This protocol is relevant for the isolation of endothelial cells from tissue samples or very small pieces of blood vessels. The main advantage of this technique is that it can be reproduced in any research laboratory and it allows to obtain a good purity of endothelial cells, even from very small samples. Begin preparing the lung parenchyma sample by washing the collected samples in a 50 milliliter tube containing PBS without calcium chloride and magnesium chloride, or PBS minus minus.
Transfer the samples into a sterile Petri dish and chop manually into small fragments of about three to four millimeters using surgical scissors. For the artery segment, wash the collected samples in a 50-milliliter tube containing PBS minus minus, and transfer it into a sterile Petri dish without chopping. To remove the large part of residual blood, give two washes of PBS minus minus to the diced lung parenchyma or the pulmonary artery segment.
Next, place the samples in 15 milliliter tubes and incubate with five milliliters of type II collagenase for 10 minutes at 37 degrees Celsius and 5%carbon dioxide. To remove the large fragments, place a sterile one-millimeter mesh-size strainer on the top of the 50 milliliter collection tube and pour the entire contents from the 15 milliliter tube onto the strainer. Next, gently massage the digested tissue with a spatula before rinsing the sample with PBS minus minus until the collection tube is filled.
To remove nonvascular endothelial cells, filter the outflow collected from digested cells in a fresh 50-milliliter tube using a 70 micrometer mesh-size cell strainer. Then using a 40 micrometer mesh-size cell strainer, collect the outflow in a fresh 50 milliliter tube labeled as Tube 1. Centrifuge the samples at 30 G for five minutes at room temperature to sediment the cell clusters.
Use a sterile pipette to transfer the supernatant to a fresh 50-milliliter tube labeled as Tube 2. Suspend the pellet in Tube 1 and fill the tube up to 50 milliliters with PBS minus minus. Similarly, fill Tube 2 up to 50 milliliters with PBS minus minus.
Once done, centrifuge the samples at 300 G for five minutes at room temperature. Suspend the pellets with two milliliters of growth medium and seed the suspension in two separate wells of a pre-coded six-well plate. It was observed that part of the treated cell aggregates, including the smooth muscle cells or SMCs and fibroblasts, were bound to long fibers.
Also, SMCs represented the majority of non-blood cell singlets. Small compact clusters of cells with diameters not exceeding 10 to 20 micrometers were without elements attributable to fragments of stromal tissue. Approximately 70%pure human lung microvascular endothelial cells, or HLMVEC, single cells were obtained.
While obtaining pure HLMVEC preparations, the sorted cells assumed the characteristic endothelial-like shape. On focal microscopy showed that almost 100%of the fat's purified cells stained positive for endothelial cell antigens, namely CD31 and the more specific von Willebrand factor. When these cells were seated in matrigel, they generated tubular structures and HUVECs, confirming their endothelial phenotype.
Due to the short incubation time during the enzymatic digestion, it is important to preheat the collagenase. And another point is that during the sedimentation of cell clusters, it is important to gently remove the supernatant without without touching the cell aggregates in the pallet. This procedure is based on the use of centrifugation and filtrations, and therefore could provide new insight to know to separate other cell types on the basis of their characteristics, such as size and clustering.
