Cell Biology > Protocols

Isolation of Mouse Lung Endothelial Cells (MLEC)/ Mouse Heart Endothelial Cells (MHEC)

MATERIALS:

• Fibronectin-coated T-75 flasks prepared by wetting for >1 hr. with 2 mL of human FN (Gibco #33016-015, 2.5ug/mL, sterile filtered, PBS, $.20/flask). One T-75 for two mice and for each passage.
• 0.1% Gelatin coated flasks can also be used for plating the cells. (Do not use
  gelatin if the cells are used for column chromatography experiments. We have found that the gelatin clogs the columns).
• Isolation Medium = Hi Glucose DMEM + 20%FCS + Pen/Strep.
• Growth Medium = same + 100ug/mL Heparin (Sigma H-3933) + 100 ug/mL
  ECGS (Biomedical Technologies, Stoughton, MA, Cat#BT-203) +1x non-essential amino acids + 2mM L-glutamine + 1x sodium pyruvate + 25mM HEPES.
• Instruments for animal dissection (sterilized in 70% EtOH): two scissors, two forceps.
• Instruments for tissue dissociation (autoclaved): small forceps, small scissors, and 6”-long 14 gauge metal cannula (Fisher #1482516N), and B-D Falcon cell strainer 70um pore size (Fisher #08-771-2).
• Type I Collagenase (Worthington) from Core, dissolved 2mg/mL (150-170 U/mg) in 25 mL of DPBS + Ca/Mg → warm at 37 C, 1 hr. → 0.22 um Sterile filter.
• Dynabeads M-450 Sheep anti-Rat IgG (Dynal #110.07) in PBS + 0.1% BSA + 0.02% NaN3. Concentration: 4 x 10⁸ beads/mL.
• Purified Rat anti-Mouse CD102 (ICAM-2) antibody (Pharmingen #553325), 1mg / 1mL.
• Purified Rat anti-Mouse CD31 (PECAM-1, clone MEC13.3) antibody (Pharmingen #553369), 0.5mg / 1 mL.
• DPBS- without Ca²⁺ and Mg²⁺

PROTOCOL:

1. Preparation of Anti-mouse ICAM-2 (PECAM-1) Dynabeads:
   
   
   1. Resuspend and aliquot of beads in 2 mL of PBS + 0.1% BSA. Mix well.
   2. Place tube on magnetic separator (e.g., Dynal MPC-S) and leave for 1-2 min.
   3. Remove supernatant and repeat wash 3x.
      
   4. Resuspend beads to original volume with PBS + 0.1% BSA.
      
   5. Add 5 uL of purified Ab for each 100 uL of beads.
      
   6. Incubate overnight on rotator at 4°C (or 2hr at RT).
      
   7. Repeat wash as in Steps 1 – 3 (4x).
      
   8. Resuspend beads in original volume with PBS + 0.1% BSA to maintain beads at 4x10⁸ beads/mL.
      
   9. Store beads at 4°C and use within 1-2 weeks.
      
   10. 0.02% sodium azide may be added as preservative, but remember to wash thoroughly before use.
       
   
   
2. Dissection:
   
   
   1. Sacrifice a young adult mouse by CO₂ inhalation.
      
   2. Soak skin with 70% EtOH.
      
   3. Nick skin above the pubis with scissors and deglove the skin to above the chest.
      
   4. Pin mouse supine on a Styrofoam board on a bench top. Remove any loose fur from gloves or field.
      
   5. Cut open anterior abdomen, and take down the anterior diaphragm with sterile scissors.
      
   6. Grasp lobes of the lung and heart and cut free from the mediastinum (with a new set of scissors and tweezers).
      
   7. Place lobes/heart in 15 mL cold Isolation Medium in a 10cm dish.
      
   8. Repeat Steps 1 – 7 for a second mouse. Combine lobes from two mice.
      
      
3. Tissue Dissociation:
   
   
   1. Transfer the dish with lung lobes to a sterile laminar flow hood for subsequent work.
      
   2. With new sterile scissors, cut off the heart (taking care to avoid as much of the aorta as possible). Cut the heart in half and place in 50 mL tube containing 40 mL of cold isolation media. Gently agitate.
      
   3. Then carefully cut out the lung lobes from any visible bronchi and mediastinal connective tissue.
      
   4. Put lung lobes in a dry 10cm dish and mince finely with scissors for 1 min.
      (Repeat for heart in a separate dish).
      
   5. Put lung pieces in 25 mL of pre-warmed, 37°C Collagenase (2 mg/mL). Wash all bits off the plate. (Repeat same for heart).
      
   6. Incubate at 37°C with gentle agitation for 45 min. (shaking waterbath or inversion in a hybrid oven).
      
   7. Using a 30 cc syringe attached firmly to the cannula, triturate the suspension 12 times, taking care to avoid frothing.
      
   8. Let chunks settle. Pipette suspension through a 70um disposable cell strainer (Falcon #35 2350) into a 50mL conical tube. Wash sieve with 10 mL of Medium.
      
   9. Spin down cell suspension at 400g (1300 rpm in GH3.7 rotor), 8 min., 4°C.
      
   10. Resuspend in 2 mL of cold PBS + 0.1% BSA, Leave behind any stubborn clumps.
       
   11. Count nucleated cells (e.g., count on a Coulter counter by adding 5 uL of suspension to 10mL of Isotonic buffer + 3 drops of Zap-globin).
       
       
4. Cell Sorting:
   
   
   1. Resuspend the heart cells in 1 ml DPBS- and lung cells in 2 ml of DPBS-. (Lung – 1ml/ mouse, Heart – 1ml/ 2 mice, 2ml/ 3 mice).
      
   2. Transfer to a 5mL polystyrene round bottom tube. Add Anti-mouse PECAM-1 Dynabeads (prepared in Section A) to cell suspension at 15 ul of beads / mL of cells suspension.
      
   3. Incubate on a rotator, RT, 10 min.
      
   4. Mount tube in a magnetic separator (e.g., Dynal MPC-S) and leave for 1-2 min.
      
   5. Remove supernatant. The beads + cells should look somewhat foamy if there is good yield of positive selection.
      
   6. Remove tube from magnetic separator and resuspend beads + cells in 3 mL Medium by vigorous trituration. Unless you are vigorous you will get many contaminating cells. (More relevent for the MLECs)
      
   7. Mount in magnetic separator for 1-2 min.
      
   8. Repeat steps 5-7 until the supernatant is clear (usually 4-5 washes).
      
   9. Resuspend in Growth Medium and plate beads + cells in one gelatin-coated T75 flask for lungs and one gelatin coated T75 flask for heart.
      
   10. The next day, rinse flask three times with isolation media to remove any loosely adherent cells.
       
       
5. Second Sort:
   
   
   1. Feed with Growth Medium on Monday, Wednesday and Friday.
      
   2. Let cells grow until approaching confluence at 5-9 days after plating. You should see nests of “cobblestone” EC comprising 10-90% cells. Other mesenchymal cells are notable for the way they pile up and don’t form monolayers.
      
   3. Detach cells with Trypsin: EDTA (0.05%:0.5M) by rinsing 3x with Ca/Mg-free buffer, and then leave new Trypsin:EDTA on only as long as needed to achieve a suspension of cells.
      
   4. Add 10mL of isolation media to inactivate Trypsin.
      
   5. Transfer cell suspension to a 15 mL tube and spin down at 1300 for 8 min.
      
   6. Remove supernatant and resuspend cell pellet in 2 mL of DPBS-.
      
   7. Add ICAM-2 coated SAR Dynabeads to the cell suspension (15 ul/ml). Incubate for 10 min. RT.
      
   8. Place in magnet and leave for 1-2 min. Remove supernatant.
      
   9. Wash gently 2x in 3 mL media.
      
   10. After final wash resuspend in 10 mL MEC media and plate in gelatin coated T75. (Split at a ratio of 1:2 if the cell recovery is good).
       
       
6. Cell Propagation:
   
   
   1. Feed with Growth Medium on Monday, Wednesday and Friday.
      
   2. Split cells 1/3 with Trypsin:EDTA when they reach 75-100% confluence.
      
   3. Replate endothelial cells on a gelatin-coated T75. It is important to maintain high density plating to avoid large numbers of senescent cells.
      
      

NOTES:

• This protocol was assembled from the collective experience of Bill Atkinson, Yaw-Chyn Lim, and Mandy Zervoglos of the Vascular Research Division, Department of Pathology, Brigham and Women's Hospital.
  
• Avoid the natural tendency to be too gentle with trituration in Step D.6. You will get overgrowth of non-endothelial mesenchymal cells!
  
• Anti-PECAM1 antibody-coated Dynabeads can also be used in the first cell sorting (step D.2), but their use in later sorts is hindered by mouse PECAM1 sensitivity to trypsin digestion.
  
• Gelatin coating of flasks works well also. Wet flasks for 1 minute with 0.1% Gelatin (in sterile water) remove, air dry for >4 hours.
  
• The washes of the initial plating of cells (Step D.10) can also be done as early as two hours after plating, but yield may suffer. Additionally, the first rinse of Step D.10 (containing non-adherent cells) can be plated on a second flask to
  increase yield, while still maintaining a fairly high % of EC.
  
• The second sort can also be performed by attaching beads to the monolayers before trypsinization. 50 uL of anti-ICAM2 beads are added to 5mL of PBS/BSA over a monolayer that has been rinsed thrice with PBS. The monolayer is kept at 4°C and rocked every 5 minutes for 30 minutes. Excess beads are then washed off with three rinses of PBS before standard trypsinization.
  
• The strength of the collagenase may vary from batch to batch. Therefore incubation times may need to be adjusted from lot to lot. This may be determined by the amount of clumping that occurs during Step C.10

June 20, 2000
Updated October, 2004

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