Terminology Cells

in
Culture
(PART 1)
 Introduction

Cell Culture:
the cultivation or growth of cells outside of the host organism

Advantage : Allows direct access a population of cells

Disadvantages : the architecture of the original tissue is lost
Cells change properties over time

Two types of cell culture

Primary culture
Secondary culture
Classification of Cell
Cultures
Primary Culture
Cells taken directly from a tissue to a
dish
Secondary Culture
Cells taken from a primary culture and
passed or divided in vitro.
These cells have a limited number of
divisions or passages. After the limit,
they will undergo apoptosis.
 Primary cultures
Cells are explanted directly from a donor organism
Capable of one or two divisions in culture
Given the right conditions, survive for some time
Do not continue to grow and eventually senesce and die

Advantages
May represent the best experimental in vitro models
May retain characteristics of normal cells from that organ

Disadvantages
Difficult to obtain
Susceptible to contamination

phase contrast micrograph of a culture of

primary neurons
Growth factors are present in cell media
so that cells will keep dividing

This is an example of how a cell culture is made from tissue.

Here, PDGF (one type of growth factor) is added--there are many
Passage number
The number of times the cells have been
removed (or split) from the plate and re-
plated.
Always write this on your plate or flask as
P#
The Hayflick Limit
 replicative senescence

Normal human fibroblasts (left) and fibroblasts showing a senescent

morphology (right).
Cells get larger, more diverse morphology
Also, telomeres gradually shorten, % of polyploid cells
increases.
 Classic example of a
continuously cultured cell line

HELA
Human cervical carcinoma cells transformed by HPV 18
Cell Lines
Cell Line
Cells that have undergone a mutation and
wont undergo apoptosis after a limited
number of passages. They will grow
indefinitely.
Transformed cell line
A cell line that has been transformed by a
tumor inducing virus or chemical. Can cause
tumors if injected into animal.
Hybrid cell line (hybridoma)
Two cell types fused together with
characteristics of each
Passaging cells (subculturing cells)
Process of diluting cell number in order
to keep cells actively growing
For adherent cells, when they cover the
tissue culture dish, they need to be
passaged
Otherwise, the cells will become unhealthy
and stop growing
Cell Culture Enemies
Cells are more susceptible to infection at certain times
When they have been stressed after recovery from liquid
nitrogen
Primary cells are often generated by enzymatic disruption and
selection procedures
Cultures prepared from live animals will often be accompanied
by micro-organisms
Splitting cells at too high a dilution can allow micro-organisms to
dominate the culture
Cells release Autocrine growth factors which condition the
medium and favour cell growth
Types of Cell
(PART 2)
 Types of cell growth
Attached cultures
-cells require a solid surface on which to grow
-plates are specially coated polystyrene
-without surface cells cant survive

Suspension cultures
flasks -Liquid cultures, cells do not adhere to plate surface
-hematopoietic cells

Proliferation Cells:
- high nutrition,
- Growth factor
plates
Differentiation Cells:
- zero/low growth factor
- intermediate nutrition
- usually need cell cycle inhibitor
Environment
for
Cells in Culture
Part 4
 Sterilization methods
Autoclave
Applies heat under high pressure; this
increases the boiling point of water to 121C
(normal boiling point of water is 100C)
15-20 min. is sufficient to kill most microbes
Filtration
Large volumes: suction filter
Small volumes: syringe filter
UV radiation
Causes mutations to form in the DNA of microbes, causing
genetic damage and eventual death
Used to sterilize surfaces (such as the surface of laminar
flow hoods)
 Asceptic technique
-execution of tissue culture procedures without the introduction
of contaminating microorganisms

Work with cells in a cell culture hood

-laminar flow hoods
-prevent airborne organisms from entering your cultures
-always use ETOH to clean hood before and after use
-always use separate sterile pipettes for each manipulation
-never sneeze directly in your culture
-work rapidly but carefully
Asceptic technique

A typical laminar flow hood

Filtered air enters the work space from the back
Do not block vents!
UV lights can be turned on after the work is finished
to sterilize surfaces.
 Incubators are required for
mammalian cell
A typical incubator for cell culture.
-internal temperature is controlled.
-CO2 incubators contain a continuous flow of
carbon dioxide-containing air.
The tanks to the right of the incubator are carbon dioxide tanks
used to provide the carbon dioxide for cell culture.
 Growing cells in culture
Place in culture dish in proper media at appropriate density
Passage cells using a dilution appropriate for the cell type
-often 1:5, 1:10 or 1:20

Remove attached cells using trypsin to break attachments

Resuspend cells in new media and put into fresh dish
Put back in incubator
 Confluency
How covered the growing
surface appears
This is usually a guess
Optimal confluency for
moving cells to a new dish
is 70-80%
too low, cells will be in lag
phase and wont proliferate
Too high and cells may
undergo unfavorable
changes and will be difficult
to remove from plate.
YEAST & BACTERIAL
CULTURE
Part 6
 Typical ingredients list in media
to grow:
E. coli bacteria Yeast
Tryptone Peptone
Peptide; source of Peptide; source of
amino acids amino acids
Yeast extract Yeast extract
Source of vitamins, Source of vitamins,
minerals, and minerals, and
nucleic acids
nucleic acids
Glucose
Glucose
Energy source
Energy source
Salts
Working with bacterial cells
Bacterial cells are grown as either
liquid or solid cultures
Solid cultures (nutrient agar plates) are used to
isolate single bacterial cells with specific
properties (more on this later)
Liquid cultures are used to scale up cell cultures
(grow larger volumes of cells)
Starting liquid cultures
Involves seeding liquid media with cells from
either another liquid culture or a colony from a
solid culture plate
Starting solid cultures
Streaking plates
Nutrient agar plates
for solid cultures of bacteria
Agar: an
unbranched
polysaccharide
obtained from
the cell walls of
some species
of seaweed
Nutrients are
added to allow
bacteria to
grow
Liquefies with
heat, solidifies
as it cools
(~60C)
 Streaking a plate
(to start a solid bacterial culture)
Use sterile loop to add cells to plate; then resterilize
loop to repeatedly spread and dilute cells on plate in
such a way as to obtain single bacterial colonies
Each colony arose from one cell Obtaining single colonies
is
the goal of this procedure
Monitoring cell growth in liquid
bacterial cultures
OD: stands for optical density
The number of cells in a bacterial culture
can be estimated by reading the
absorbance at 600 nm (OD600).
Want to maximize cell
density while keeping
cell cultures in growth
phase
Yeast
Yeast are small, unicellular eukaryotic cells
Grow at 30C (sometimes lower when expressing
certain proteins)
Most common yeast
species in BT/BM is
Saccharomyces
cerevisiae (S. cerevisiae)
S. cerevisiae is also
known as brewers
yeast or bakers yeast
S. cerevisiae are
budding yeast--they
grow by budding off
a daughter cell from
the mother cell
Working with yeast cell cultures
Similar in many ways to working
with bacterial cultures
Cells can be grown as liquid or
solid cultures, and lab technicians
frequently go from one type of
culture to the other
Seed a liquid culture with a colony from a plate
Streak a plate from a liquid culture
Example of a plasmid used to produce
recombinant proteins in E. coli
Bacterium Cell containing
1Plasmid
isolated gene of interest
DNA
2
Gene isolated
3Inserted
BacterialPlasmid into plasmid
chromosome
Recombinant DNA DNA
(plasmid) Gene of
Plasmid put interest
4
into cell
Recombinant
bacterium
Cell
5
multiplies with
gene of interest

Copies of gene Copies of protein

Gene for pest Clone of cells Protein used

resistance to make snow
inserted into form at higher
plants temperature

Gene used to alter bacteriaProtein used to dissolve blood

for cleaning up toxic waste clots in heart attack therapy