Cell isolation: Procedure and Troubleshooting

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Cell isolation Procedure and Troubleshooting

• Sepideh Khoshnevis

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Why do we need to isolate these cells?

• We need to have cells in culture in order to
  gather data on HSP expression and cell death
• Cells from normal canine tissue are not
  commercially available
• This process is called primary culture
• There are many challenges as they would be
  explained

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Procedure

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Culture of epithelial cells

• Acquisition of tissue
• Isolation of cells
• Primary culture
• Subculture
• Freezing and thawing cells

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Acquisition of tissue

• Quality of tissue
• Viability
• Tissue transportation to the laboratories
• Media
• HBSS
• Saline
• Cell appropriate media
• Temperature
• Oxygenation

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Isolation of cells

• Enzymatic method
• Washing steps
• Centrifugation
• RCF
• duration
• Mincing step
• Temperature
• Media
• size
• Collagenase step
• Objective is to dissolve/digest intercellular
  matrix without damaging the cells
• Composition of collagenase solution
• This is potentially the most stressful step on
  the cells
• Rocking step
• Oscillations per minute

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Primary culture issues

• Coated vs non-coated surface
• Collagen I
• Collagen IV
• Media that is friendly to the cells
• No guidelines are available to define the
  best/appropriate media composition
• Small changes in composition can have large
  consequences for cell survival

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Example media compositions
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Subculture to get enough cells to conduct
experiments

• Trypsinization step to remove cells from culture
  surface
• Trypsin concentration
• Duration
• Trypsin inactivation
• Incubation step
• Temperature
• CO2 concentration

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Troubleshooting

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Non-adherent cells

• Surface coating of the slide
• Choice of ECM elements
• Coating protocol
• Different concentration
• Drying procedure
• sterilization
• Use of elements that promote surface adhesion
• Testosterone

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Non-adherent cells

• The cells are apoptotic
• Trypsinization step
• 0.05 trypsin VS 0.25 trypsin
• Incubation condition
• CO2 concentration
• Acquisition of tissue
• Transport of whole tissue VS tissue pieces
• Composition of transport media

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Non-adherent cells

• Choice of media
• Toxicity
• Composition
• Isolated cell types
• Stromal VS epithelial
• Characterization of epithelial cells

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Characterization of epithelial cells

• Morphology
• Immunostaining
• Anti-cytokeratin antibody
• Anti-vimentin antibody
• Anti-SMA antibody

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Cos7 in epithelial environment

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Prostate stromal cells

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Conclusion

• Still can not isolate prostatic epithelial cells
  successfully and satisfactorily
• Possible causes
• Problem with collagenase step
• Lack of essential nutrients in collagenase
  solution
• Problem with incubation
• Too high CO2 concentration
• Amongst a very large number of coupled variables
  there are no other apparent variable contributing
  to the problem at hand

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Future steps

• Do another cell isolation with the following
  changes
• Change of the protocol
• Change of the collagenase solution
• adding serum and media to the solution
• Using lower concentration of collagenase
• Longer incubation in collagenase solution
• Use lower concentration of trypsin
• Improve incubation condition
• By correcting the CO2 concentration
• Cell characterization at the end of isolation
• By performing immunofluorescence studies on the
  cell smear using anti-cytokeratin, anti-SMA and
  anti-vimentin antibodies
• No final success yet but significant progress