Cell Culture Techniques

Question 1

What is cell/tissue culture?

Answer 1

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

Question 2

What are the advantages of cell culture techniques?

Answer 2

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

Question 3

What is the physiochemical environment?

Answer 3

pH, temperature, osmolarity

Question 4

What are the physiological conditions?

Answer 4

Hormone and nutrient levels

Question 5

What is the microenvironment of the cells?

Answer 5

Matrix, cell-cell interactions and cell substrate attachment

Question 6

What is cryptopreservation?

Answer 6

Cells can be stored in liquid nitrogen for long periods

Question 7

What are the disadvantages of cell/tissue culture?

Answer 7

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

Question 8

What are the methods of isolation?

Answer 8

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

Question 9

What are the characteristics of primary tissue cells?

Answer 9

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

Question 10

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

Answer 10

Retain morphological characteristics

Question 11

What are the methods of mechanical dissociation?

Answer 11

Mincing,
Sieving
Pipetting

Question 12

What enzymes are used to isolate primary tissue cells?

Answer 12

Trypsin
Collagenase
Hyaluronidase
Protease 
DNAase

Question 13

How do you separate blood cells?

Answer 13

Density centrifugation

Question 14

Give some examples of primary non-haematopoietic cells

Answer 14

Liver, 
Endothelial cells
Muscle
Skin
Nerves
Fibroblasts
Prostate

Question 15

Give some examples of primary haematopoietic cells

Answer 15

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

Question 16

What are the characteristics of cell lines?

Answer 16

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

Question 17

What are the methods of production of cell lines?

Answer 17

Isolated from cancerous tissues
Immortalisation of healthy primary cultures
Genetic manipulation

Question 18

How can you genetically manipulate cell lines?

Answer 18

Elongate telomeres

Question 19

How do you elongate telomeres?

Answer 19

Introducing telomerase and inhibiting tumour suppressor proteins

Question 20

What is the method for telomeric elongation?

Answer 20

SV40s T-antigen interacts with p53 and pRb

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

Question 21

What do some cells need for immortalisation?

Answer 21

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

Question 22

How are only the colonies with resistance able to survive?

Answer 22

Selection pressure is applied

Question 23

What are 3D cell cultures?

Answer 23

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

Question 24

What are the disadvantages of 2D cell cultures?

Answer 24

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

Question 25

What are the advantages of 2D cell cultures?

Answer 25

Simple, well established | Affordable

Question 26

What are the advantages of 3D cell culture?

Answer 26

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

Question 27

What are the disadvantages of 3D cell cultures?

Answer 27

More complex | More expensive

Question 28

What are the two types of 3D cultures?

Answer 28

Spheroids and organoids

Question 29

What are spheroids generated from?

Answer 29

Cell lines

Question 30

What may spheroids exhibit?

Answer 30

Enhanced physiological responses

Question 31

What do spheroids not do?

Answer 31

Undergo differentiation or self-organisation

Question 32

What are organoids derived from?

Answer 32

PSCs, neonatal stem cells or adult stem cells

Question 33

What do organoid cells spontaneously do?

Answer 33

Self organise into properly attenuated functional cell types and progenitors

Question 34

What do organoids recap?

Answer 34

At least some function of the organ

Question 35

What do patient derived organoids allow?

Answer 35

The study of cancer drug resistance

Question 36

What is cell transfection?

Answer 36

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

Question 37

What are the methods of cell transfection?

Answer 37

Lipofection Electroporation Nucleofection Viral infection/transfection

Question 38

What are the steps of lipofection?

Answer 38

``` 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 ```

Question 39

Why does lipofection work?

Answer 39

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

Question 40

Why are liposomes potential drug carriers for drug delivery?

Answer 40

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

Question 41

How does electroporation work?

Answer 41

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

Question 42

What is nucleofection a combination of?

Answer 42

Electroporation and lipofection

Question 43

What does viral infection/transduction exploit?

Answer 43

Mechanism of viral infection

Question 44

What viruses are used for viral infection/transduction?

Answer 44

Retrovirus Adenovirus Most commonly lentivirus

Question 45

What do viral transduction target cells need to do?

Answer 45

Express the viral receptor

Question 46

What are the steps of viral transduction?

Answer 46

``` 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 ```