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MAT300 > Cell culture > Flashcards

Cell culture Flashcards

1
Q

What is cell culture used in?

A
  • Stem cell biology
  • Cancer research
  • Drug discovery
  • Antibody production (CHO cells)
  • Cell therapy
  • And much more
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2
Q

What are the pros of cell culture?

A

1) Well established, reproducible methods
2) Reduction in animal testing
3) Able to test human cells
4) Can adjust environment (eg. ECM, O2)
5) Can look at single cell properties
6) Many endpoints possible (gene expression, secreted factors, phenotype, function)

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3
Q

What are the cons of cell culture?

A

1) Very different to in vivo environment
2) Limited culture time
3) Many cells are cancerous or “immortal”
4) Artificial environment, often 2D
5) Cells usually exist in complex multicellular environment

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4
Q

What are the different methods of cell culture?

A 
Pipettes 
Media components 
Aseptic technique 
Tissue Culture plastic 
Biosafety cabinets
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5
Q

What is the cell culture media and what can it consist of?

A
  • Many different types
  • Formulated for optimal cell growth
  • Salts, amino acids, vitamins, glucose etc.
  • Phenol red: pH indicator
  • Antibiotics
  • Antifungals
  • Serum- contains proteins and lipids – usually fetal calf serum (FCS)
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6
Q

What are serum free alternatives?

A

– Human platelet rich plasma
– Bovine pituitary extract
– Growth factors
– Animal product free (limited cell types)

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7
Q

What are some sterilisation methods?

A
  • Autoclave
  • Ethylene oxide
  • γ irradiation
  • UV
  • Alcohol (70%)
  • Peracetic acid
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8
Q

What were tissue culture surfaces made from and what are they made from now?

A

Initially glass and pyrex
(in 1950s, improved with rat tail collagen)
(in 1960s polystyrene, modified to improve hydrophilicity and cell attachment
• Poly-D-lysine coatings used for some cell types
• ECM proteins eg. fibronectin, laminin and collagen

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9
Q

What is cell dettachement?

A

• Trypsin EDTA
• Inhibited by protein
EDTA = calcium chelator (Ca2+ inhibits trypsin) Trypsin = protease
• Non-enzymatic dissociation solutions available but not as effective

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10
Q

What are the types of cells used?

A
  • Primary cell Eg. Human dermal fibroblast
  • Cancer cell line Eg. HeLa
  • Immortalised cell line Eg. HaCaT (Many others immortalised with SV40 viral oncogene or hTERT)
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11
Q

What are primary cells?

A

Isolated directly from tissue Physiologically relevant
Limited life span in culture
High variability
Commercially available

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12
Q

What are cell lines?

A

Immortalised cells
Relevant for cancer research
Manipulated to be immortal (or cancerous)
Reproducible between labs Commercially available

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13
Q

What are the key areas of cell behaviour?

A
  • Confluency
  • Contact inhibition
  • Adherent vs non-adherent
  • Passage number
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14
Q

What are Cell culture end points?

A
  • Viability and growth
  • Secreted factors
  • Gene and protein expression
  • Imaging
  • Functional assays
  • Cell phenotype and cell surface markers
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15
Q

What is Viability and cell growth?

A
  • Trypan blue exclusion
  • MTT assay (MAT2530 practical)
  • Resazurin assays (AlamarBlue®)
  • Cell counts
  • Proliferation assays (eg. With CellTrace™)
  • ATP assays
  • Apoptosis and necrosix
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16
Q

What are considered more complex cell culture?

A
  • 3D culture
  • 3D microenvironment
  • Co-culture (multiple cells types)
  • Dynamic culture
17
Q

Before cells (or any new medical intervention) can be used for human treatment, what two bodies must it be approved by?

A
  1. Regulator/regulatory body UK examples include the Medicines and Health Regulatory Authority and The Human Tissue Authority.
  2. Ethic Committee UK examples include NHS Research Ethics Committee.
18
Q

What does ‘The Regulators’ do?

A

protect patients. They are made up of professionals who determine what can be done in a legal framework. They perform a preclinical Risk Assessment and analyse preclinical data

19
Q

What does ‘the ethics committee do?

A

consider risk vs. benefit. They are made up of a mix of professionals and lay people. They look at the protocol to determine places where the patient could be treated “unethically”. Considerations include: data security, informed consent, patient identification, treatment procedures, handling of adverse effects and reporting of results

MAT300 (14 decks)

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