Plant and Mammalian Tissue Culture

1
Plant and Mammalian Tissue Culture

• Culture Systems and Aseptic Technique

2
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.

3
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.

4
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

5
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

6
Adherent Cells

• Flasks are commonly used to carry and expand
  cells.
• Either vented or non-vented tops.

7
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.

8
Adherent Cells

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

9
Adherent Cells
10
Adherent Cells

• Chamber Slides
• Used to prepare cells for microscope studies.

11
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

12
Suspension Cells

• Spinner Flasks
• Require special variable speed magnetic stir
  plate.
• Erlenmeyer Flasks
• Require platform shaker

13
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

14
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

15
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.

16
Aseptic Technique

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

17
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

18
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.

19
Aseptic Technique

• Always use separate sterile pipettes for each
  manipulation
• Never cough, sneeze, or yawn directly in your
  culture
• Work rapidly but carefully

20
Incubator

• Cell Culture Incubator 
• Internal temperature is controlled. 
• CO2 incubators contain a continuous flow of
  carbon dioxide containing air. 

21
Visualizing Cells

• Inverted Microscope
• Large stage so plates and flasks can be used.
• Magnification 4X, 10X, 20X, 40X

22
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.

23
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

24
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

25
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 8.0
pH 7.2
pH 6.5
26
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

27
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)

28
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