Isolation and Primary Culture of Mouse Aortic Endothelial Cells

Summary

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

Abstract

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

Introduction

The vascular endothelium is not only a barrier layer that separates blood and tissue, it is considered a vast endocrine gland that stretches over the entire vascular tree with a surface area of 400 square meters¹. The well-being of the endothelium is essential to vascular homeostasis. The dysfunctional endothelium participates in various cardiovascular disorders, including atherosclerosis, vasculitis and ischemia/reperfusion injuries, etc. ^2-4. To date, the specific cellular and molecular mechanisms involved in these disease settings are not well understood due to the diffused anatomic nature of endothelium.

The mouse is an important model for research because genetic manipulation techniques are more developed in mice than in any other mammalian species. However, the isolation of primary murine aortic endothelial cells is considered particularly difficult because the small size of the aorta makes enzymatic digestion of endothelium impractical. Some reported procedures to isolate and purify ECs require ^5-7.

The goal of this protocol is to use a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. In this protocol, the freshly isolated aorta is cut into small segments and seeded onto a matrix with the endothelium facing down to allow for endothelial sprouting. After segments are removed, endothelial cells are expanded in endothelium-favored medium and are ready for experiments after two or three passages. The advantages of the described method are that: 1) considerably high numbers of endothelial cells are harvested from a single aorta; 2) cell viability is well preserved; and 3) no special equipment or technique is needed. It provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell pathophysiology. For those who are interested in studying primary cultured endothelial cells from either gene knock-out mice, gene knock-in mice, or a murine disease model, this protocol is very useful and easy to practice.

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Protocol

1. Isolation of Aorta from Mice

All the procedures described here were approved by the Institutional Animal Care and Use Committee of Wayne State University.

1. Put the mouse into the induction chamber of the anesthesia machine. Set the isoflurane flow to 4% in conjunction with 25% fresh air and 75% O₂. Anesthetize the mouse by inhalation of isoflurane (4% for induction, ± 1.0% for maintenance) in conjunction with 25% fresh air and 75% O₂ via induction chamber followed by a dedicated nose cone. NOTE: Anesthesia with isoflurane can be delivered in 75% oxygen/25% air or in 100% oxygen. 
2. Check the mouse every 5 min until the appropriate level of anesthesia is reached (lack of withdrawal to toe pinch).
3. Take the mouse out of the induction chamber and continue isoflurane inhalation though the dedicated nose cone that is connected to the anesthesia machine. Switch the isoflurane flow to 1% to maintain anesthesia.
4. While it is under anesthesia, put the mouse onto a surgery pad, positioned on its back. Use laboratory tape to secure the limbs.
5. Use a heating lamp to keep the mouse warm. Keep the lamp in an appropriate distance from the mouse so that the mouse will not over-heat.
6. Spray the chest with 70% ethanol.
7. Use dissection scissors to open the abdomen from the midline, and expose the abdominal aorta. Open the chest cavity and expose the heart and lungs.
8. Cut the abdominal aorta at the middle with dissection scissors to release the blood.
9. Fill a 1 mL syringe (with 25 G needle) with 1 mL of PBS containing 1,000 U/mL of heparin. Inject PBS containing 1,000 U/mL of heparin to the left ventricle and perfuse the aorta. Push the heart and the lung with forceps at the great arteries to the right side of the mice to expose the thoracic aorta.
10. Quickly remove the thoracic aorta using micro-dissection forceps and put it in ice-cold 1x PBS (sterile), then transport the container into a laminar airflow hood.
11. Insert a 1 mL syringe fitted with 25 G needle into one end of the aorta, and gently flush the aorta with ice-cold PBS to remove the blood.
12. Use micro-dissection forceps to remove as much of the attached adipose tissue and small lateral vessels as possible.
13. Immediately transfer the aorta to endothelial growth medium.
14. Cut the aorta into 1 mm rings using a sterile scalpel blade. Harvest about 8 - 10 rings per aorta.
15. Open each aortic ring using a pair of micro-dissection scissors.

2. Seed the Aortic Segments on Matrix

1. When not in use, keep the growth factor-reduced matrix in -20 °C to prevent solidification. Put the growth factor-reduced matrix in 4 °C at least overnight to allow a complete thaw.
2. Precool a 6-well plate and pipet tips to -20 °C for at least 10 min. Put the 6-well plate on ice and coat one well of the plate with 1 mL of matrix without introducing any air bubbles. Place the plate in a 37 °C incubator for 20 min to allow the matrix to solidify.
3. Implant the aortic pieces onto the solidified matrix using sterile micro-dissection forceps. Place the pieces lumen-side-down on the matrix without touching the endothelium. Place 3 - 4 aortic segments close to each other on the matrix.
4. Add just enough endothelial cell growth medium to keep segments wet (~ 200 µL).
5. Incubate the plate at 37 °C under 5% CO₂ for 4 to 6 h. At the end of the day, add just enough medium to cover the aortic segments.
6. Observe the aortic segment sprouting periodically under a phase-contrast microscope. Check medium level and cell growth each day. Add medium if necessary to keep aortic segments covered.
7. On the 4^th day, gently remove the medium and remove the aortic segments from the matrix using a sterile needle without interrupting the growing endothelial cells.
8. Add 2 mL of new endothelial cell growth medium and allow the endothelial cells continue to proliferate on matrix for 2 - 3 days.

3. Initial Passaging of the Mouse Aortic Endothelial Cells

1. Coat a new T12.5 flask with gelatin (0.1%), incubate at 37 °C for 30 min.
2. Wash the matrix plates carefully with sterile 37 °C PBS.
3. Add 2 mL of neutral proteinase (50 U/mL) to the matrix plates and incubate at room temperature on platform rocker with occasional shaking. Check the cells under a phase-contrast microscope to make sure the majority of cells are detached.
4. Add 2 mL of D-Val to inactivate the neutral proteinase.
5. Collect the supernatant carefully in a 15 mL centrifuge tube. Wash the plate with 2 mL D-Val and collect the supernatant.
6. Centrifuge the cell suspension at 900 x g for 5 min at room temperature.
7. Re-suspend the cell pellet in 4 mL of endothelial cell growth medium and plate in the T12.5 flask coated with gelatin. Incubate the cells at 37 °C, 5% CO₂ for 2 h.
8. Replace the medium. Incubate the cells at 37 °C, 5% CO₂ until they are 85% - 90% confluent.

4. Passaging of the Mouse Aortic Endothelial Cells

1. Coat two new T12.5 flasks with gelatin (0.1%), incubate at 37 °C for 30 min. Pre-heat Trypsin-EDTA (0.25% Trypsin, 0.02 EDTA in PBS) and sterile PBS at 37 °C for about 15 min.
2. Wash the cells carefully with sterile 37 °C PBS.
3. Add 0.5 mL of Trypsin-EDTA (0.25% Trypsin, 0.02 EDTA in PBS) and incubate at 37 °C for ~ 1 min or until the majority of the cells turn round.
4. Add 2 mL of endothelial cell growth medium to stop the digestion. Pipette the cell suspension up and down within the flask several times. If necessary, use a cell scraper to collect the cells.
5. Collect the cell suspension in a 15 mL centrifuge tube. Centrifuge the cell suspension at 900 x g for 5 min at room temperature.
6. Re-suspend the cell pellet in 4 mL of endothelial cell growth medium and plate in 2 T12.5 flasks coated with gelatin. Incubate the cells at 37 °C, 5% CO₂ for 2 h.
7. Replace the medium. Incubate the cells at 37 °C, 5% CO₂ until they are 85% - 90% confluent.
8. Use the mouse aortic endothelial cells after 2 - 3 passages.

5. Characterization of the Mouse Aortic Endothelial Cells

1. Re-plate the cells.
   1. Coat a 6-well plate with gelatin (0.1%), incubate at 37 °C for 30 min. Rinse the plate with PBS.
   2. Pre-heat the Trypsin-EDTA and sterile PBS to 37 °C for about 15 min.
   3. Wash the cells with sterile PBS 3 times. Add 0.5 mL of Trypsin/EDTA to the cells, incubate for ~1 min at 37 °C or until the majority of the cells turn round.
   4. Add 2 mL of endothelial cell growth medium to stop the digestion. Pipette the cell suspension up and down within the flask several times. If necessary, use a cell scraper to collect the cells.
   5. Collect the cell suspension in a 15 mL centrifuge tube. Centrifuge the cell suspension at 900 x g for 5 min at room temperature.
   6. Discard the supernatant, re-suspend the cells in endothelial cell growth medium. Seed the cells in 6-well plate coated with gelatin at the density of 3 x 10⁵/well.
   7. Incubate the cells overnight at 37 °C, 5% CO₂ to allow the cells to attach to the culture surface.
2. Dil-ac-LDL uptaken and Ulex-lectin binding.
   1. Store the 1,1'-dioctadecyl-3,3,3',3'- tetramethylindo-carbocyanine perchlorate-labeled acetylated LDL (Dil-ac-LDL) at -20 °C. Before staining, thaw Dil-ac-LDL on ice. The stock Dil-ac-LDL solution is 1 mg/mL. Store the FITC-labeled Ulex europaeus agglutinin (Ulex-Lectin, 1 µg/mL) at 4 °C. The stock Ulex-Lectin solution is 1 mg/mL. Store Hoechst 33342 at 4 °C. The stock Hoechst 33342 solution is 100 µg/mL.
   2. Add 10 µL of Dil-ac-LDL stock solution to 1 mL of culture medium to make a final concentration of 10 µg/mL.
   3. Prior to staining, remove the old medium and wash the cells in each well with new medium.
   4. Remove the new medium and add the Dil-ac-LDL staining medium.
   5. Incubate the cells at 37 °C, 5% CO₂ for 1 h.
   6. Wash the cells with sterile 1x PBS 3 times.
   7. Add 1 mL of 10% formalin to fix the cells. Put the plate in room temperature for about 30 min.
   8. Wash the cells with sterile 1x PBS 3 times. Avoid light.
   9. Add 1 mL sterile 1x PBS that contains 10 µL of Ulex-Lectin.
   10. Incubate the cells at room temperature in the dark for 1 h.
   11. Remove the Ulex-Lectin by washing the cells with sterile 1x PBS 3 times.
   12. Add Hoechst 33342 to final concentration of 1 µg/mL, incubate 10 min in the dark.
   13. View the fluorescence of Dil-ac-LDL (red, excitation wavelength of 576 nm), Ulex-Lectin (green, excitation wavelength of 519 nm) and Hoechst (blue, excitation wavelength of 361 nm) with an inverted fluorescent microscope.
3. Fluorescent staining for CD31, VEGFR2, eNOS, VE-Cadherin and Calponin.
   1. Store FITC-conjugated anti-mouse CD31 antibody, eNOS antibody, VE-Cadherin antibody, FITC-conjugated anti-rabbit IgG at 4 °C in the dark. Store VEGFR2 antibody in -20 °C.
   2. Wash the cells with sterile 1x PBS 3 times.
   3. Add 1 mL of 10% formalin to fix the cells. Put the plate in room temperature for about 30 min.
   4. Wash the cells with sterile 1x PBS 3 times.
   5. To stain CD31, add 1 mL of sterile 1x PBS that contains 10 µL of CD31-FITC antibody. To stain either VEGFR2, eNOS, VE-Cadherin or calponin, add 1 mL of sterile 1x PBS that contains 4 μL of VEGFR2, eNOS, VE-Cadherin or calponin primary antibody, respectively.
   6. Incubate the cells on ice for 1 h in the dark.
   7. Remove the antibodies by washing the cells with sterile PBS 3 times.
   8. For the staining of VEGFR2, eNOS, VE-Cadherin or calponin, add 1 mL of sterile 1x PBS that contains 10 μL of FITC-conjugated anti-rabbit IgG. Incubate the cells on ice for 1 h in the dark. Remove the IgG by washing the cells with sterile PBS 3 times.
   9. Add Hoechst 33342 to final concentration of 1 µg/mL, incubate 10 min in the dark.
   10. View the fluorescence of CD31, VEGFR2, eNOS, VE-Cadherin, calponin (green, excitation wavelength of 518-535 nm) and Hoechst (blue, excitation wavelength of 361 nm) with an inverted fluorescent microscope.

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Results

Endothelial Cell Sprouting

Spontaneous endothelial cell sprouting started from a mouse aorta segment. The mouse aorta segment was allowed to grow on a growth factor-reduced matrix then in endothelial cell growth medium for 4 days. The endothelial cell sprouting usually appears in 2 - 4 days. Photomicrographs were taken on day 4 (Figure 1). As shown in the pictures, numerous endothelial cells migrate away from the segment. The newly formed sprouts continue to e...

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Discussion

This study demonstrates a simple method to isolate and culture endothelial cells from a mouse aorta without any special equipment. The immunofluorescence staining indicated that the majority of cells were endothelial cells after the second passage. It is suggested that cells at second to third passage are suitable for in vitro and in vivo experiments to study endothelial biology.

The Key Notes from the Present Protocol

There are fi...

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Materials

Name	Company	Catalog Number	Comments
4- or 6-week-old mice	Jackson Laboratory	#000664
Sterile 1x phosphate-buffered saline (PBS)	Gibco	#10010-023
Sterile 1x PBS containing 1,000 U/mL of heparin	Sigma Aldrich	H3149
Endothelial cell growth medium (Dulbeccos’ Modified Eagle’s Medium [DMEM] with 25 mM HEPES [Gibco, #12320-032], supplemented with 100 μg/mL endothelial cell growth supplement from bovine neural tissue [ECGS, Sigma, #2759], 10% fetal bovine serum [FBS, Gibco, #10082-147], 1,000 U/mL heparin [Sigma Aldrich, H3149], 10,000 U/mL penicillin and 10 mg/mL streptomycin [Gibco, #15140-122])
Growth factor reduced matrix	BD Biosciences	356231
Neutral proteinase (Dispase, 1 U/mL, Fisher Scientific, #CB-40235) and D-Val medium (D-Valine, 0.034 g/L, Sigma, #1255), in Dulbeccos’ Modified Eagle’s Medium, low glucose (Gibco, #12320-032)
1,1'-dioctadecyl-3,3,3',3'- tetramethylindo-carbocyanine perchlorate-labeled acetylated LDL (Dil-ac-LDL)	Life Technologies	L3484
FITC-labeled Ulex europaeus agglutinin (Ulex-Lectin)	Sigma	L9006
Anti-mouse CD31-FITC conjugated antibody	BD Biosciences	553372
Anti-mouse vascular endothelial growth factor receptor 2 antibody	Cell Signaling	9698
Anti-mouse endothelial nitric oxide synthase	Abcam	ab5589
Anti-mouse vascular endothelium-cadherin	Abcam	33168
Anti-mouse calponin	Abcam	700
FITC-conjugated anti-rabbit IgG	Sigma	F6005
1 mL syringe fitted with 25-G needle	Fisher Scientific	50-900-04222
100 mm Peri dishes	Fisher Scientific	07-202-516
Six-well cell culture plates	Fisher Scientific	08-772-1B
T12.5 culture flask	Fisher Scientific	50-202-076
Scissors, forceps, microdissection scissors and forceps, Scalpel blade	Fine Science Tools, Inc.
Anesthesia machine with isoflurane	Webster Veterianary Supply	07-806-3204
heating lamp
Centrifuge machine
Inverted phase-contrast microscope
inverted fluorescence microscope