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Mammalian and Plant Cell Culture Culture Systems and Aseptic Techniques Lecture Handout Culture Vessels 

Mammalian and Plant Cell Culture

Culture Systems and Aseptic Techniques Lecture Handout

Culture Vessels

and Aseptic Techniques Lecture Handout Culture Vessels  Mammalian cells can be grown in a variety

Mammalian cells can be grown in a variety of containers.

The choice of container is typically dependent upon cell growth characteristics and the number of cells required.

Most tissue culture container are disposable, made of polystyrene, and have been radiation-sterilized.

Untreated plastic is usually fine for suspension cells

Most adherent cells grow better on treated plastic.

Treated Plastic

Permanent modification to the polystyrene surface

Causes a net charge on the surface of the plastic

Modifier used include:

• Proteins

• Plasma

• Amino Acids

Some cells types require a specific attachment substrate be added to the culture dish.

Common examples are extracellular matrix proteins

Collagen

Fibronectin

Laminin

Adherent Cells

Flasks are commonly used to carry and expand cells.

Either vented or non-vented tops.

Dishes commonly used for specific experiments

Scraping cells for SDS-PAGE and Western Blotting

Fixing and staining cells for protein localization and interactions.

Multi-well plates

6, 12, 24, 96, 384 wells

Allow for multiple replicates of experiments effectively

Different Growth Areas for each size

Chamber Slides

Used to prepare cells for microscope studies.

Suspension Cells

Suspension cultures are usually grown either:

In magnetically rotated spinner flasks or shaken Erlenmeyer flasks

• This actively keeps cells suspended in medium

In stationary culture vessels such at T-flasks and bottles

• Dont need to agitate because they are unable to attach firmly to the surface

Spinner Flasks

Require special variable speed magnetic stir plate.

Erlenmeyer Flasks

Require platform shaker

Types of Cells

Cultured cells are usually described based upon their morphology.

Epithelial-like cells

• Attached to substrate and flattened in shape

Lymphoblast-like cells

• Cells that do not attach to a substrate and have a spherical shape

Fibroblast-like cells

• Cells that are attached to a substrate and appear elongated and bipolar frequently forming swirls in heavy culture

Handling Cell Cultures

Adherence to good laboratory practice when working with cell cultures is essential for two reasons:

reduce the risk of exposure of the worker to any potentially infectious agent(s) in the cell culture

Mammalian and Plant Cell Culture Culture Systems and Aseptic Techniques Lecture Handout  to prevent

Mammalian and Plant Cell Culture

Culture Systems and Aseptic Techniques Lecture Handout

Culture Systems and Aseptic Techniques Lecture Handout  to prevent contamination of the cell culture with

to prevent contamination of the cell culture with microbial or other animal cells

 Aseptic Technique Mammalian and Plant Cell Culture Culture Systems and Aseptic Techniques Lecture Handout

Aseptic Technique

Mammalian and Plant Cell Culture

Culture Systems and Aseptic Techniques Lecture Handout

Aseptic Technique

and Aseptic Techniques Lecture Handout Aseptic Technique  Refers to a procedure that is performed under

Refers to a procedure that is performed under sterile conditions.

This includes medical and laboratory techniques, such as with microbiological cultures.

For Cell and Tissue culture this is the execution procedures without the introduction of contaminating

microorganisms

Work with cells in a biological safety cabinet

laminar flow hood

prevent airborne organisms from entering your cultures

always use ETOH to clean hood before and after use Laminar Flow Hood

A typical laminar flow hood

Filtered air enters the work space

from the from above

Do not block vents!

UV lights can be turned on after the work is finished to sterilize surfaces.

Always use separate sterile pipettes for each manipulation

Never cough, sneeze, or yawn directly in your culture

Work rapidly but carefully

Cell Culture Incubator

 Work rapidly but carefully  Cell Culture Incubator Incubator  Internal temperature is controlled. 

Incubator

Internal temperature is controlled.

CO 2 incubators contain a continuous flow of carbon dioxide containing air.

Inverted Microscope

Visualizing Cells

Large stage so plates and flasks can be used.

Magnification; 4X, 10X, 20X, 40X

Contamination

The presence of microorganisms can inhibit cell growth, kill cells, and lead to inconsistent results.

It is not a question of if, but when, your cells become contaminated.

Contamination is both observed under microscope and only by other tests.

Cultures can be infected through:

only by other tests.  Cultures can be infected through:  Poor handling  From contaminated

Poor handling

From contaminated media, reagents, and equipment (e.g., pipets)

From microorganisms present in incubators, refrigerators, and laminar flow hoods

From skin of the worker and in cultures coming from other

laboratories

Bacteria, yeasts, fungi, molds, mycoplasmas, and other cell cultures are common contaminants in animal cell culture.

Cloudiness (due to large cells in suspension) or filaments from mold are obvious signs

Microbial Contamination

The presence of an infectious agent sometimes can be detected by turbidity and a sharp change in the pH of the medium (usually indicated by a change in the color of the medium), and/or cell culture death.

Mammalian and Plant Cell Culture Culture Systems and Aseptic Techniques Lecture Handout Contamination  Mycoplasma

Mammalian and Plant Cell Culture

Culture Systems and Aseptic Techniques Lecture Handout

Contamination

Systems and Aseptic Techniques Lecture Handout Contamination  Mycoplasma – grow slowly and do not kill

Mycoplasma grow slowly and do not kill cells but will likely alter their behavior. Mostly tested by PCR for specific mycoplasma genes or using kits based on staining of growth in cytoplasm of cells

Some labs will test every 6 months for this kind of contamination

Contamination

Cross-culture contamination: multiple cells growing together based on doubling rate, one cell may take over the other as the dominant population

Up to 60% of cultured lines are contaminated (NIH 2009)

How to get rid of contamination?

Contamination

AVOID at all costs: sterile techniques, clean and properly maintained hood and incubator, clean room.

Laziness or familiarity are most common causes.

Antibiotics may help reduce contamination but may also alter cell functions

Clearing contamination only for novel cell lines, can be done with some agents.

• Wash cells to reduce contaminant burden

• Use sub-lethal doses of fungacide or antibiotic

be done with some agents. • Wash cells to reduce contaminant burden • Use sub-lethal doses
be done with some agents. • Wash cells to reduce contaminant burden • Use sub-lethal doses
be done with some agents. • Wash cells to reduce contaminant burden • Use sub-lethal doses