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