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Week 2 Flashcards

1
Q

What is cell culture ?

A

The process of growing prokaryotic, eukaryotic, or plant cells under controlled conditions

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2
Q

What is tissue culture?

A

Removal of cells, tissues, or organs from an animal and placing them in an artificial environment for growth.

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3
Q

Define animal cell culture.

A

Culture of animal cells outside the tissue they were obtained from (in vitro).

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4
Q

List 3 reasons for culturing cells.

A
  • Production of monoclonal antibodies and proteins
  • Viral vaccine production
  • Drug activity investigations

(also valid: cell therapies, clinical investigations)

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5
Q

Name 3 commonly used cell lines and their origins.

A

CHO: Chinese hamster ovary

3T3: Mouse fibroblasts

Vero: Kidney cells from African green monkey

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6
Q

What is CHO-K1 and why is it important?

A

A continuous CHO cell line with short doubling time (15 hrs), used in biotech due to its adaptability.

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7
Q

What are MDCK cells used for?

A

Vaccine production, e.g., FLUCELVAX, the first mammalian-cell based flu vaccine (2012).

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8
Q

Why are Vero cells important in virology?

A

They are interferon-deficient and FDA-approved for vaccine production (e.g. rabies, polio).

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9
Q

What are HeLa cells and why are they famous?

A

First human immortal cell line from cervical cancer (Henrietta Lacks); widely used in research.

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10
Q

Who is considered the father of cell culture and what did he do?

A

Harrison (1907): Cultivated frog nerve cells in vitro for several weeks.

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11
Q

What did Lewis & Lewis contribute in 1911?

A

Developed the first liquid media using seawater, serum, and embryo extracts.

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12
Q

What breakthrough occurred in 1913 by Carrel?

A

Introduced strict aseptic techniques to extend culture periods.

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13
Q

What is the role of trypsin in cell culture?

A

Used as a proteolytic enzyme to subculture adherent cells (Rous & Jones, 1916).

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14
Q

What advancement did Gey make in 1952?

A

Established the first human continuous cell line: HeLa.

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15
Q

What is EMEM and who developed it?

A

Eagle’s Minimum Essential Medium; developed by Eagle in 1955 for nutrient optimisation.

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16
Q

What is serum-free media and who pioneered it?

A

Media without animal serum, developed by Sato in 1978 using hormone/growth factor cocktails.

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17
Q

What are the two main methods of initiating cell culture?

A

Explant culture and enzymatic dissociation

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18
Q

What happens during explant culture?

A

Tissue is placed in a culture vessel with medium; cells migrate out and begin to proliferate.

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19
Q

What is used in enzymatic dissociation to isolate cells?

A

Proteolytic enzymes like trypsin.

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20
Q

What is the general flow of cell culture from tissue to experiment?

A

Tissue → Isolation → Culture → Passage → Freeze or Use (experiment/assay).

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21
Q

Why is freezing part of the workflow?

A

To preserve cells in a viable state for future use.

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22
Q

What are the 3 basic cell morphologies?

A

Fibroblastic, epithelial-like, and lymphoblast-like.

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23
Q

Describe fibroblastic cell morphology.

A

Elongated, bipolar/multipolar, attachment-dependent.

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24
Q

Describe epithelial-like cells.

A

Polygonal, regular shape, grow in patches.

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25
Describe lymphoblast-like cells.
Spherical, grow in suspension.
26
What does anchorage-dependent mean?
Cells require a surface to grow on.
27
Give an example of anchorage-independent cells.
Blood cells.
28
What kind of cell line can grow both ways?
Transformed cell lines (e.g., CHO).
29
What is the role of CAMs in cell adhesion?
CAMs help cells attach to substrates for survival and growth.
30
What triggers subculture in adherent cells?
Reaching confluence (full surface coverage).
31
What is contact inhibition?
Cells stop dividing once the surface is covered.
32
How do suspension cells grow?
Free-floating in medium without a surface.
33
How do you know it’s time to subculture suspension cells?
Medium turns turbid due to cell clumping.
34
Why is subculturing important?
To maintain healthy and proliferative cells.
35
When should subculture happen?
At 80–90% confluency.
36
What enzyme is commonly used for passaging adherent cells?
Trypsin (with EDTA).
37
How is subculturing of suspension cells done?
Remove a portion and add fresh medium.
38
When should you use cells for subculturing?
During log (exponential) growth phase.
39
How should you handle cells during subculture?
Gently, with minimal exposure to enzymes.
40
What does seeding density depend on?
Growth rate — low for fast-growing, high for slow-growing.
41
What is the Hayflick limit?
The number of times normal cells can divide (typically 50–100 passages).
42
What is cellular senescence?
When cells stop dividing permanently.
43
Characteristics of senescent cells?
Large, SA-β-GAL activity, resist apoptosis.
44
What are the 4 phases of cell growth?
Lag, Log (growth), Stationary, Death.
45
When is subculturing ideally done?
During the logarithmic (exponential) phase.
46
What are primary cells?
Cells directly from tissue, not yet subcultured.
47
What is a key limitation of primary cells?
Finite lifespan.
48
How are cell lines formed?
From subculturing primary cells that can proliferate.
49
Can cell lines be anchorage-independent?
Yes, depending on the type.
50
What are continuous cell lines?
Transformed cells that can divide indefinitely.
51
What’s a major drawback of continuous cell lines?
They often lose original tissue characteristics.
52
Name 3 characteristics of transformed cell lines.
Fast growth, altered chromosome number, less adherence.
53
Do continuous lines express tissue-specific genes?
No, they often stop expressing them.
54
What are the essential conditions needed to create a "HAPPY" environment for cell culture?
Solid phase (substrate), liquid phase (medium), gaseous phase, temperature control, and aseptic environment.
55
What are some common basal media used in cell culture?
DMEM, EMEM, MEM, RPMI1640, HAM F12.
56
What components does EMEM contain?
Balanced salt solution, non-essential amino acids, glucose, sodium bicarbonate.
57
What are the pH indicator colors for DMEM?
Pink (neutral), yellow (acidic), purple (alkaline).
58
What is essential for most cell cultures aside from the basal medium?
Serum, growth factors, glutamine, additional amino acids.
59
What are key pieces of equipment required for cell culture?
Laminar flow hood, incubator, fridge/freezer, centrifuge, microscope.
60
What is chemical contamination in cell culture?
Hard to detect; caused by endotoxins, plasticisers, metal ions, disinfectant residues.
61
What are signs of biological contamination in cell culture?
Turbid medium, abnormal pH, cell lysis, vacuolization, poor attachment.
62
What is special about mycoplasma contamination?
High concentration, invisible to the naked eye, no visible turbidity.
63
Name 5 types of planar culture vessels.
T-flasks, multilayer plates, cell factories, roller bottles, well plates.
64
What are scalable culture systems for suspension and adherent cells?
Suspension: free-floating; Adherent: require microcarriers for attachment.
65
Name 4 types of bioreactors.
Airlift, Hollow Fibre, Stirred Tank, Packed Bed.
66
How does Trypan Blue help in manual cell counting?
Stains dead cells with compromised membranes blue; live cells remain unstained.
67
What is acridine orange and how does it work?
It's a nucleic acid-selective fluorescent cationic dye that interacts with DNA/RNA and is useful for cell cycle determination.
68
What are the limitations of automated cell counting?
It relies on software estimations and assumes homogeneity in the cell population.
69
What does a merged fluorescence image show in automated counting?
It combines acridine orange (green for live cells) and DAPI (blue for dead cells) signals to identify and count cells.
70
What are some indirect methods of monitoring cell growth and viability?
Luminescent ATP monitoring Fluorescent live/dead staining Fluorescent proliferation assays (e.g. Presto Blue, Alamar Blue) Colorimetric proliferation assays (e.g. XTT, MTT)
71
How does Calcein-AM work in live/dead fluorescent staining?
It's converted into green fluorescent Calcein by intracellular enzymes in live cells.
72
What does Ethidium Homodimer indicate in live/dead staining?
It enters dead cells with damaged membranes and binds nucleic acids, producing bright red fluorescence.
73
Why is animal cell culture important to healthcare?
It plays a key role in developing proteins, bio-therapeutics, and vaccines.
74
How long did it take to develop modern cell culture techniques?
Almost a century, from 1885 to 1978, and they are still evolving.
75
What is critical to maintain when culturing cells?
Key parameters to keep cells "HAPPY," like pH, temperature, medium, and aseptic conditions.
76
What are the two key properties of stem cells?
Self-renewal – Ability to replicate or go through many cycles of cell division. Potency – Ability to differentiate into specialised cells.
77
What are 4 applications of stem cells?
Cell therapy In vitro drug testing Models for growth & birth defects Complex delivery agents
78
What is a totipotent cell?
A cell that can form all specialised cells and extra-embryonic cells (amnion, placenta).
79
What are zygote, morula, and blastocyst?
Zygote: Cell formed when sperm joins egg Morula: Solid ball of ~4 undifferentiated cells Blastocyst: Early embryo with ~50–100 cells
80
Difference between pluripotent and multipotent cells?
Pluripotent: Can form all specialised cells (not extra-embryonic) Multipotent: Can form a limited range of specialised cells
81
Stem cells by maturity/potency?
Pluripotent: Embryonic stem cells Multipotent: Adult stem cells (HSCs, MSCs)
82
Stem cells by source?
Autologous: Same donor and recipient Allogeneic: Different individuals Xenogeneic: Different species

Biotechnology (3 decks)

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