Cell culture

Question 1

What types of contamination are there in cell culture?

Answer 1

• chemical = incorrect reagent, excessive reagent conc, or accidentally introd chemicals
• human cell contam = indiv cell lines have unique characteristics, eg. specific mutations, so when culturing multiple lines can introduce one to another
• viral = need specific testing to confirm presence, often have little impact, but severe infections can change cell morphology/behaviour, when culturing human blood or tissue samples from patients risk of transmission of pathogenic viruses to user is higher, need good practise plus additional control measures, eg. PPE and specialised cell culture hoods
• bacterial = most common and easily identified as small dark cylindrical cells, causes physiological stress to cells, can inc antibiotics but not substitute for good practise
• ic bacteria, mycoplasma = no cell wall, can fuse membrane w/ host cell and compete for resources, v small so need PCR etc to detect
• fungal = yeast (small, uniform) or other fungi and moulds (large growths w/ hyphae)

Question 2

How is aseptic technique achieved in cell culture?

Answer 2

• laminar flow hoods –> pull outside air down and away from work surfaces, then pass t/ HEPA filter to prevent contaminants reaching work area
• must spray and wipe everything that goes in w/ 70% ethanol (inc gloves)

Question 3

What is req for maintaining cells in culture?

Answer 3

• media w/ electrolytes, AAs, vitamins, sugars and add supplements
• phenol red often in media, pH indicator to assess acidity of growing culture
• FBS, for prots, hormones, GFs to stim growth/prolif
• passage cells when become confluent, to provide excess of space and nutrients again

Question 4

Role of trypsinisation in passaging?

Answer 4

• detach adherent cells from flask

Question 5

Role of FBS in cell culture?

Answer 5

• stim growth and prolif
• quenches trypsin
• provide prots, hormones, GFs

Question 6

How is cell conc and viability determined?

Answer 6

• haematocytometer to count cells
• conc = no. x 10^4 cells/ml
• dead cells take up Trypan Blue as membrane more permeable, so can calc viability as total live/total cells x100

Question 7

Why must cultures be checked for confluency?

Answer 7

• optimum balance between the health and number of cells in a culture is achieved
• after this point, as cell numbers increase, health of cells will begin to be
  negatively affected

Question 8

What are primary cells? Adv and limitations?

Answer 8

• taken directly from an organism
• cannot divide indefinitely, ie. non-immortal
• adv = “normal”, non culture adapted, more physiologically comparable than established cell lines, easily obtained from tissue samples
• limitations = success rate of isolation is variable, variability between samples, access to samples, contamination, limited growth capacity, not suitable for LT experiments, non-immortal

Question 9

What are immortalised cells, advs and limitations?

Answer 9

• derived from normal or cancer cells
• adv: divide indefinitely, allows LT/large scale experiments, diverse range available, maintains consistency between experiments
• limitations: have to immortalise, increased genomic instability, culture adaptation, often derived from tumours, not ‘normal’,

Question 10

What is the Hayflick limit?

Answer 10

• no. of times a cell pop will divide before cell division stops
• 40-60 divisions

Question 11

What is the adv of lymphoblastoid production?

Answer 11

• continuous source of patient DNA –> if new test could carry it out on stored sample
• avoid senescence and are semi-immortal
• cytogenetically normal
• serve as bio-repositories of patient data
• comparable to parent organism