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 Plastic Permanent 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 Collagen Fibronectin Laminin

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 plates 6, 12, 24, 96, 384 wells  Allow for multiple replicates of experiments effectively  Different Growth Areas for each size

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 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 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 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.

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. CO 2 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 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