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Cell isolation

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					Cell isolation: Procedure and
       Troubleshooting
      Sepideh Khoshnevis
  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
Procedure
         Culture of epithelial cells

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


• Quality of tissue
   – Viability
• Tissue transportation to the laboratories
   – Media
      • HBSS
      • Saline
      • Cell appropriate media
   – Temperature
   – Oxygenation
                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
       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
   Example media compositions
                           Peehl           Walden                                 Lang           Eaton               Terracio

                           MCDB 105        90% MCDB 153, 10% RPMI 1640            Ham's F12 DME w 4.5g/l glucose     Ham's F12

cholera toxin              10 ng/ml        10 ng/ml                               20 ng/ml
EGF                        10 ng/ml        10 ng/ml                               10 ng/ml
bovine pituitary extract   10 ug/ml        10 mg/ml
phosphoethanolamine        0.1 mM
hydrocortisone             1 ug/ml                                                1 ug/ml
selenous acid              30 nM
all-trans retinoic acid    0.01 ng/ml
insulin                    4 ug/ml         5 mg/ml                                10 ug/ml       (+)
alpha-tocopherol           2.3 uM
gentamycin                 +/- 100 ug/ml
oleic acid                                 0.025% w/v/bovine serum albumin (BSA
dexamethasone                              1 mg/ml
heparin                                    50 mg/ml
Fungizone                                  25 ng/ml                                                                  1.25 ug/ml
transferrin                                                                       5ug/ml
selenium                                                                          1ng/ml
anti/anti                                                                                   1%
penicillin                                                                                       100 U/ml            100 U/ml
streptomycin                                                                                     100 U/ml            100 ug/ml
NaHCO3                                                                                                       7.50%
FBS                                                                                              (+)
hourse serum                                                                                                                      10%
 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
Troubleshooting
          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
          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
          Non-adherent cells
• Choice of media
  – Toxicity
  – Composition
  – Isolated cell types
     • Stromal VS epithelial
        – Characterization of epithelial cells
 Characterization of epithelial cells
• Morphology
• Immunostaining
  – Anti-cytokeratin antibody
  – Anti-vimentin antibody
  – Anti-SMA antibody
   Cos7 in epithelial environment


Day 1      Day 2    Day 3




Day 4     Day 5     Day 6
        Prostate stromal cells



Day 1              Day 2




Day 3             Day 4
                     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
                      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

				
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posted:4/25/2011
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