Cell Culture History, Basics, and Making Medium Flashcards
Ross Harrison
1907- used a hanging drop technique to observe neurological activity
used frog spinal cord in frog lymph fluid
Alexis Carrel
1910-1913- used plasma clots instead of lymph fluid, first to subculture by transferring tissue to new clot
used chicken embryo extract which is similar to FBS
1923
first glass cell culture flasks were developed by Carrel
1940’s
the phase contrast and inverted microscopes were developed
Wilton Earle
1940- developed many new techniques, developed T flask, first cloned cell line from mouse subcutaneous tissue
George Gey
1951- developed immortalized human cell line: HeLa cells
HeLa cells original medium
chicken plasma clot
human spinal cord serum
bovine embryo extract
saline
Reasons for HeLa cells becoming immortal
Gey’s wife
aseptic technique
cell culture room had filtered air and was sterilized with steam
all surfaces and glassware was sterilized
1950’s
synthetic media was developed
antibiotics started being used in cell culture
cyropreservation was successful with 10-15% viability
1952
trypsin used for the first time in cell culture
1956
mycoplasma detected in cell culture
1959
Eagle developed Eagle’s Minimal Essential Medium (MEM), later modified by Dulbecco —> DMEM
Finite culture characteristics
usually diploid
retain some in vivo characteristics
recently out of the body
contact inhibited (don’t like to touch one another)
limited lifespan (about 30 cell divisions)
very anchorage dependent
Continuous culture characteristics
unlimited lifespan alterations in cytomorphology increase in heteroploidy and aneuploidy increase in tumorigenicity loss of tissue-specific markers reduction in serum dependence increased growth rate to 12-36 hours reduction in anchorage dependence and contact inhibition
Suspension cells morphology
free floating cells or clumps of cells that need to be continuously rolling
usually derived from blood, spleen, or bone marrow and some malignant tumors
Adherent cells morphology
form a monolayer on a culture vessel
usually derived from solid tissue
attachement is essential for proliferation
cease growing when confluency is reached
Epithelial cells derived
ectoderm and endoderm
Epithelial cells morphology
cobble-stone appearance with clear, sharp boundaries, flattened
Epithelial cells tissue
cells that cover the out and inner body surfaces and cavities
Endothelial cells derived
mesoderm
Endothelial cells morphology
cobble-stone appearance with clear, sharp boundaries, flattened
Endothelial cells tissue
lines blood vessels
Fibroblast cells derived
mesoderm
Fibroblast cells morphology
spindle shaped (bipolar) or stellate (multipolar)
Fibroblast cells tissue
connective tissue
Limitations of cell culture
expertise genetic and phenotypic instability cellular spatial interactions are lost proliferation cellular metabolism does not reflect the in vivo situation consumables environmental control
Requirements for media
sterile
supply nutrients
maintain pH- indicated by phenol red
Media contents
water
amino acids (essential + nonessential)
vitamins (group B essential)
inorganic salts (Ca2+, Na+, K+)
carbohydrates (glucose)
proteins and peptides (albumin, transferrin, fibronectin)
fatty acids and lipids (cholesterol and steroids)
Sera definition
a complex mixture of proteins, growth factors, growth inhibitors, hormones, attachment and differentiation factors, and many undefined components
Animals sera comes from
fetal bovine, fetal calf, newborn calf, mature calf, adult bovine, horse
Positives of sera
increase the buffering capacity, bind and neutralize toxins, protect against mechanical damage (all done by albumin)
provide components necessary for cell function
contains protease inhibitors
supports growth of many cell types
Negatives of sera
contains factors which can negatively influence cell characteristics increase risk of contamination batch to batch variation quality control limited shelf life availability must be tested with cells expensive
Buffers
way to counterbalance the lactic acid produced from cellular metabolism
use sodium bicarbonate
Way peristaltic pump works
sucks up media and then pushes it through a filter, creates positive pressure which increases the pH
