Growing Cells in Culture

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Growing Cells in Culture

• Part 1 Terminology

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Cell Culture
The maintenance of cells outside of the living
animal (in vitro) for easier experimental
manipulation and regulation of controls.

• Pros
• Use of animals reduced
• Cells from one cell line are homogenous and have
  same growth requirements, optimizing growing
  patterns.
• In vitro models allow for control of the
  extracellular environment
• Able to monitor various elements and secretions
  without interference from other biological
  molecules that occurs in vivo

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• Cons
• Removal of cells from their in vivo environment
  means removing the cells, hormones, support
  structures and various other chemicals that the
  cells interact with in vivo.
• It is nearly impossible to recreate the in vivo
  environment. The artificial conditions could
  cause cells to de-differentiate which will cause
  them to behave differently and produce proteins
  other than it would in vivo.
• Genotype the genetic make-up of the cell
• Phenotype the appearance and behavior of a cell
  as a result of their genotype. Most often,
  scientists are looking at phenotypic changes in
  their analysis of cells in culture

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Classification of Cell Cultures

• Primary Culture
• Cells taken directly from a tissue to a dish
• Secondary Culture
• Cells taken from a primary culture and passed or
  divided in vitro.
• These cells have a limited number of divisions or
  passages. After the limit, they will undergo
  apoptosis.
• Apoptosis is programmed cell death

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Cell Lines

• Cell Line
• Cells that have undergone a mutation and wont
  undergo apoptosis after a limited number of
  passages. They will grow indefinitely.
• Transformed cell line
• A cell line that has been transformed by a tumor
  inducing virus or chemical. Can cause tumors if
  injected into animal.
• Hybrid cell line (hybridoma)
• Two cell types fused together with
  characteristics of each

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This weeks cells

• 3T3-L1 murine embryonic fibroblasts
• Fibroblasts are ubiquitous in the body, forms all
  elements of connective tissue
• Connective tissue includes bone, cartilage,
  tendons, dermis, adipose
• An immortal cell line, but not tumorogenic, will
  reach contact inhibited state
• Can produce triglycerides
• Can undergo and adipose-like conversion
• Especially with higher serum content
• Not known to harbor an agent known to cause
  disease in humans but has not been screened for
  Hep B or HIV.

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Growing Cells in Culture

• Part 2 Understanding Cell Behavior

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Confluency

• How covered the growing surface appears
• This is usually a guess
• Optimal confluency for moving cells to a new dish
  is 70-80
• too low, cells will be in lag phase and wont
  proliferate
• Too high and cells may undergo unfavorable
  changes and will be difficult to remove from
  plate.

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Contact Inhibition

• When cells contact each other, they cease their
  growth.
• Cells arrest in G0 phase of the cell cycle
• Transformed cells will continue to proliferate
  and pile upon each other

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Anchorage Dependence

• Cells that attach to surfaces in vivo require a
  surface to attach to in vitro.
• Other cells or specially treated plastic or other
  biologically active coatings
• Blood cells are primary exception.
• Transformed cells may not require attachment

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Passage number

• The number of times the cells have been removed
  (or split) from the plate and re-plated.
• Always write this on your plate or flask as P

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Growing Cells in Culture

• Part 3 Solutions used in cell culture

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Phosphate Buffered Saline - Ca2 Mg2 Free
(PBS-CMF)

• Used to wash/remove excess serum that inhibits
  the function of TRED.
• Calcium will also inhibit the function of TRED.
• Must be warmed in the water bath before use so
  cells are not shocked by cold liquid.

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Trypsin EDTA (TRED)

• An enzyme used to detach the cells from a culture
  dish.
• Trypsin cleaves peptide bonds (LYS or ARG) in
  fibronectin of the extracellular matrix.
• More about fibronectin and the ECM next week
• EDTA chelates calcium ions in the media that
  would normally inhibit trypsin.
• Trypsin will self digest and become ineffective
  if left in water bath more than 20 minutes.
• Trypsinizing cells too long will reduce cell
  viability

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Trypan Blue

• An exclusion dye
• Living cells cannot take up the dye and will
  appear bright and refractile.
• Dead cells with broken membranes will absorb the
  dye and appear blue.
• Usually add 200 ml of trypan blue to 200 ml of
  cell suspension in eppendorf tube

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Bleach

• Used to destroy any remaining cells in dishes and
  tubes before they are tossed in the trash can.
• Add enough to change media to clear,
• wait 5 minutes,
• rinse solution down sink
• throw away the dish/flask/plate in the trash can.