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Isolation of Primary Epithelial Cells:

Isolation of Primary Epithelial Cells

 

Digestion: In Collagenase Medium

 

  1. Sacrifice mouse with CO2 inhalation and cervical dislocation.
  2. Put whole mouse in 70%ETOH.
  3. Dissect out 3rd, 4th and 5th pair of MG, (avoid muscle and tendons) with sterile instruments.  Remove lymph nodes in 4th glands.
  4. Collect MG in warm DME/F12 media.
  5. After collecting glands from all mice, put MG (from about 5/6 animals) on flat sterile surface.
  6. Mince tissue with 2 X sterile scalpel for approximately 10-15 minutes.
  7. Collect homogenate to warm collagenase solution in 50ml sterile falcon tube.  Use 50ml for up to 6 or 7 mice.  Prepare two 50 ml collagenase solutions for 8+ mice.
  8. Incubate @ 37degree C with constant shaking for 3 hours at 100 rpm.

 

Purification: In PBS+5%ABS

 

  1. Centrifuge at 1000 rpm for 5min @ R.T.
  2. Re-suspend the pellet in 4ml DMEM-F12
  3. Centrifuge at 800 rpm for 5min @ R.T.
  4. Re-suspend the pellet in 4ml DMEM-F12 containing 2U/ml DNAse
  5. Shake gently for 2 minutes.
  6. Add 4ml DMEM-F12 with 4% FBS to stop DNAse activity.
  7. Centrifuge at 1000 rpm, 10min @ R.T.
Do part 9-15 in 50ml tubes

 

**(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.

 

  1. Remove supernatant and re-suspend in 7-10ml  PBS+ 5% ABS
  2. Pulse to 1500 rpm and stop quickly (15sec) with breaks “on” on centrifuge.
  3. Remove supernatant between each pulse and check under the scope for clarity of prep.
  4. Repeat step #17-18 for 7-8 times until you get clean prep: 90% at least purity of individual epithelial cells or organoids 100 cells, free of red blood cells and fibroblasts. As a control, check 10 ul of the supernatant after 5th or 6th time to make sure there are no organoids left in the supernatant (if yes then stop pulsing and plate).

Do part 16-19 in 15ml tubes

 

 

Culture: In Growth Medium

 

·        Counting: Pre-dilute Crystal violet at 1/10. In a small eppendorf  put ½ crystal violet and ½ 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 Banking :

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.

 

 

Cell Banking :

 

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).

 

________________________________________________________________________

 

SOLUTIONS AND MEDIA

 

DIGESTION MEDIUM:

 

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)

DMEM-F12 (sigma)

 

Step2: Add and shake in warm bath at 37°C for 10minutes. Filter on the hood.

2mg/ml Collagenase A (Boehringer)

 

DNAse SOLUTION:

4ml DMEM-F12

2U/ml DNAse (Sigma)

 

PBS SOLUTION: (can be prepare day before)

5% Adult Bovine Serum (Atlanta biological)

PBS

 

GROWTH MEDIUM: (can be prepared weeks before). To make on the hood with everything sterile.

5ng/ml EGF (Becton Dickinson)

10ug/ml Insulin

5mg/ml BSA (fraction V of bovine serum albumin) (sigma)

5ug/ml Linoleic acid complex (sigma)

50ug/ml Gentamycin

20U/ml Nystatin

DMEM-F12

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.

Filter

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

Filter

Aliquot in 40ml portions and store at -20°C.

 

 

Dispase solution:

By Dispase 1X ready to use from Boehringer (cat # 295-825).

 

Trypsin solution:

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.