cell isolation: procedure and troubleshooting sepideh khoshnevis
TRANSCRIPT
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
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/mlEGF 10 ng/ml 10 ng/ml 10 ng/mlbovine pituitary extract 10 ug/ml 10 mg/mlphosphoethanolamine 0.1 mMhydrocortisone 1 ug/ml 1 ug/mlselenous acid 30 nMall-trans retinoic acid 0.01 ng/mlinsulin 4 ug/ml 5 mg/ml 10 ug/ml (+)alpha-tocopherol 2.3 uMgentamycin +/- 100 ug/mloleic acid 0.025% w/v/bovine serum albumin (BSAdexamethasone 1 mg/mlheparin 50 mg/mlFungizone 25 ng/ml 1.25 ug/mltransferrin 5ug/mlselenium 1ng/mlanti/anti 1%penicillin 100 U/ml 100 U/mlstreptomycin 100 U/ml 100 ug/mlNaHCO3 7.50%FBS (+)hourse serum 10%
Peehl
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