cell culture techniques

Question 1

describe what a cell culture is and advantages

Answer 1

lab method - in vitro - where cells are grown outside of their natural environment

advantages

• control physiochemical environment/conditions
• cells easily categorised by cytological or immune staining techniques and visualised using imaging techniques
• cells easily quantified
• reduce use of animal testing
• cheaper to maintain

Question 2

describe primary tissue cells

Answer 2

from tissue/patient, good for personalised medicine
finite lifespan
cells divide and differentiate
cells carry out normal functions

Question 3

how do we isolate primary tissue cells

Answer 3

1. cells migrate out of the explant
2. mechanical or enzymatic dissociation occurs
   EXCEPT: haemopoietic cells = don’t need to be disaggregated, they already are as individual cells circulating in the blood

Question 4

how do we isolate haemopoietic cells

Answer 4

density centrifugation

Question 5

disadvantages of primary tissue cells

Answer 5

inter-patient variation
limited number
finite lifespan and hard to maintain 
different molecular manipulation 
phenotypic instability
variable contamination

Question 6

describe cell lines

Answer 6

immortalised cells
less limited in number of cell divisions or unlimited
phenotypically stable, defined population
limitless availability
easy to grow
good reproducibility and model for basic science

Question 7

describe methods of production for cell lines

Answer 7

isolate from cancerous tissues
immortalisation of healthy primary cultures = genetic manipulation > regulate growth and ageing
as cells divide = telomeres shorten = cell division stops

Question 8

describe oncoprotein

Answer 8

SU40 = interacts with P53 and pRb = increased growth without the loss of function of these proteins

e6 = degradation of p53

e7 = inactivates pRb

cell lines with e6/7 oncoprotein are believed to maintain a differentiated phenotype

Question 9

list pros and cons of 2D culture

Answer 9

x - forced apical basal polarity
x- high stiffness
x- limited communication with other cells
x- no diffusion gradient
x- results not relevant to human physiology
BUT

• simple, well established
• affordable

Question 10

list pros and cons of 3D culture

Answer 10

x- more complex
x- added expense
BUT

adhesion
no forced polarity 
variable stiffness
diffusion gradient of nutrients and waste products 
more relevant to human physiology

Question 11

describe what spheroids and organoid are

Answer 11

spheroid = 3d, from cell lines, 1 or more cell type

organoid = 3D, from primary tissues, derived from stem cells

Question 12

define transfection

Answer 12

foreign DNA, introduced to eukaryotic cells via non-viral methods in the lab e.g. plasmids

Question 13

describe methods of cell transfection

Answer 13

1. liposuction = liposomes introduce DNA, interacts with cell, endocytosis, released from endosome, transported to nucleus.
2. electroporation
3. nucelofection = combination of electroporation and lipofection. increased efficiency esp of non dividing cells.
4. viral transduction = exploits mechanisms of viral infection, high transfection efficiency