bcbt 478 introduction to mammalian cell culture 2009

8/9/2019 BCBT 478 Introduction to Mammalian Cell Culture 2009

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BCBT 478:

Plant and Mammalian Tissue Culture

Introduction to Mammalian Tissue Culture



Tissue Culture

Tissue Culture:

The general term for the removal of cells,

tissues or organs from an animal or plant

and their subsequent placement into an

artificial environment conducive to growth.



Organ Culture

Organ Culture

The culture of whole organs or intact organ

fragments with the intent of studying their

continued function or development.



Cell Culture

Cell Culture

When cells are removed from the organ

fragments prior to, or during cultivation,

thus disrupting their normal relationships

with neighboring cells.



Mammalian Cell Culture

Mammalian Cell Culture

Cell culture of

mammalian cells.

Eukaryotic cells are

much more difficult to

culture than most

prokaryotes.

They demand complex

media

They are very

susceptible to

contamination and

overgrowth by microbes

such as bacteria, yeastsand fungi.



Cell Culture

Two types of cell culture

Primary Culture

Cell Line Culture



Primary Culture

Come from the outgrowth of migrating

cells from a piece of tissue or from tissue

that is disaggregated by enzymatic,chemical, or mechanical methods.

Formed from cells that survive thedisaggregation process, attach to the cell

culture vessel (or survive in suspension),

and proliferate.



Primary Culture

Primary cells are morphologically similar

to the parent tissue.

These cultures are capable of only a

limited number of cell divisions, after

which they enter a nonproliferative statecalled senescence and eventually die

out.



Primary Culture

Primary cells are considered by many

researchers to be more physiologically

similar to in vivo cells.

Primary cell culture is generally more

difficult than culture of continuous celllines.



Primary Culture

Advantages

They are thought to

represent the bestexperimental models

for in vivo situations.

Have the same

karyotype as the

parent tissue normal

or abnormal.

DisadvantagesDifficult to obtain.

Relatively short lifespan in culture.

Very susceptible to

contamination



Finite Cell Lines

Finite cell cultures are

formed after the first

subculturing (passaging)

of a primary cell culture.

These cultures will

proliferate for a limited

number of cell divisions,after which they will

senesce.

The factors which

control the replication of

such cellsin vitro

arerelated to the degree of

differentiation of the cell



Finite Cell Lines

The cells will proliferate for an extended time,

but usually the culture will eventually cease

dividing, similar to senescent primary cells.

Use of such cells is sometimes easier than use

of primary cell cultures, especially for

generation of stably transfected clones.



Finite Cell Lines

MRC5 cells

Human embryoniclung fibroblasts

Undergo between60-70 doublings

before senescence.



Finite Cell Lines

Advantages

Can obtain a large

population of similar cells.

Most cellular

characteristics are

maintained

Disadvantages

Cells have a

tendency todifferentiate over

time in culture.

Over time the culture

tends to select for

aberrant cell



Continuous Cell Line

A cell line that hasdemonstrated thepotential to besubculturedindefinitely.

Infinite cell line

Immortal cells line

Immortalized celllines are also knownas transformedcells:

Cells whose growthproperties have beenaltered.




Finite cell cultures will eventually either die out

or acquire a stable, heritable mutation that gives

rise to a continuous cell line that is capable of unlimited proliferative potential.

This alteration is commonly known as in vitro

transformation or immortalization and frequentlycorrelates with tumorigenicity.




Continuous cell lines are generally

easier to work with than primary or finite

cell cultures.

These cells have undergone genetic

alterations and their behavior in vitromay not represent the in vivo situation.



HeLa Cells

Classic example of

an immortalized cell

line.

These are human

epithelial cells from a

fatal cervical carcinomatransformed by human

papillomavirus 18

(HPV18).




Advantages

Easy to maintain in

culture.

Easy to obtain large

population of cells.

Typically easy to

manipulate gene

expression.

Disadvantages

The more aggressive

the cell line the moreit changes over time

in culture.

Not clear how the

function of these

cells relates to that of

other cells, healthy

or diseased.



Transformed Cells

Transformed Cells

Changed from normal cells to cells with many of

the properties of cancer cells.

Some of these cell lines have actually been

derived from tumors or are transformed

spontaneously in culture by mutations.

No matter how transformation occurred, the result

is a cell with altered functional, morphological,

and growth characteristics.



Know Your Cells

The more you know about the cells and

the more finely attuned you are to the

cell¶s quirks, the quicker and more clear the interpretation of results will be.



Know Your Cells

The more differentiated the cell line, the slower

it will grow.



Know Your Cells

Adherently cultured transformed cells

are usually highly anchorage-

independent and adhere lightly even totissue culture dishes.

Wash these cells very carefully, as theloose monolayer can be inadvertently

aspirated away.



Characterization by Cell

Growth

Growth characteristics are functional

descriptions and are dependent upon

cell type.

Suspension and adherent growth are

properties of the cell as well as of theculture conditions




Growth

Attachment CulturesTo survive and grow,

most cells require asurface to which theycan attach

Without the surface

attachment these

cells cannot survive




Growth

Anchorage-Dependent and Anchorage-

IndependentGrowth

A subdivision of adherent growth




Growth

Anchorage-Dependent

Require attachment to the

surface for cell

proliferation

Anchorage-Independent

Do not require

attachment for cell

proliferation

Growth of cells in tissue

culture dishes looks more

haphazzard than thegrowth of anchorage

dependent cells with cells

only loosely attached to

the surface.




Growth

The advantages of adherent growth is

the ability of the cells to adhere and

spread on surfaces such as coverslips,making microscopy, hydribidizations,

and functional assays more easily

performed.




Growth

SuspensionCultures

Some cells cansurvive and dividewhile beingsuspended in a fluidmedia and stirred or

shaken.

Flasks

Spinner Cultures

Shaker Cultures

A limited number of cell types can bemaintained and

grown in either format.

Can be switchedbetween formats to

meet experimentalneeds.




Growth

The advantages of suspension growth

are the large numbers of cells that can

be achieved, and the ease of harvesting.

bcbt 478 introduction to mammalian cell culture 2009

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