Plant and Mammalian Tissue Culture

Culture Systems and Aseptic Technique

Culture Vessels

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.

Culture Vessels

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.

Culture Vessels

Treated PlasticPermanent modification to the polystyrene surface

Causes a net charge on the surface of the plastic

Modifier used include:• Proteins• Plasma• Amino Acids

Culture Vessels

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

Common examples are extracellular matrix proteins CollagenFibronectinLaminin

Adherent Cells

Flasks are commonly used to carry and expand cells.

Either vented or non-vented tops.

Adherent Cells

Dishes commonly used for specific experiments Scraping cells for SDS-

PAGE and Western Blotting

Fixing and staining cells for protein localization and interactions.

Adherent Cells

Multi-well plates6, 12, 24, 96, 384 wells

Allow for multiple replicates of experiments effectively

Different Growth Areas for each size

Adherent Cells

Adherent Cells

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

• Don’t need to agitate because they are unable to attach firmly to the surface

Suspension Cells

Spinner FlasksRequire special

variable speed magnetic stir plate.

Erlenmeyer FlasksRequire 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

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

Aseptic Technique

Aseptic Technique Refers to a procedure that is performed

under sterile conditions. This includes medical and laboratory

techniques, such as with microbiological cultures.

Aseptic Technique

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

Aseptic Technique

Work with cells in a biological safety cabinet laminar flow hoodprevent 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.

Aseptic Technique

Always use separate sterile pipettes for each manipulation

Never cough, sneeze, or yawn directly in your culture

Work rapidly but carefully

Incubator

Cell Culture Incubator 

Internal temperature is controlled. 

CO2 incubators contain a continuous flow of carbon dioxide containing air. 

Visualizing Cells

Inverted Microscope

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.

Contamination

Cultures can be infected through:

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

Contamination

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.

pH 7.2

pH 6.5

pH 8.0

Contamination

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)

Contamination

How to get rid of 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 functionsClearing 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