Cell Culture History, Basics, and Making Medium

Question 1

Ross Harrison

Answer 1

1907- used a hanging drop technique to observe neurological activity
used frog spinal cord in frog lymph fluid

Question 2

Alexis Carrel

Answer 2

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

Question 3

1923

Answer 3

first glass cell culture flasks were developed by Carrel

Question 4

1940’s

Answer 4

the phase contrast and inverted microscopes were developed

Question 5

Wilton Earle

Answer 5

1940- developed many new techniques, developed T flask, first cloned cell line from mouse subcutaneous tissue

Question 6

George Gey

Answer 6

1951- developed immortalized human cell line: HeLa cells

Question 7

HeLa cells original medium

Answer 7

chicken plasma clot
human spinal cord serum
bovine embryo extract
saline

Question 8

Reasons for HeLa cells becoming immortal

Answer 8

Gey’s wife
aseptic technique
cell culture room had filtered air and was sterilized with steam
all surfaces and glassware was sterilized

Question 9

1950’s

Answer 9

synthetic media was developed
antibiotics started being used in cell culture
cyropreservation was successful with 10-15% viability

Question 10

1952

Answer 10

trypsin used for the first time in cell culture

Question 11

1956

Answer 11

mycoplasma detected in cell culture

Question 12

1959

Answer 12

Eagle developed Eagle’s Minimal Essential Medium (MEM), later modified by Dulbecco —> DMEM

Question 13

Finite culture characteristics

Answer 13

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

Question 14

Continuous culture characteristics

Answer 14

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

Question 15

Suspension cells morphology

Answer 15

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

Question 16

Adherent cells morphology

Answer 16

form a monolayer on a culture vessel
usually derived from solid tissue
attachement is essential for proliferation
cease growing when confluency is reached

Question 17

Epithelial cells derived

Answer 17

ectoderm and endoderm

Question 18

Epithelial cells morphology

Answer 18

cobble-stone appearance with clear, sharp boundaries, flattened

Question 19

Epithelial cells tissue

Answer 19

cells that cover the out and inner body surfaces and cavities

Question 20

Endothelial cells derived

Answer 20

mesoderm

Question 21

Endothelial cells morphology

Answer 21

cobble-stone appearance with clear, sharp boundaries, flattened

Question 22

Endothelial cells tissue

Answer 22

lines blood vessels

Question 23

Fibroblast cells derived

Answer 23

mesoderm

Question 24

Fibroblast cells morphology

Answer 24

spindle shaped (bipolar) or stellate (multipolar)

Question 25

Fibroblast cells tissue

Answer 25

connective tissue

Question 26

Limitations of cell culture

Answer 26

``` expertise genetic and phenotypic instability cellular spatial interactions are lost proliferation cellular metabolism does not reflect the in vivo situation consumables environmental control ```

Question 27

Requirements for media

Answer 27

sterile supply nutrients maintain pH- indicated by phenol red

Question 28

Media contents

Answer 28

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)

Question 29

Sera definition

Answer 29

a complex mixture of proteins, growth factors, growth inhibitors, hormones, attachment and differentiation factors, and many undefined components

Question 30

Animals sera comes from

Answer 30

fetal bovine, fetal calf, newborn calf, mature calf, adult bovine, horse

Question 31

Positives of sera

Answer 31

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

Question 32

Negatives of sera

Answer 32

``` 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 ```

Question 33

Buffers

Answer 33

way to counterbalance the lactic acid produced from cellular metabolism use sodium bicarbonate

Question 34

Way peristaltic pump works

Answer 34

sucks up media and then pushes it through a filter, creates positive pressure which increases the pH