Isolation of Primary Epithelial Cells
Digestion: In Collagenase Medium
Purification: In PBS+5%ABS
**(Pre coat all tubes, tubes and pipettes with sterile 5% ABS/PBS before using them this will help you avoid loosing cells that attach to the sides of the tubes and pipettes.
Do part 16-19 in 15ml tubes
· Counting: Pre-dilute Crystal violet at 1/10. In a small eppendorf put ˝ crystal violet and ˝ of cell suspension vortex hard for 15 to 20 seconds to disrupt the cells and free the nuclei. Count the nuclei in a hemocytometer. The cell concentration is calculated by correction for the crystal violet dilution.
· Plate cells at density of 50 000/cm2 in growth medium (Medina: 2X106 cells/ 100mmdish, Virginia: 12 wholes plate with 5/10 organoids per whole in 1ml medium). Add 2% ABS and 5-10% collagen I (optional – it is hard to remove collagen when passaging cells) to allow cells to attach at the bottom of the plate. After 1-2 days, remove the medium with ABS and add serum-free medium. If you still a lot of fibroblasts, you can add dispase (boehringer) for 5 to 10 minutes and then put serum free media. Cells survive for 8-9 days but the medium needs to be changed every 2-3 days.
· Passaging: Try first dispase only (less damage to cell membrane than trypsin) at 2.4mg/ml for 20-30 minutes. If dispase does not work, try a collagenase solution of DMEM/F12 and 2mg/ml collagenase for 15-30minutes. If this doesn’t work, add trypsin (0.025% final concentration). Split ratios very low at the beginning (1:1,1:2 or 1:3) then, once cell line grows readily in culture change ratio to 1:5 or 1:10. (Virginia said put dispase + trypsin and no split in passaging first one)
· To differentiate epithelial cells: Keratins (8, 18, 19 for epithelial cells and 5,14 for myoepithelial), morphology and responsiveness to hormone (casein)
· To transplant cells, need 1X105 to 7.5X105 cells/site. Better success for transplant if passage 1 or 2 then later.
Tissue must be clean, lymph node removed.
Mince tissue in 1mm-sized pieces.
Place tissue in solution containing 10%DMSO and 90% FBS (Medina: 7% DMSO, 2% ABS in DMEM-F12). Put aliquots (2 aliquots/ fat pad) in eppendorf tubes. Use cryogenic tube.
Slowly freeze aliquots at a rate of 1°C per minute using Nalgene freezing container (cat# 5100-0001) field with isopropanol.
Freeze at –70°C for few months.
Frozen tissues are recovered by quickly melt frozen aliquots by submersing vial in a 37°C water bath. Add 9ml of growth medium DMEM+2%FBS to 1ml aliquots. Centrifuge 5min at 800rpm and then begin digestion protocol with collagenase.
Remove cells (when just confluent) using dispase.
Dilute with equal volume of DMEM-F12+ 2% serum. Centrifuge 1000rpm for 5min and remove supernatant, save pellet.
Resuspend pellet is medium with 10% DMSO, 90% FBS (Medina: 7% DMSO, 2% ABS in DMEM-F12). Use cryogenic tube.
Slowly freeze aliquots lowering temperature at the rate of 1°C per minute using Nalgene freezing container (cat# 5100-0001) field with isopropanol. Freeze at -80°C.
Frozen cells are recovered by quickly melt frozen aliquots by submersing vial in a 37°C water bath. Dilute with growth medium up to 6 times the volume of the frozen stock. Centrifuge (100rpm for 5min), remove supernatant, resuspend pellet and plate in growth medium (one aliquot/100mm dish).
Step1: mix together (can be prepare day before)
5ug/ml Insulin (Sigma chemical)
Antibiotics: 600U/ml nystatin (Sigma), 100U/ml penicilin/streptomycin and 5 ug/ml gentamycin (Gibco)
Step2: Add and shake in warm bath at 37°C for 10minutes. Filter on the hood.
2mg/ml Collagenase A (Boehringer)
2U/ml DNAse (Sigma)
PBS SOLUTION: (can be prepare day before)
5% Adult Bovine Serum (Atlanta biological)
GROWTH MEDIUM: (can be prepared weeks before). To make on the hood with everything sterile.
5ng/ml EGF (Becton Dickinson)
5mg/ml BSA (fraction V of bovine serum albumin) (sigma)
5ug/ml Linoleic acid complex (sigma)
5 to 10% of Collagen I (Ask Virginia for this product) can be added to the solution to have better protection. Leave for 1hour at 37°C unshaken. Adjust pH at 7.4
Linoleic Acid Complex:
Dissolve 5g of LA-Albumin (Sigma L8384) and 95g BSA in PBS to have final concentration of LA of 2.5mg/ml.
Aliquot in 2ml portions and store at -20°C. Protect from light.
Fatty acid free BSA:
Mix 50 mg of fatty acid free BSA in 250ml of standard medium
Stir slowly (avoid foaming) for 30min @RT
Aliquot in 40ml portions and store at -20°C.
By Dispase 1X ready to use from Boehringer (cat # 295-825).
From UCSF (#GP110-2FL02), trypsin 0.05% diluted in DMEM X2.
To coat dishes with thin collagen layer (only for cell line):
Dilute 1ml of collagen solution (2mg/ml) with 39 ml of 0.02N acetic acid (1ml of glacial acetic acid in 870ml distilled water).
Pipette diluted collagen solution (50ug/ml) onto dishes to be coated. Use at least 5ug/cm2; therefore a 60mm dish requires 3ml, 100mm require 8ml.
Incubate @RT for 1Hr.
Aspire off liquid.
Rinse dishes with PBS.