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BIOT 2922. Tissue Culture Methods. Objectives. Work aseptically. Maintain mammalian cells in healthy, uncontaminated state for 2-4 weeks. Manipulate mammalian cells. Cell lines. Chinese hamster ovary (CHO) cells Introduced in the 1960s. Used in a cultured monolayer in culture flasks.

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biot 2922

BIOT 2922

Tissue Culture Methods

objectives
Objectives
  • Work aseptically.
  • Maintain mammalian cells in healthy, uncontaminated state for 2-4 weeks.
  • Manipulate mammalian cells.
cell lines
Cell lines
  • Chinese hamster ovary (CHO) cells
    • Introduced in the 1960s.
    • Used in a cultured monolayer in culture flasks.
    • Used in studies of genetics, toxicity screening, nutrition and gene expression particularly expression of recombinant proteins.
  • Mouse myeloma cells
    • Cancer cell line.
    • Grow in clumps.
working with cell lines
Working with cell lines
  • Each student has own media and cells.
  • Make complete and incomplete media and test for sterility.
  • Obtain cells/monitor cell growth.
  • Use of inverted microscope.
  • Prepare laminar flow hood. Monitor CO2 incubator (Fyrite procedure for level).
  • Handle cells, split cells, count cells, freeze down cells.
cho cells1

CHO cells

10% confluent

~100% confluent

Confluency: coverage or proliferation that the cells are allowed over or throughout the culture medium

first day
First Day
  • 2 students make 1 L incomplete media, split into two 500 mL bottles.
  • Prep hood.
  • Each student makes 50 mL complete media (do this in hood).
  • Test both for sterility (Fri-Mon/Tues).
  • Get cells, make observation (notebook): color of media, confluency, shape of cells etc.
second day tuesday jan 22
Second Day (Tuesday, Jan 22)
  • Check cells.
  • Check sterility of media.
  • Decide on and take action.
when ready to passage split cho cells
When ready to passage/split CHO cells
  • Get hood ready
  • Warm media
  • Follow protocol on page 9
  • Make appropriate notes in notebook