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 cellsDisadvantages : the architecture of the original tissue is lost

Cells change properties over time

Two types of cell culturePrimary cultureSecondary 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

phase contrast micrograph of a culture of primary neurons

Cells are explanted directly from a donor organismCapable of one or two divisions in culture Given the right conditions, survive for some timeDo not continue to grow and eventually senesce and die

AdvantagesMay represent the best experimental in vitro modelsMay retain characteristics of normal cells from that organ

DisadvantagesDifficult to obtainSusceptible to contamination

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 others

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 morphologyAlso, telomeres gradually shorten, % of polyploid cells increases.

Classic example of a continuously cultured cell line

HELAHuman cervical carcinoma cells transformed by HPV 18

• Cell Line – Cells that have undergone a mutation and

won’t 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

Cell Lines

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 growthAttached cultures

-cells require a solid surface on which to grow-plates are specially coated polystyrene-without surface cells can’t survive

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

Proliferation Cells: - high nutrition, - Growth factor

Differentiation Cells:- zero/low growth factor- intermediate nutrition- usually need cell cycle inhibitor

plates

flasks

Environment for

Cells in CulturePart 4

Sterilization methods• Autoclave

– Applies heat under high pressure; this increases the boiling point of water to 121ºC (normal boiling point of water is 100ºC)

– 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

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

-execution of tissue culture procedures without the introductionof contaminating microorganisms

Asceptic technique

A typical laminar flow hoodFiltered air enters the work space from the backDo not block vents!UV lights can be turned on after the work is finishedto 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 densityPassage cells using a dilution appropriate for the cell type-often 1:5, 1:10 or 1:20

Remove attached cells using trypsin to break attachmentsResuspend cells in new media and put into fresh dishPut 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 won’t 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• Tryptone

– Peptide; source of amino acids

• Yeast extract– Source of vitamins,

minerals, and nucleic acids

• Glucose– Energy source

• Salts

Yeast• Peptone

– Peptide; source of amino acids

• Yeast extract– Source of

vitamins, minerals, and nucleic acids

• Glucose– Energy source

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 (~60ºC)

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

isthe 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 30ºC (sometimes lower when expressing

certain proteins)• Most common yeast

species in BT/BM is Saccharomyces cerevisiae (S. cerevisiae)

• S. cerevisiae is also known as brewer’s yeast or baker’s 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

Bacterialchromosome

Plasmid

1Plasmidisolated

3Gene

Inserted into plasmid

2DNAisolated

Cell containing gene of interest

DNAGene ofinterest

Recombinant DNA(plasmid)

4Plasmid putinto cell

Recombinantbacterium

5Cell multiplies withgene of interest

Copies of proteinCopies of gene

Clone of cellsGene for pestresistanceinserted intoplants

Gene used to alter bacteriafor cleaning up toxic waste

Protein used to dissolve bloodclots in heart attack therapy

Protein used to make snow form at highertemperature