Download Cell culture and bioassay

Cell culture and bioassay
R S Mason, Physiology
Cell culture:
Cell types
Requirements for culture
Infections
Cell preparation and manipulations
Other aspects:
Culture surfaces
Differentiating agents
Organotypic cultures
Bioassay:
Requirements
Advantages and disadvantages
Cell types:
Non- attached eg lymphocytes, hybridoma cells
Attached cells eg fibroblasts, keratinocytes
Cell lines – transformed, immortalised
Primary cells
Cell culture requirements:
1) source of cells
2) sterile environment
3) nutrients
4) gas exchange
5) pH balance
6) temperature control
Sterile environment:
Laminar flow or biohazard hood
Sterile disposables (flasks, filters, gloves, pipettes)
Autoclaved instruments
Alcohol solutions 70% in water
Aseptic techniques: flaming
pipette not pour
hand movements in hood
Nutrients:
Culture medium:
Isoosmotic (260-320 mOsm/kg)
pH balanced – often bicarbonate/CO2 system
Salts
Carbohydrate source
Amino acids
Vitamins
Others: cholesterol, essential fatty acids, growth
factors, hormones etc
Fetal bovine serum:
used at 2-10%
“undefined”
source of protein, hormones, growth factors
charcoal stripping to remove hormones
heat inactivation
Serum substitutes:
Protein – eg bovine serum albumin
Growth factors eg insulin
Transferrin
Selenium
Specific factors eg specific growth factors
Gas exchange:
O2 in – CO2 out
To atmosphere – filtered
Facilitation of diffusion – eg shallow liquid levels
pH balance:
pH optimum around pH 7.2 – 6.8-7.4
pH indicator eg phenol red
bicarbonate/CO2 buffer system (cheap but pK 6.1)
CO2
(5% in air)

----------------------------------------------------------------------CO2 + H2O  H2CO3  H + + HCO3 –

NaHCO3  Na+ + HCO3
Other buffers eg HEPES
–
Temperature:
Generally 37 o C
Temperature sensitive mutants
Low temperature to  metabolism
eg tissue storage
particular experimental studies
Cell storage in liquid nitrogen
Controlled CO2 and temperature provided by incubator
Infections:
Much easier to prevent than treat
Obvious – bacterial and fungal
Not obvious – mycoplasma
Sources:
Operator
Instruments
Media
Use of antibiotics:
Often penicillin/streptomycin combinations for
prevention
Infection present – what to do
Mostly: throw away cells (note disposal procedures)
Treat with antibiotics –
Add physical measures eg new flasks
Cell preparation and manipulations:
From tissues: dissociation
– mechanical
- enzymatic
- combinations
eg
skin sample:
wash alcohol, betadine
dispase overnight (breaks down dermal-epidermal
junction)
mechanically separate epidermis
trypsin digestion
plate cells : media to select for cell type
fibroblast, keratinocyte, melanocyte
Cell preparation and manipulations cont:
Passaging:
Non-attached cells
– centrifuge, resuspend, dilute, place in flasks
Attached cells
– EDTA, trypsin, mechanical removal
- centrifuge, resuspend, dilute, replate
Freezing and thawing cells:
- problem of ice crystal formation
- 10% dimethylsulphoxide
- controlled rate freezing
- store in liquid nitrogen
- cells fragile on thawing
Other aspects:
Culture surfaces:
Normal plastic
Coated surfaces
Other surfaces eg Thermanox
Differentiating agents:
eg for bone cells – phosphate
ascorbic acid
dexamethasone
Organotypic cultures:
3 dimensional multilayers for bone cells
confluent epithelial cells for transport studies
(culture well inserts)
skin equivalents: collagen with fibroblasts
multilayered keratinocytes
melanocytes
actual tissue pieces – eg long bones
fracture calluses
Bioassays
Factor - target – response
Can use: whole animals
cell cultures
requirements:
reliable source of target material
cell lines
inbred animals
identical conditions
age of animal, housing conditions etc
passage number of cells, density
establish normal range
degree of variability
for each assay – positive and negative controls
other standards
advantages:
useful for non-defined agents
(cant raise antibody for measurement assay)
examines biological activity (not just immune
binding)
disadvantages:
cumbersome
often large variability assay to assay
generally low sensitivity, low precision