Microvascular and Macrovascular Endothelial Cell Isolation and Purification from Lung-Derived Samples

Summary

Although challenging, the isolation of pulmonary endothelial cells is essential for studies on lung inflammation. The present protocol describes a procedure for the high-yield, high-purity isolation of macrovascular and microvascular endothelial cells.

Abstract

The availability of cells isolated from healthy and diseased tissues and organs represents a key element for personalized medicine approaches. Although biobanks can provide a wide collection of primary and immortalized cells for biomedical research, these do not cover all experimental needs, particularly those related to specific diseases or genotypes. Vascular endothelial cells (ECs) are key components of the immune inflammatory reaction and, thus, play a central role in the pathogenesis of a variety of disorders. Notably, ECs from different sites display different biochemical and functional properties, making the availability of specific EC types (i.e., macrovascular, microvascular, arterial, and venous) essential for designing reliable experiments. Here, simple procedures to obtain high-yield, virtually pure human macrovascular and microvascular endothelial cells from the pulmonary artery and lung parenchyma are illustrated in detail. This methodology can be easily reproduced at a relatively low cost by any laboratory to achieve independence from commercial sources and obtain EC phenotypes/genotypes that are not yet available.

Introduction

The vascular endothelium lines the inner surface of the blood vessels. It plays key roles in regulating blood coagulation, vascular tone, and immune-inflammatory responses^1,2,3,4. Although the culture of endothelial cells (ECs) isolated from human specimens is essential for research purposes, it must be remarked that the ECs from different blood vessels (arteries, veins, capillaries) have specific functions. These cannot be fully recapitulated by human umbilical vein endothelial cells (HUVEC), which are easily available and widely used in studies on vascular endothelium pathophysiology^5,6. For instance, human lung microvascular endothelial cells (HLMVECs) play key roles in lung inflammation by controlling leukocyte recruitment and accumulation^4,7. Thus, an experimental setting aimed at reproducing lung inflammation with high fidelity should include HLMVECs. On the other hand, EC dysfunction can be observed in several pathologies; therefore, ECs from the patient are fundamental to building a reliable in vitro model of the disease. For instance, the isolation of fragments of ECs from the pulmonary artery (HPAECs), dissected from the explanted lungs of people affected by cystic fibrosis (CF), have enabled us to uncover mechanisms of endothelial dysfunction in this disease^8,9.

Thus, protocols aimed at optimizing the isolation of ECs from different sources/organs also in disease states are essential to provide investigators with valuable research tools, particularly when these tools are not commercially available. HLMVEC and HPAEC isolation protocols have been previously reported^{10,11,12,13,14,15,16,17,18,19}. In all cases, the enzymatic digestion of the lung specimens resulted in mixed cell populations, which were purified using ad hoc selective media and magnetic beads- or cytometric-based cell sorting. Further optimizations of these protocols must address two main issues in EC isolation: (1) cell and tissue contamination, which should be resolved at the earliest possible culture passages to minimize EC replicative senescence²⁰; and (2) the low yield of primary EC isolates.

This study describes a new protocol for the high-yield, high-purity isolation of HLMVECs and HPAECs. This procedure can be easily applicable and give virtually pure macrovascular and microvascular ECs in a few steps.

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Protocol

This study was approved, and the protocol followed the guidelines of the human research ethics committee of the University of Chieti-Pescara (#237_2018bis). Figure 1 illustrates the isolation of endothelial cells from segments (1-3 cm long) of pulmonary parenchyma or pulmonary artery from deidentified human subjects (with written consent) undergoing thoracic surgery for various reasons, such as pneumothorax or lobectomy. In this latter case, the surgeons also collected a pulmonary artery segment. Notably, the surgeons were accurately instructed to collect cancer-free samples. The presented protocol was optimized to obtain the greatest possible yield and purity.

1. Experimental preparation

1. Collagenase reconstitution
   1. Dissolve collagenase powder in phosphate-buffered saline without CaCl₂ and MgCl₂ (PBS⁻⁻, see Table of Materials) at a 2 mg/mL concentration, and filter the solution using a 0.22 µm pore filter.
   2. Prepare 5 mL aliquots, and store them at −20 °C. Thaw and preheat the aliquots to 37 °C prior to use.
2. Plate coating
   1. For plate coating, pipette 1.5% gelatin solution or 50 µg/mL fibronectin (see Table of Materials) into the culture plate (500 µL is enough to cover the surface of each well of a 6-well plate), and incubate for 1 h at 37 °C.
   2. After incubation, remove the excess gelatin, and wash the wells with PBS⁻⁻. Aspirate the PBS⁻⁻, and let the plate dry in a sterile hood.
      NOTE: For gelatin reconstitution, dissolve the powder in water to make a 1.5% solution, then autoclave it, and store at 4 °C. Gelatin at 1.5% is not liquid at 4 °C; therefore, it must be warmed up prior to plate coating. Alternatively, any commercial gelatin solution suitable for cell cultures can be used for the plate coating.

2. Sample preparation

1. Lung parenchyma
   1. Wash the collected samples by immersing them in a 50 mL tube containing PBS⁻⁻.
   2. Transfer the samples into a sterile Petri dish, and manually chop the sample using surgical scissors (optimal size: >2 cm) into small fragments of about 3-4 mm each.
2. Artery segment(s)
   1. Wash the collected samples by immersing them in a 50 mL tube containing PBS⁻⁻.
   2. Transfer it into a sterile Petri dish without chopping, as fragmentation will increase the surface area of the cross-section of the artery segment, thus increasing the possibility of isolating non-ECs.

3. Enzymatic digestion

1. Wash the diced lung parenchyma or the pulmonary artery segment/s twice with PBS⁻⁻. This step will remove a large part of the residual blood.
2. Place the samples in 15 mL tubes, and incubate with 5 mL of type 2 collagenase (see Table of Materials) for 10 min at 37 °C and 5% CO₂. During this incubation, the degradation of the extracellular matrix releases single cells and cell aggregates.

4. Recovery of the digested cells

1. Place a sterile steel/metal strainer (i.e., a tea strainer with a ~1 mm mesh size) on the top of a 50 mL collection tube.
2. Pour the entire contents from the 15 mL tube onto the strainer, gently massage the digested tissue with a spatula, and rinse the sample with PBS⁻⁻ until the collection tube is filled.
   ​NOTE: This step will eliminate large tissue fragments on the millimeter scale, ensuring greater efficiency of the subsequent filtration steps.

5. Non-EC removal by filtration

1. Filter the outflow collected in step 4.2 using a cell strainer with a 70 µm mesh size, and collect the outflow in a fresh 50 mL tube.
2. Then, use a cell strainer with a 40 µm mesh size, and collect the outflow in a fresh 50 mL tube. Label this tube as "tube 1".
   ​NOTE: These filtrations in step 5.1 and step 5.2 will remove large cell aggregates, which are often composed of mixed cell populations.

6. Sedimentation of cell clusters and cell seeding

1. Centrifuge the samples at 30 x g for 5 min at room temperature to sediment the cell clusters.
2. Carefully remove the supernatant using a sterile pipette (do not pour), and place it in a fresh 50 mL tube ("tube 2").
3. Suspend the pellet in "tube 1", and fill the tube up to 50 mL with PBS⁻⁻. Fill "tube 2" up to 50 mL with PBS⁻⁻.
   NOTE: From this step onward, both tubes are treated in the same way.
4. Centrifuge the samples at 300 x g for 5 min at room temperature.
5. Suspend the pellets with 2 mL of growth medium (see Table of Materials), and seed the suspension in two separate wells of a pre-coated 6-well plate (step 1.2).
   ​NOTE: From this step, feed the cells every 2 days with the growth medium until they reach confluency (approximately 1 week, depending on the sample size) in order to obtain a reasonable number of cells for sorting.

7. Cell expansion

1. Wash the cells with 2 mL of PBS with CaCl₂ and MgCl₂ (PBS⁺⁺, see Table of Materials) to remove the residual blood cells (using PBS⁺⁺ is important to avoid cell detachment).
2. Remove the PBS⁺⁺, and add fresh culture medium.
3. Repeat step 7.1 and step 7.2 until the cells reach confluency (approximately 1 week, depending on the sample size).

8. Cell sorting

1. Detach the cells using 500 µL of trypsin-EDTA (0.05%, see Table of Materials), centrifuge, and resuspend the pellet with 190 µL of PBS⁻⁻.
2. Add 10 µL of a fluorescent conjugate anti-human CD31-FITC antibody (1:20 dilution, see Table of Materials), and incubate the cell suspension at 4 °C for 30 min.
3. Wash the cells using 10 mL of PBS⁻⁻, centrifuge at 300 x g for 5 min at room temperature, and resuspend the pellet in 300 µL of PBS⁻⁻.
4. Isolate and collect CD31 positive (CD31+) cells by fluorescence-activated cell sorting (FACS), using a 100 μm nozzle, and collect them in a tube.
   1. As per the gating strategy, first define the cell morphology using the side scatter area parameters, SSC-A and FSC-A. Then, select single cells using an FSC-Area/FSC-Height or SSC-Area/SSC-Height dot plot, define the positive cells in the marked sample versus the unstained control sample, and direct the fluorescent cells into the collection tube²¹.
5. Centrifuge the cell suspension at 300 x g for 5 min at room temperature, and suspend the pellet in 2 mL of growth medium for seeding in the pre-coated wells of a 6-well plate.

9. Post-sorting expansion

1. Two days after cell sorting, replace the conditioned medium with fresh growth medium.
2. Repeat step 7.1 and step 7.2 every 2 days for cell expansion.

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Results

HLMEC isolation
The main problem during the isolation of HLMVECs is the presence of contaminating cells since the microscopic capillaries cannot be easily separated from the stromal tissue. Therefore, achieving the highest possible purity at the earliest stages of the isolation process is crucial in order to reduce the culture passages and, thus, the cell aging. Likewise, an optimal isolation protocol should give the highest possible yield of pure HLMVECs. To achieve these goals, a new procedure wa...

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Discussion

The multiple roles played by vascular endothelial cells in human pathophysiology make these cells an indispensable tool for in vitro pathogenetic and pharmacological studies. Since ECs from different vascular sites/organs display peculiar features and functions, the availability of healthy and diseased ECs from the organ of interest would be ideal for research purposes. For instance, HLMVECs are essential for studies on lung inflammation; therefore, a methodology for the high-yield, high-purity isolation of thes...

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Materials

Name	Company	Catalog Number	Comments
0.05% trypsin-EDTA 1X	GIBCO	25300-054	Used to detach cells from the culture plates
Anti CD31 Antibody, clone WM59	Dako	M0823	Used for CD-31 staining in immunocytochemistry. Dilution used: 1:50
Anti vWF Antibody	Thermo Fisher Scientific	MA5-14029	Used for von Willebrand factor staining in immunocytochemistry. Working dilution: 1:100
Autoclavable surgical scissors	Any		Used for chopping specimens
Cell strainers 40 µm	Corning	431750	Used during the second filtration
Cell strainers 70 µm	Corning	431751	Using during the first filtration
Collagenase, Type 2	Worthington	LS004177	Type 2 Collagenase used for enzymatic digestion. Working concentration: 2 mg/mL
Conjugated anti CD31 Antibody	BD Biosciences	555445	Used for cell sorting (1:20 dilution)
Dulbecco′s Phosphate Buffered Saline (PBS) with  CaCl2 and MgCl2	Sigma-Aldrich	D8662	Used for cell washing before medium change
Dulbecco′s Phosphate Buffered Saline (PBS) without CaCl2 and MgCl2	Sigma-Aldrich	D8537	Used for washing surgical specimens and cells before trypsinization
Endothelial Cell Growth Medium MV	PromoCell	C-22020	HLMVEC growth medium
Fibronectin	Sigma-Aldrich	F0895	Fibronectin from human plasma used for plate coating. Working concentration: 50 µg/mL
Gelatin from porcine skin, type A	Sigma-Aldrich	G2500	Used for plate coating
Type A gelatin	Sigma-Aldrich	g-2500	Gelatin from porcine skin used for plate coating. Working concentration: 1.5%