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 Mincing step 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
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 3 Day 1 Day 2 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