Plant and Mammalian Tissue Culture

Mammalian Cell 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.

• Can be used to prepare finite or continuous cell cultures

• Will be similar biochemically and physiologically to parent tissue

Organ Culture

Organ CultureThe culture of whole organs or intact organ

fragments with the intent of studying their continued function or development.

• Can be maintained for hours or days by perfusion with oxygenated blood or serum.

• Used for metabolism and drug studies• Provides most accurate reflection of organism’s

physiology

Organ Culture

Organ Culture• U of Minnesota 2007 – heart perfusion w/stem cells

created a “new heart”. (Click here for movie)

Cell Culture

Cell CultureWhen cells are removed from the organ

fragments prior to, or during cultivation, thus disrupting their normal relationships with neighboring cells.

• General term explaining non-”in vivo” experiments

• Non-tissue growth of plant and animal 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, yeasts and fungi.

Cell Culture

Two types of cell culturePrimary CultureCell Line Culture

• AKA – finite / continuous / established / secondary / subclone / immortalized cell 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 the disaggregation 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 state called 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 cell lines.

Primary Culture

AdvantagesThey are thought to

represent the best experimental models for in vivo situations.

Have the same karyotype as the parent tissue normal or abnormal.

Not “dedifferentiated”

DisadvantagesDifficult to obtain.

Relatively short life span in culture.

Very susceptible to contamination

May not fully act like tissue due to complexity of media

Considerable variation in population and between preparations

Primary Culture Tumor Primary Cell Culture

Easier to create as tumors cell cycle / growth regulators have been altered

Tumor cells often produce own growth factors (autocrine)

Mechanically disrupted tissue easily plates, binds and can thrive

Seeding density often must be high

for primary culture of tumors.

Finite Cell Lines

AKA – secondary or subclone culture

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 cells in vitro are related 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 embryonic lung fibroblasts

Undergo between 60-70 doublings before senescence.

Finite Cell Lines

AdvantagesCan obtain a large

population of similar cells.

Most cellular characteristics are maintained

Can transform cells to grow indefinatly

DisadvantagesCells have a

tendency to differentiate over time in culture.

Over time the culture tends to select for aberrant cell

Continuous Cell Line

A cell line that has demonstrated the potential to be subcultured indefinitely.

Infinite cell line

Immortal cells line

Immortalized cell lines are also known as transformed cells:

Cells whose growth properties have been altered.

Continuous Cell Line

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 frequently correlates with tumorigenicity.

Continuous Cell Line

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 vitro may not represent the in vivo situation.

HeLa Cells

Classic example of an immortalized cell line.

These are human epithelial cells from a fatal cervical carcinoma transformed by human papillomavirus 18 (HPV18).

Continuous Cell Line

AdvantagesEasy to maintain in

culture.

Easy to obtain large population of cells.

Typically easy to manipulate gene expression.

DisadvantagesThe more aggressive

the cell line the more it 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, Infinite or Established CellsChanged 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.

Chemical or gamma ray treated cells can become infinite with loss of growth factors

Viral infection with SV40 T antigen can insert oncogenes and lead to p58 and RB gene alteration

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.

Categories of cell cultures based on origins. Origin Similarity to original

tissueEase of maintenance Doublings

Primary cells Animal tissue, fetal or adult

Representative Difficult 0 - 1

Finite cell lines Animal tissue, usually fetal

Representative Difficult Fetal: 20-80; adult tissue: very limited

Continuous cell lines Spontaneous tranformation of primary or finite cell lines

Not very representative; cell are less differentiated

Easy Indefinite, with selection for higher growth rate

Transformed cell lines Tumor tissue, spontaneous or viral transformation of continuous cell line

Not very representative; less differentiated than parent

Easy Indefinite, with selection for higher growth rate

Hybridoma (monoclonal antibody production)

Fusion of antibody secreting B cells and myeloma cells

Not representative of either cell type

Difficult Limited

Know Your Cells

Adherent or monolayer cells – must bind to solid surface to survive and propagate.

Suspension cells – may need stirring or simply placed in flask.

Know Your Cells

Wash these cells very carefully, as the loose monolayer can be inadvertently aspirated away.

Adherently cultured transformed cells are usually highly anchorage-independent and adhere lightly even to tissue culture dishes.

Characterization by Cell Growth

Attachment Cultures To survive and grow,

most cells require a surface to which they can attach

Without the surface attachment these cells cannot survive

Anchorage-Independent

Do not require attachment for cell proliferation

Growth of cells in tissue culture dishes looks more haphazard than the growth of anchorage dependent cells with cells only loosely attached to the surface.

Characterization by Cell 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.

Characterization by Cell Growth

Suspension CulturesSome cells can survive and divide while being suspended in a fluid media and stirred or shaken.

• Flasks• Spinner Cultures• Shaker Cultures

A limited number of cell types can be maintained and grown in either format.

Characterization by Cell Growth

The advantages of suspension growth are the large numbers of cells that can be achieved, and the ease of harvesting.

Growing Cells in Culture

Place cells in a culture dish. Give them nutrients, growth

factors, keep them free from bacterial.

Cells will grow to cover the surface of the dish.

Can take cells out of this culture and start a new culture.

Splitting cells from one dish to another is a passage.

Number of Cell Divisions

This ability to split cells and have them continue to divide is not without limits however.

Normal cells have a limit to the number of times which they can be passed in culture.

This number does vary from cell type to cell type, but commonly the limit is between 40 and 60 passages.

Hayflick’s Phenomenon

Cells will continue to grow and divide normally for a limited number of passages

When they get to a certain point even if they are given the appropriate nutrients, they simply stop dividing and will eventually die.

Cel

l Num

ber

Passage Number

Hayflick’s Phenomenon and Aging

There appears to be a correlation between the maximal number of passages and aging.

The number of passages decreases when cells are harvested from older individuals. Age (years)

Pa

sse

s

70

0-0.5 100

Progeria

A collection of defects which causes premature aging.

Genetic disorder which causes physical symptoms like gray hair, wrinkled skin, hair loss, muscle degeneration.

Child of age 4 or 5 appears like they are 80.

Cells from these individuals show dramatically decreased passage number.

Contact Inhibition

The phenomenon observed in normal animal cells that causes them to stop dividing when they come into contact with one another.

Cells in a culture flask with the appropriate nutrients and the cells grow and divide.

Continues until the cells are covering the entire surface.

At that point they stop dividing.

Contact Inhibition

These cells can be triggered to begin dividing again by giving them more room.

The cells now being in an environment where they are not in contact with one another begin to divide again.

Passage Number and Cancer

Cancer cells appear to be immortal. In the early 1950’s cervical cancer cells were

removed from a woman.HeLa cells.Helen Lang, Henrietta Lack.

These cells have been grown in culture and used extensively in science.

HeLa cells have been passed well over 1,000 times and show no sign of slowing their grow rate.

Contact Inhibition

Cancer cells do not display contact inhibition. Put them in a culture dish, they will grow to create a

single layer of cells Then they will continue to grow multiple layers and

create piles of cells.

GROWTH CYCLE IN ATTACHEMENT CULTURE

Eukaryotic cells in attachment culture have a characteristic growth cycle similar to bacteria.

The growth cycle is typically divided into three phases.Lag PhaseLog PhasePlateau Phase

GROWTH CYCLE IN ATTACHEMENT CULTURE

Days (in culture)

Cells/Well

Average #

of Cells

Standard

Deviation

Day 1

115000115000105000105000

110000 5774

Day 2

117500155000152500132500

139375 17722

Day 3

190000237500145000175000

186875 38588

Day 4

380000227500287500320000

303750 63656

Day 5

642500532500547500690000

603125 75674

Day 6

675000710000885000770000

760000 92105

Lag Phase

Feed

Subculture

PlateauPhase

Lag Phase

This is the time following subculture and reseeding during which there is little evidence of an increase in cell number.

It is a period of adaptation during which the cell replaces elements of the glycocalyx lost during trypsinization, attaches to the substrate, and spreads out.

During spreading the cytoskeleton reappears and its reappearance is probably an integral part of the spreading process.

Log Phase

This is the period of exponential increase in cell number following the lag period and terminating one or two doublings after confluence is reached.

The length of the log phase depends on the seeding density, the growth rate of the cells, and the density at which cell proliferation is inhibited by density.

In the log phase the growth fraction is high (usually 90%-100%) and the culture is in its most reproducible form.

Log Phase

It is the optimal time for sampling since the population is at its most uniform and viability is high.

The cells are, however, randomly distributed in the cell cycle and, for some purposes, may need to be synchronized.

Plateau Phase

Toward the end of the log phase, the culture becomes confluent All the available growth surface is occupied

and all the cells are in contact with surrounding cells.

Following confluence the growth rate of the culture is reduced, and in some cases, cell proliferation ceases almost completely after one or two further population doublings.

Plateau Phase

At this stage, the culture enters the plateau (or stationary) phase, and the growth fraction falls to between 0 and 10%.

There may be a relative increase in the synthesis of specialized versus structural proteins and the constitution and charge of the cell surface may be changed.

The dilemma – passage number or how long can I use these cells?

High number of subculturing will change cell biochemical and molecular properties, morphology, response to agonists, growth rates and other responses

May be a cell response to changed conditions from tissue

When doubling times significantly change, you may check other functions of the cells to see if these are now a “different strain” than you started with.