Isolation and Culture of Primary Endothelial Cells from Canine Arteries and Veins

Podsumowanie

Novel isolation methods of primary endothelial cells from blood vessels are needed. This protocol describes a new technique that completely inverts blood vessels of interest, exposing only the endothelial side to enzymatic digestion. The resulting pure endothelial cell culture can be used to study cardiovascular diseases, disease modelling, and angiogenesis.

Streszczenie

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study vasculogenesis, (tumor) angiogenesis, and atherosclerosis. The current standard for in vitro research on human endothelial cells (ECs) is the use of Human Umbilical Vein Endothelial Cells (HUVECs) and Human Umbilical Artery Endothelial Cells (HUAECs). For canine endothelial research, only one cell line (CnAOEC) is available, which is derived from canine aortic endothelium. Although currently not completely understood, there is a difference between ECs originating from either arteries or veins. For a more direct approach to in vitro functionality studies on ECs, we describe a new method for isolating Canine Primary Endothelial Cells (CaPECs) from a variety of vessels. This technique reduces the chance of contamination with fast-growing cells such as fibroblasts and smooth muscle cells, a problem that is common in standard isolation methods such as flushing the vessel with enzymatic solutions or mincing the vessel prior to digestion of the tissue containing all cells. The technique we describe was optimized for the canine model, but can easily be utilized in other species such as human.

Wprowadzenie

Dogs are used as large animal model for cardiovascular disease research and can also suffer from inborn (genetic) vascular abnormalities^{1, 2}. To study these diseases commercial endothelial cell lines are often used to assess endothelial cell (EC) functionality. For dogs there is one commercial endothelial cell line available (CnAOEC), derived from canine aorta. This cell line is mostly used in studies as control normal ECs^3-5. In human cardiovascular research the most commonly used endothelial cell lines are Human Umbilical Vein Endothelial Cells (HUVECs) and Human Umbilical Artery Endothelial Cells (HUAECs) derived from human umbilical cord vein and artery, respectively. HUVECs have been used as the golden standard in vascular research since the 1980s⁶. They are considered to be the classic model system to study endothelial function and disease adaptation. Endothelial cells isolated from different blood vessels vary in appearance and functionality due to genetic background and exposure to the microenvironment⁷. In addition, HUVECs and HUAECs are derived from umbilical cord, a developmental vascular structure that might not fully mimic adult blood vessels with respect to the conditions they are exposed to and response to disease. Hence, translating results found in HUVECs and HUAECs to cardiovascular disease in general is inadequate.

When studying adaptation and behavior of adult ECs, primary ECs from the vessel of interest should be used as a more direct approach. To isolate these cells, several methods have been reported. A widely described method, which is also used for HUVECs, is flushing the vessel with an enzymatic digestion solution⁸. This often results in contamination with non-ECs such as smooth muscle cells and fibroblasts⁹. Another frequently used method for isolation is enzymatic digestion of minced vessel tissue followed by fluorescence-activated cell sorting (FACS) based on endothelial cell marker Cluster of Differentiation (CD)31^{7, 8}. FACS sorting and subsequent cell culture requires relatively large amounts of cells and is therefore not suitable for the isolation of endothelium from small blood vessels. We therefore aimed at developing a new robust method for isolating a pure endothelial cell population from various canine blood vessels with high purity. To test the efficiency of the new isolation method, we isolated and obtained pure Canine Primary Endothelial Cell (CaPEC) cultures from different canine arteries and veins, both large and small. This method also enables the culture of endothelial cells originating from diseased and/or aberrant vessels such as inborn intra- or extra-hepatic portosystemic shunts, a common disease in dogs². The method allows the isolation of additional relevant cell types of the same vessel such as vascular smooth muscle cells since most of the vessel remains intact during the procedure.

Protokół

Ethics statement: Blood vessels used in this study were harvested as surplus material obtained from fresh canine cadavers (n= 4) from healthy dogs euthanized for other unrelated research (University 3R policy). Aberrant blood vessels (intra- and extrahepatic portosystemic shunts, n= 1 each) were harvested post-mortem after informed consent of the owners from dogs presented to the University Clinic for Companion Animals of Utrecht University.

1. Isolation and Culture of Primary Canine Endothelial Cells

1. Pre-coat 6-well plates with 2 ml/well 0.5% w/v gelatin and leave for 2 hr in an incubator with a humidified atmosphere of 5% CO₂ at 37 °C. Remove excess gelatin solution prior to seeding the primary cells.
2. Aseptically remove the blood vessel(s) of interest (e.g., aorta, vena cava, vena porta) from a fresh canine cadaver (Figure 1A). Maintain the anatomy of the vessel system by placing straight forceps on both ends of the vessel of interest. Avoid unnecessary manipulation of the tissue with forceps to prevent damage to the endothelial cells.
   1. Cut the clamped vessel on both ends with surgical scissors and remove from the cadaver. Transport in Hank's Balanced Salt Solution (HBSS) on ice.
      NOTE: A vessel length of approximately 5 cm is preferred for this procedure, but those that measure 1 cm will also provide enough cells to culture.
3. Transfer the blood vessel to a Petri dish filled with ice-cold HBSS. Using surgical scissors, remove any adherent tissue and fat from the outside of the vessel, keeping the vessel itself intact (Figure 1B). Make sure the vessel is clearly visible at all times when cutting: pull aside the surrounding tissue with a clamp or forceps for optimal view.
   1. Close any branches of the vessel with ligatures (e.g., polyglactin 3-0) and subsequently remove them with surgical scissors or a scalpel.
4. Carefully enter a vessel end with a curved Halsted mosquito forceps, clamp the tip of the forceps at the other end of the vessel and then retract, slowly inverting the vessel until it is completely inside out (Figure 1C-G). The outside now consists of the endothelial cell layer. If any difficulty is encountered upon inverting the vessel, submerge in HBSS again to reduce friction.
5. Place purse-string sutures at both ends of the vessel to close it completely and to prevent exposure of any non-endothelial vascular tissue to the digestion procedure (Figure 1H). Use the ligature ends to manipulate the inverted vessel.
6. If digestion and culture is performed later, cryopreserve the inverted vessel prior to the digestion protocol (step 1.7).
   1. Place the inverted vessel in a cryovial and fill with cell culture freezing medium. Freeze down to -80 °C using a freezing container. Store at -80 °C if vessels are to be used within one week. For long term storage, place cryovials at -180 °C. When performing the EC isolation, thaw the cryovials rapidly in a water bath (37 °C) and immediately place in ice-cold HBSS. Proceed as indicated in 1.7.
      NOTE: Take into account that the total yield of ECs after isolation will be lower due to loss of viability during the freezing process.
7. Transfer the vessel to a 50 ml tube and rinse it twice in HBSS to remove erythrocytes (or residual freezing medium in case of thawing) (Figure 1I).
8. Digest the vessel in a 50 ml tube with a solution of 30 ml collagenase type II (0.15 U/ml) and dispase (0.15 U/ml) in Canine Endothelial Cells Growth Medium (CECGM) for 1 hr at 37 °C with intermittent gentle agitation.
9. Remove the vessel from the tube and centrifuge the cell suspension for 5 min at 250 x g.
10. Resuspend the cell pellet in CECGM and seed 2 ml cell suspension per well (1 - 3 wells depending on vessel size). Culture the cells at 37 °C in a humidified atmosphere with 5% CO₂ in air and change the culture medium twice a week.
11. Passage the cells when a confluency of 70 - 80% is reached.
    1. Wash the cells with pre-warmed HBSS to remove dead or non-attached cells. Add 200 µl recombinant cell-dissociation enzyme (1x) to each well and place back in the incubator for 5 min or until all cells are detached.
    2. Transfer the cells to a 15 ml tube and stop trypsinization by adding 10 ml of culture media (CEGM) including 10% Fetal Calf Serum (FCS). Centrifuge for 5 min at 250 x g. Continue the culture in a gelatin pre-coated plate or flask.

2. Characterization

1. Culture Canine Aortic Endothelial Cells (CnAOECs) and compare the morphology of the primary and commercial cell lines twice weekly with a microscope.
   NOTE: The typical growing pattern of ECs in patches can best be observed when a confluency of > 70% is reached.
   1. Culture CnAOECs in CECGM on a 0.5% w/v gelatin pre-coated T75 flask. Passage cells once a week when the confluency is 70 - 80%.
      1. For passaging, wash the cells once with pre-warmed HBSS and add 1 ml recombinant cell-dissociation enzyme (1x). Place the flask back in the incubator for 5 min or until all cells are detached. Inactivate trypsin by adding 10 ml culture medium (CEGM) including 10% FCS. Centrifuge the cell suspension for 5 min at 250 x g, discard the supernatant and resuspend the cells in 1 ml culture medium.
      2. Take a 10 µl aliquot of the cell suspension and dilute 1:1 with 0.4% trypan blue. Count the cells using an automated cell counter, and plate out 4.0 x 10⁵ viable cells in a new T75 flask. Add 10 ml of pre-warmed CECGM to the flask and culture at 37 °C in a humidified atmosphere with 5% CO₂.
2. Isolate RNA from passage 1 of the cultured CaPECs and CnAOECs.
   1. Collect at least 1 x 10³ cells as a pellet, add 20 µl of sample preparation reagent (SPR) and incubate for 1 min to lyse the cells. After incubation, carefully collect the cell lysate containing total RNA and store at -70 °C.
3. Convert mRNA to cDNA using a cDNA synthesis kit (e.g., iScript) following the instructions of the manufacturer. Store cDNA at 4 °C up to one week, or at -20 °C for long term until ready to perform the qPCR.
4. Measure gene expression of the endothelial marker CD31 to confirm endothelial cell origin. Perform experimental setup and quantification using the MIQE précis guidelines¹⁰. For normalization, measure expression of reference genes GAPDH, RPS19, and B2MG¹¹.
   1. Prepare a 10-fold dilution of the obtained cDNA in nuclease free water.
   2. Prepare a 4-fold dilution from pooled cDNA samples for the standard line and use nuclease free water as a non-template control. Dilute the samples five times to reach a 50-fold total dilution for qPCR reactions. Pipette 10 µl reactions in duplicate in a 384-well format using 4 µl cDNA and 6 µl of the fluorophore mixed with 20 pmol of reverse and forward primer (Table 1).
   3. Set the program to 95 °C for 5 min for Taq polymerase activation, followed by 39 cycles of 10 sec at 95 °C for denaturation, and 30 sec at Tm for annealing and elongation. The Tm for each primer set is shown in Table 1.
   4. Perform melting curve analyses following every run to ensure only one product is amplified. Normalize the expression levels of the samples using the average relative amount of the reference genes, and calculate the ΔCt if reaction efficiency is between 95% and 105%.
5. Assess EC functionality with an angiogenesis assay.
   1. Add 10 µl of extracellular matrix to each well of a pre-cooled angiogenesis slide and spread with a pipette tip to cover the surface of the well. Keep the extracellular matrix on ice and avoid the introduction of air bubbles into the gel. Solidify the gel by placing the slide in a Petri dish with water soaked paper towels in an incubator at 37 °C with 5% CO₂ for 30 min.
   2. Add 1.0 x 10⁴ primary ECs in 50 µl Endothelial Growth Medium per well. Incubate the slide for 6 hr at 37 °C with 5% CO₂.
   3. After 6 hr take photographs with a 20X magnification, making sure to include the whole well in the image.

Wyniki

Different blood vessels were successfully subjected to the described isolation protocol (Figure 2). It was possible to dissect and invert aorta, vena cava, vena porta, and coronary artery from healthy dogs (all vessels from each dog, n= 4). With the same approach ECs were isolated from two congenital portosystemic shunts (extrahepatic and intrahepatic, n= 1 each). Although aorta was easily inverted, thoracic aorta segments were more challenging than abdominal aorta. In th...

Dyskusje

In studies focusing on canine ECs the CnAOEC primary line is used to model the endothelial lineages of the dog^{3, 12, 13}. In human studies, the HUVEC culture is still considered the gold standard. Clearly, focusing merely on ECs derived from umbilical cord is a firm restriction in cardiovascular research. Endothelial cells have a specific gene expression pattern determining arteriovenous specification. In order to account for these differences in postnatal vessels we present this novel isolation method based on...

Materiały

Name	Company	Catalog Number	Comments
Collagenase type II	Life Technologies	17101-015
Dispase	Life Technologies	17105-041
DMEM (1X) + GlutaMAX	Life Technologies	31966-021
Hank's Balanced Salt Solution	Life Technologies	14025-050
Canine Endothelial Cells Growth Medium	Cell Applications	Cn211-500
CnAOECs	Cell Applications	Cn304-05
Fetal Calf Serum (FCS)	GE Healthcare	16000-044
TrypLE Express	Life Technologies	12604-013
SPR	Bio-Rad	170-8898
iScript synthesis kit	Bio-Rad	170-8891
SYBR green super mix	Bio-Rad	170-8886
Recovery Cell Freezing Medium	Gibco/Life Technologies	12648-010	Keep on ice prior to use
Freezing container, Nalgene Mr. Frosty	Sigma-Aldrich	C1562
Gelatin	Sigma-Aldrich	G1890
Surgical scissors (Mayo or Metzenbaum)	B. Braun Medical	BC555R
Mosquito forceps	B. Braun Medical	FB440R
Mosquito forceps curved	B. Braun Medical	FB441R
polyglactin 3-0	Ethicon	VCP311H
Trypan blue	Bio-Rad	145-0013
Automated counting chamber	Bio-Rad	145-0102
Counting Slides, Dual Chamber	Bio-Rad	145-0011
Matrigel	BD Biosciences	BD356231	Slowly thaw on ice
µ-Slide Angiogenesis	Ibidi	81501
Endothelial Growth Medium	Lonza	CC-3156
EGM-2 SingleQuot Kit	Lonza	CC-4176