q

What are the disadvatages of using primary cells for cell culture?

A

Isolation is labour intensive

May produce a heterogeneous population from the sample obtained

Cells have a limited lifespan

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14
Q

What can be done to remove the disadvantage of having a heterogeneous primary cell population?

A

Different cells may contaminate the sample

Characterisation is necessary

This will allow you to isolate the cell type you want to study

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15
Q

Why do primary cells have a limited lifaspan?

A

Genetic programme is still in place

Leads to limited culture period

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16
Q

Describe the technique used to isolate primary cells

A

Excise the terminally differentiated cells from a well-identified tissue

Culture the cells

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17
Q

What tissue is used to obtain the primary cells?

A

Normal healthy tissue

Tumour tissue

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18
Q

What techniques are used to culture primary cells?

A

As explant

Into single cell suspension following dissociation from ECM by enzyme digestion or gradient centrifugation

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19
Q

What are the advantages of primary cells?

A

Retain many native cellular functions in vitro

Cells retain normal morphology, cellular function, growth characteristics, cellular markers, signalling and genetic integrity when propagated in culture

Have varied donor characteristics

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20
Q

Why is having donor-to-donor variability in primary cells an advantage?

A

Makes the results of the study conducted on primary cells reflective of the whole population

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21
Q

Why has using primary cells become more popular?

A

Some cases are mandated to use primary cells in order to receive data approval

More reliable cell culture tool

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22
Q

What is fibrosis?

A

Formation of excess fibrous connective tissue in an organ

Marked by quantitative and qualitative changes in the composition of hepatic ECM

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23
Q

What happens when stellate cells become activated?

A

Produce a massive amount of ECM

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24
Q

What are characteristics of activated stellate cells?

A

Highly proliferative

Highly contractile

Highly motile

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25
Q

What causes the stellate cells to become activated?

A

Autocrine loops:

  • PDGF
  • TGF-b
  • ET-1
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26
Q

Describe the steps of obtaining hepatic stellate cells from liver tissue

A

Liver is digested using enzymes

Capture all the cells

Centrifuge the sample

The cells have different viscocities and produce a gradient

Obtain the HSC from the centrifuged sample

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27
Q

What are safety precautions one must consider when handling biomaterial?

A

May contain pathogenic agents that use humans as hosts

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28
Q

What are safety precautions must the operator follow when working in a lab?

A

Wear PPE:

  • gloves
  • laboratory coat

Refrain from eating or drinking

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29
Q

Were are cell lines derived from?

A

Primary cells

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30
Q

What characteristic must primary cells have to become cell lines?

A

Immortal

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31
Q

How are primary cells immortalized?

A

Spontaneously by genetic mutation

By transformation vectors

  • viruses
  • plasmids
32
Q

What is an immortal primary cell?

A

Single cell type that has gained the ability of infinite growth

Can be serially propagated for an unlimited number of cell divisions

33
Q

What are the advatages of using cell lines?

A

Show increased growth rate to primary cells

Will have massive amounts of data under controlled conditions

34
Q

What are the disadvatages of using cell lines?

A

Genetically unstable

Stop expressing tissue specific genes

Reduced serum and anchorage dependence - grow in suspensions different to the donor tissue

35
Q

What are the different types of primary cells used to form cell lines?

A

HeLa - epithelial

BAE-1 - endothelial

SH-SY5Y - neuronal

36
Q

What differences exist between different cell lines?

A

Morphology

Functional characteristics

37
Q

What are characteristics of HeLa?

A

Attached to a substrate

Appears flattened

Polyglonal in shape

38
Q

What are characteristics of lymphoblast like- cells?

A

Do not attach to a substrate

Remain in cell suspension

Spherical in shape

Isolated from lymphoid tissue

39
Q

What are characteristics of fibroblast like- cells?

A

Attached to a substrate

Elongated

Bipolar

40
Q

What are common uses of cell line cultures?

A

Undestanding of cancer biology

Assessment of drug sensitivities

41
Q

What is needed in order to use cell lines to understand cancer biology?

A

Maximal molecular similarity between a primary tumour site and the model

42
Q

What is a way to investigate the molecular similarity of a cell line to the tissue it is trying to model?

A

Next generation sequencing technologies

Enabled the comparison of primary tumours and their cell lines on a genomic scale

Unmasked the inadequacy of some models and confirmed the reasonable genomic similarity in other

43
Q

What are the required conditions to create an optimal cell environment for cell culture experiments?

A

Cell culture flask or vessel

Nutrients

Appropriate environment

Sterility

Adequate pH and osmolarity

Oxygen tension maintained at atmospheric levels

44
Q

Examples of appropriate cell culture flasks or vessels to perform cell cultures

A

Petri dishes

Culture plates

45
Q

Which nutrients are needed to provide an appropriate culture media?

A

Inorganic salts

Carbohydrates

Amino acids

Vitamins

Fatty acids and lipids

Proteins and peptides

Serum

Trace elements

46
Q

What, in terms of the environment, is needed for the cell environment to be adequate?

A

Appropriate

  • temperature
  • carbon dioxide concentration
  • humidity
47
Q

What is the main role of the cell environment?

A

Acts as the building blocks for the cells

48
Q

What are the components of serum?

A

Serum is a complex mix of

  • albumins
  • growth factors
  • growth inhibitors
49
Q

Why is it important to screen batches of serum before being used in cell culture?

A

Quality, type and concentration of different plasma components differ between batches

50
Q

What is a subculture?

A

Transfer of cells from a previous culture into a fresh growth medium

51
Q

Why are subcultures important?

A

Enables the propagation of cell lines

52
Q

What are the 4 stages of cell growth in culture?

A

Lag phase

Stationary phase

Log phase

Death phase

53
Q

What is the lag phase?

A

Cells adapt to their new media

Cells are metabolically active

Cell proliferation is minimal

54
Q

What is the log phase?

A

Cells expand exponentially

Cells are metabolically active

55
Q

What happens to cell proliferation if the cultured cells are not placed in a subculture?

A

Cell proliferation will decline

Cells will go into apoptosis

56
Q

What are the two conditions that causes cell proliferation to cease?

A

The cells in adherent cultures have no room left for expansion

The cells in suspension cultures exceed the capacity of the medium to support further growth

57
Q

What is trypsinization?

A

Process by which you can detach the cells from the adherent substrate

Various proteolytic enzymes are used

58
Q

What is the main enzyme used for trypsinization?

A

Trypsin

59
Q

From which family does trypsin originate?

A

From the serine protease family

60
Q

Which cells release trypsin?

A

Exocrine cells of the pancreas

61
Q

Exocrine cells of the pancreas release trypsin

TRUE or FALSE

A

FALSE

Exocrine cells of the pancreas release trypsinogen

62
Q

What is the optimum temperature for trypsin?

A

37 degrees

63
Q

How does trypsin cause cell death?

A

Strips the surface of the cells of their proteins

64
Q

When is a combination of trypsin and EDTA used?

A

Weaken the interactions between cell-cell and cell-matrix

65
Q

The same concentration of trypsin is always used using trypsinization

TRUE or FALSE

A

FALSE

Concentration used changes on cell type

Strongly adherent cell lines = 2.5%

Studies which require cell surface protein integrity = 0.5%

66
Q

What concentration of trypsin is used in strongly adherent cell lines?

A

2.5%

67
Q

What concentration of trypsin is used in studies which require cell surface protein integrity?

A

0.5%

68
Q

Why is phenol red used in cell cultures?

A

Used to indicate the pH of the cell culture

Should be red in normal conditions

Metabolites build up in cell cultures following proliferation

This lowers the pH, causing the colour to change to an orange-yellow shade

69
Q

What is the major source of trypsin?

A

Porcine

Sourced from bovines

70
Q

What is often used as a source of trypsin to avoid animal or microbial products contaminating the sample?

A

Trypzean solution

Obtained from corn

71
Q

Name two instruments used to count cells

A

Coulter counter

Hemocytometer

72
Q

What is Trypan blue used for?

A

Used to determine the number of viable cells present in a cell suspension

73
Q

What feature does Trypan blue exploit in order to identify dead cells?

A

Live cells possess intact cell membranes that exclude the entry of certain dyes, including Trypan blue

Dead cells become stained due to their broken membrane

74
Q

What chemical is used to quantify the effect of a drug on cell proliferation?

A

BrdU

75
Q

Why can BrdU be used to quantify the effect of a drug during cell proliferation?

A

Cells can naturally incorporate BrdU into their cells during cell division

Makes it an excellent marker of cell cycle and proliferation

76
Q

What is BrdU a marker for?

A

Cell cycle

Proliferation

77
Q

How is BrdU used to quantify proliferation of a cell?

A

Anti-BrdU antibody is used to analyses the BrdU concentration

Concentration is detected through immunofluorescent staining and colorimetric assay

Techniques of Molecular Medicine (12 decks)

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