Introduction to Cell Culture

1
Introduction to Cell Culture

• Biotechnology I

2
Cell Culture

• Definition the in vitro growth of cells isolated
  from multi-cellular organisms
• Process Cells will continue dividing until they
  fill up the container cell to cell contact stops
  cell division
• Uses vaccines, research of all kinds including
  stem cell, recombinant DNA, production of
  antibodies

3
Types of Cell used

• Mammalian cells are commonly used but they do
  grow slower and are more fragile than bacterial
  cells. Also have more complex nutrient
  requirements
• Bacterial cells were first used to make
  genetically engineered products and they
  reproduce very quickly

4
Growing Bacterial Cells

• Choose bacteria E. coli most common
• Make media
• Petri plates use agar in media (Luria Broth,
  nutrient agar)
• Liquid cultures use broth (LB, nutrient broth)
• Sterilize media in autoclave
• Pour media plates
• Innoculate media
• Grow cells in incubator (37oC)

5
Equipment Required for Mammalian Cells

• CO2 incubator for proper atmospheric conditions
• Laminar flow hood for sterility
• Hemocytometer for cell counting
• Inverted Microscope for viewing cells on
  hemocytometer

6
Cell Culture Process

• Contaminating the cell culture is area of concern
• To avoid contamination mfg. techs do the
  following
• Sterilize all containers used
• Change the media frequently
• Media is the source of nutrition, pH indicator
  often times included in media to help monitor pH
  which is critical to cell survival

7
Cell Culture Process

• Count cells
• Using a hemocytometer the cells are counted to
  determine if they are ready to harvest
• Can stain cells with trypan blue to determine if
  they are still alive. All cells which exclude
  the dye are viable. All stained cells are dead.

Live cell
Dead cell
8
Cell Culture Process

• View cells
• Inverted microscope is used to determine cell
  growth and activity
• Adherent cells are ones which are stuck to the
  flask or vessel
• Cell counting video for cell culture