sterile technique
1. working area far from drafts and traffic
2. clean working area
3. wipe down working area with cleaning agent before use
4. pipettes and bottles should be sterile
5. minimize hand movements
6. have everything you need assembled in 1 place
7. wear gloves
Technique tips
1. minimize: distance, exposure, motion, pouring
2. open bottles and point @ 45 degree angle
3. Cap/lid face down on clean surface
4.flame glass pipettes and open bottles before use
5. DO NOT flame plastic
6. pipette gently/don't swirl bottles
7. don't le
Biosafety cabinet
vents air up and out
Laminar flow hood / Class 2 Biosafety cabinet
recycles filtered air
working in a biosafety cabinet
1. verify hood is on and air is circulating
2. lower the sash to the calibrationmark
3. do not block airflow
4. secure papers and other lightweight material
5. wipe down surface w/ 70% ethanol
6. put sterile caps face down
7. minimize hand movements
8. DO
Types of culture and cell lines
1. primary cells: animal tissue (fetal or adult)
2. finite cell lines: animal tissue (usually fetal)
3. continuous cell lines: spontaneous
transformation of primary or finite cell lines
4. transformed cell lines: tumor tissue
5. hybridomas: fusion of plas
Cell cultures are described in 2 ways:
1. origin of the cells (cell lines)
2. manner of growth (suspension or adhesion)
primary cell lines
Cell culture grown from cells taken directly from a tissue
continuous cell lines
cells manipulated to express a particular and needed phenotype
transformed cell lines
derived from tumors or transformed spontaneously in culture via mutation (cancer):
1. grows to high cell density
2. lower requirement for growth factors and serum
3. more anchorage-independence
4. ability to proliferate indefinitely
manner of growth
1. suspension cells
2. adhesion cells
suspension cells
1. proliferate in the medium
2. floating
3. round
ex: B/T cells
adhesion cells
1. grow in a monolayer
2. attached to surfaces of culture vessel
3. derived from ecto- or endodermal layers
4. have various shapes (can be flat)
ex: fibroblasts
anchorage dependent growth
The requirement that to divide/grow, a cell must be attached to a solid surface.
anchorage independent growth
A trait exhibited by transformed (cancer) cells, which grow well when they are freely suspended in a liquid or semisolid medium
Observing Cells physical condition
1. check medium color (pH indicator)
2. cloudiness (overgrown culture)
3. clumped cells (suspended culture)
4. peeling cells (adherent culture)
5. contamination
when pH of cell culture medium is acidic:
yellow / orange color in media
when pH of cell culture medium is basic:
pink / purple color in media
Observing adherent cells
1. flat cells, or shapely
2. pattern of growth like a street, swirls, random growth, or growing on top of each other
3. can see dark, round shadow of nucleus
4. cytoplasm visible
Observing suspension cells
1. spherical shaped upon observation
2. floating
Culturing frozen cells
- usually frozen in medium w/ serum (FBS) + freezing additive (DMSO) @ 196C
1. warm in 37C water bath, thaw it fast
2. 70% ethanol the thawed beaker
3. use
Cell maintenance
1. Feed: with fresh medium
2. Split: original culture into multiple cultures to prevent overgrowth
3. Freeze
Feeding and passaging (antibiotics)
penicillin/streptomycin to prevent contamination by mycoplasma (PCR can be used to detect contaminants)
Mycoplasma
can silently contaminate entire cell cultures and labs, but is easily detectable (LOOK OUT FOR THIS)
Standard medium: DMEM
has nutrients but NO growth factors
Serum used for cell cultures
fetal bovine serum 10% (FBS) = growth factors
asynchronized growing
cells are in a bunch of different cell cycle phases
synchronized growing
DMEM (standard medium) purpose: all cells growing in same cell cycle phase
-CELLS DO NOT DIVIDE UNTIL YOU ADD GROWTH FACTOR
phenol red
pH indicator
complete growing medium
1. DMEM (media)
2. 10% FBS
3. 1% P/S (penicillin/streptomycin)
free-down medium
1. DMEM (media)
2. 20% FBS
3. 10% P/S (penicillin/streptomycin)
4. DMSO (freezing factor)
DMSO
- culture should be frozen slowly
- prevents freeze fracturing of cell culture in media
- DMSO added and washed off
293T cells
human embryonic kidney cells (adhesion cells)
NIH3T3 cells
fibroblast of mouse, embryo (adhesion cells)
confluence
how many cells you see
splitting adherent cells
- single plate split into multiple plates to prevent overgrowth (ex: 1 split into 4 plates in a 1:8 ratio)
- once attached, media DOES NOT add volume, it's feeding the cells
- growth limited by surface area
splitting suspension cells
- grow in the media
- DILUTE the volume differences to stimulate growth
-keep track of volume ratios though
splitting 293Ts w/ Dr D steps:
1. Aspirate medium from cells
2. Add warm PBS gently
3. pipette medium to dish wall, not directly onto cells, to spread PBS all over adherent cells
4. aspirate PBS/medium wash off of cells
5. add trypsin just enough to cover cells
6. add DMEM to inhibit t
Trypsin
- digests proteins
- gets rid of extracellular attachments (integrins) of adhesion cells so they no longer stick to the plate (now float)
extracellular component broken up by trypsin
integrins, proteins
HEC293
human kidney cells used to make retroviruses
most common tissue culture conditions
- 30C
- 5-10 % CO2 (to maintain physiological pH)
- humidified to not lose media volume (bacteria is not humidified)
Patient derived xenografts (PDX)
tissue from 1 animal to another
Freezing cells
1. use freezing medium (w/ DMSO)
2.dispense into freezer boxes
3. store in liquid nitrogen tank/freezer
How to recognize contamination
1. cloudiness
2. medium color change
3. smell!
4. presence of other organisms
CO2 incubator (5%CO2)
-used to regulate pH of the cell medium
- 37C
CO2 tanks
- tank of compressed CO2 attached to incubator, delivers CO2
- chained upright to wall
- high pressure: regulator reduces pressure (2)