BIOT 2922

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. – 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.

CHO cells

Myeloma cells

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.

Laminar Flow Hood

CHO cells

10% confluent ~100% confluent

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

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)

• Check cells.

• Check sterility of media.

• Decide on and take action.

When ready to passage/split CHO cells

• Get hood ready

• Warm media

• Follow protocol on page 9

• Make appropriate notes in notebook