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MBD/MI (Wothaya) > Cell Culture Techniques > Flashcards

Cell Culture Techniques Flashcards

1
Q

What is cell/tissue culture?

A

Lab method by which cells are grown under controlled conditions outside their natural environment

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2
Q

What are the advantages of cell culture techniques?

A

Control of the physiochemical environment and physiological conditions
Control of the micro-environment of the cells
Cells can be easily characterised by cytological and immune staining techniques and visualised using imaging techniques
Cells can be stored in liquid nitrogen for long periods
Cells can be easily quantified
Reduces use of animals in scientific experiments
Cheaper to maintain

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3
Q

What is the physiochemical environment?

A

pH, temperature, osmolarity

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4
Q

What are the physiological conditions?

A

Hormone and nutrient levels

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5
Q

What is the microenvironment of the cells?

A

Matrix, cell-cell interactions and cell substrate attachment

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6
Q

What is cryptopreservation?

A

Cells can be stored in liquid nitrogen for long periods

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7
Q

What are the disadvantages of cell/tissue culture?

A 
Inter-patient variation
Limited number (at high cost) 
Finite lifespan and hard to maintain
Difficult molecular manipulation
Phenotypic instability
Variable contamination
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8
Q

What are the methods of isolation?

A

Cells allowed to migrate out of an explant
Mechanical dissociation
Enzymatic dissocitation

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9
Q

What are the characteristics of primary tissue cells?

A

Cells derived directly from tissues/ patients
Finite lifespan
Cells divide and/or differentiate
Cells carry out normal functions

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10
Q

Why is it good if the cells are allowed to migrate out of an explant?

A

Retain morphological characteristics

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11
Q

What are the methods of mechanical dissociation?

A

Mincing,
Sieving
Pipetting

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12
Q

What enzymes are used to isolate primary tissue cells?

A 
Trypsin
Collagenase
Hyaluronidase
Protease 
DNAase
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13
Q

How do you separate blood cells?

A

Density centrifugation

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14
Q

Give some examples of primary non-haematopoietic cells

A 
Liver, 
Endothelial cells
Muscle
Skin
Nerves
Fibroblasts
Prostate
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15
Q

Give some examples of primary haematopoietic cells

A 
Stem, progenitor cells
T and B cells
Monocyte
Osteoblasts
Dendritic cells
Neutrophils
Erythrocytes
Megakaryocytes
Platelets
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16
Q

What are the characteristics of cell lines?

A

Immortalised cells
Less limited (or unlimited) number of cell divisions
Phenotypically stable, defined population
Limitless ability
Easy to grow
Good reproducibility
Good model for basic science

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17
Q

What are the methods of production of cell lines?

A

Isolated from cancerous tissues
Immortalisation of healthy primary cultures
Genetic manipulation

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18
Q

How can you genetically manipulate cell lines?

A

Elongate telomeres

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19
Q

How do you elongate telomeres?

A

Introducing telomerase and inhibiting tumour suppressor proteins

20
Q

What is the method for telomeric elongation?

A

SV40s T-antigen interacts with p53 and pRb

E-6 targets p53 for degredation and E7 binds to pRb, inactivating it

21
Q

What do some cells need for immortalisation?

A

Introduction of the telomerase gene and inactivation of the pRb/p53

22
Q

How are only the colonies with resistance able to survive?

A

Selection pressure is applied

23
Q

What are 3D cell cultures?

A

Artificially created environment in which cells are permitted to grow or interact with their surroundings in all three dimensions

24
Q

What are the disadvantages of 2D cell cultures?

A 
Forced apical-basal polarity
High stiffness
Limited communication with other cells
No diffusion of gradients
Results not relevant to human physiology
25
Q

What are the advantages of 2D cell cultures?

A

Simple, well established

Affordable

26
Q

What are the advantages of 3D cell culture?

A 
Adhesion in all three dimensions
No forced polarity
Variable stiffness
Diffusion gradients of nutrients and waste products 
More relevant to human physiology
27
Q

What are the disadvantages of 3D cell cultures?

A

More complex

More expensive

28
Q

What are the two types of 3D cultures?

A

Spheroids and organoids

29
Q

What are spheroids generated from?

A

Cell lines

30
Q

What may spheroids exhibit?

A

Enhanced physiological responses

31
Q

What do spheroids not do?

A

Undergo differentiation or self-organisation

32
Q

What are organoids derived from?

A

PSCs, neonatal stem cells or adult stem cells

33
Q

What do organoid cells spontaneously do?

A

Self organise into properly attenuated functional cell types and progenitors

34
Q

What do organoids recap?

A

At least some function of the organ

35
Q

What do patient derived organoids allow?

A

The study of cancer drug resistance

36
Q

What is cell transfection?

A

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

37
Q

What are the methods of cell transfection?

A

Lipofection
Electroporation
Nucleofection
Viral infection/transfection

38
Q

What are the steps of lipofection?

A 
Interaction with the cell membrane
Taken up by endocytosis
Release from the endosome
Transport to the nucleus
Entry to the nucleus is inefficient and may need mitosis
39
Q

Why does lipofection work?

A

Liposomes have a net positive charge and plasma membranes are negatively charged

40
Q

Why are liposomes potential drug carriers for drug delivery?

A

Can carry hydrophobic or hydrophilic drugs by attaching tissue specific antigens to the surface of the liposome which allows for targeted drug delivery

41
Q

How does electroporation work?

A

Electric field applied to cells which increases their permeability - opens pores in the plasma membrane

42
Q

What is nucleofection a combination of?

A

Electroporation and lipofection

43
Q

What does viral infection/transduction exploit?

A

Mechanism of viral infection

44
Q

What viruses are used for viral infection/transduction?

A

Retrovirus
Adenovirus
Most commonly lentivirus

45
Q

What do viral transduction target cells need to do?

A

Express the viral receptor

46
Q

What are the steps of viral transduction?

A 
Create viral plasmid
Carry out non-viral transfection to insert viral DNA into cell line
Collect first supernatant
Refrigerate
Collect second supernatant
Collect viral pellet after centrifuging
Use for transduction or save in freezer

MBD/MI (Wothaya) (57 decks)

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