Microvascular endothelial cells have been isolated previously from a variety of organs including human lung, however we present the first report of a method to successfully isolate such cells from emphysematous lung tissue. This method allows ex vivo study of a cell population which may be key in the pathogenesis of emphysema. Importantly, the method we have developed allows the isolation and culture of large numbers of human LMVECs (Table 1) with a high success rate (71%). Furthermore, the cells could be successfully expanded, cryopreserved and later re-animated for use in future studies. Cells showed stability of phenotype to passage 7.
Certain steps were critical to the success of this method. The tissue could be stored for up to 24 hours from the time of transplant until histopathological processing, however once processing began, cell isolation had to follow immediately otherwise it was unsuccessful. Careful dissection of the large vessels and removal of pleura to prevent overgrowth by contaminating mesothelial cells was also vital. Daily observation of cell numbers and doubling time was also required in order to determine the optimal time for bead separation as time between each passage differed between donors and did not appear related to passage number or disease severity.
Endothelial cell extraction employed bead separation with magnetic dynal beads for CD31 (endothelial cell surface marker) and UEA-1 (an endothelial based lectin). Other researchers have previously reported difficulties when using CD31 dynal beads, hypothesising that disruption of cell surface CD31 by beads inhibited the cell to cell interactions required for successful growth in culture . We did not encounter such problems, although doubling time immediately post bead separation was more prolonged.
By passage 4–6, cells appeared free from contaminating spindle shaped cells and were characterised according to a standard protocol developed using commercially available cells. The commercially available cells were used both to set a standard against which the cells isolated could be characterised and to ensure that precious isolated primary cells were not used for optimisation of experimental protocols. Comparing the cells isolated from patients with emphysema to those isolated from lung resection operations and commercially available cells provided a further control.
The immunocytochemical detection of cell surface markers via confocal microscopy confirmed the isolated cells were endothelial, staining positively for the endothelial marker CD31 with weak/absent staining for mesenchymal markers. This was further confirmed by flow cytometry, with cells staining positively for the endothelial cell marker CD31 and negatively for the fibroblast marker CD90. These approaches proved very cell efficient, requiring only small numbers of cells for full characterisation (~1×106), thus preserving large numbers of cells for use in future studies.
Plant derived lectins have previously been employed to differentiate between microvascular and macrovascular endothelial cells . We encountered difficulties with non-specific binding of the lectins Griffonia (Bandeiraea) simplicifolia and Helix pomatia previously used to differentiate between microvascular and macrovascular endothelial cells respectively both on single cells and on paraffin embedded tissue. As a result, we investigated E-selectin (CD62E) expression as an alternative method to differentiate between microvascular and macrovascular endothelial cells. E-Selectin (CD62E) and P-Selectin (CD62P) are receptor molecules for monocytes and neutrophils that are expressed on activated endothelial cells . CD62E, in contrast to CD62P which is stored in Weibel-Palade bodies in endothelial cells, is transcriptionally induced on microvascular endothelial cells in response to cytokine stimulation . Capillaries are not thought to express CD62E [23, 24]. Thus quiescent microvascular cells do not express CD62E but following activation intraacinar arterioles and venules express CD62E, while capillaries remain negative for CD62E. In our studies, the isolated CD31 positive cells showed very low (<5%) staining for CD62E at baseline. In response to stimulation with TNFα, there was inducible staining in around 30-50% of the isolated cells at 1 hour. Inducible CD62E in response to TNFα suggests these endothelial cells are microvascular. Furthermore, the presence of a subpopulation that was endothelial (i.e. positive for CD31) but did not up-regulate CD62E in response to TNFα suggests that the cells in this subpopulation are pulmonary capillary endothelial cells. Importantly this subpopulation was greater in the cells isolated from patients with emphysema compared with commercially available cells from Promocell used in the optimisation experiments. These pulmonary microvascular endothelial cells may therefore provide a more appropriate model than the current commercially available cells.
Infection is undoubtedly the major challenge to successful isolation and investigation of human LMVECs. Due to the inherent risks of infection whilst isolating the cells, the lobe of lung was placed in media containing 1% PSA prior to processing. 1% PSA was included in all MV2 media used in cell culture, the risk of infection being deemed greater than any adverse effect on growth kinetics the antimicrobials may have. In spite of this, a number of cell cultures were lost to infection, mostly around passage 6. With cell aging, growth kinetics reduced, with greater time to confluence. Cells therefore spent a longer time in culture with each successive passage, increasing the likelihood of infection. Amphotericin was included as we encountered more fungal infections than bacterial infections.
As with all ex vivo cell culture systems, inherent limitations are associated. Cells were passaged 3–5 times prior to obtaining pure cobblestone cultures which were characterized as endothelial. Cells therefore have a protracted culture period, with possible associated increased senescence and change in cell characteristic. Cells were grown in MV2 media (Promocell) which included 5% fetal calf serum supplementation and other survival factors such as hydrocortisone (0.2 ug/ml), recombinant human epidermal growth factor (5 ng/ml) fibroblast growth factor (10 ng/ml) vascular endothelial growth factor (0.5 ng/ml) and insulin like growth factor (Long R3) (20 ng/ml). The addition of hydrocortisone to cell culture media has been a contentious matter due to concerns over increased cell stress and how this may change cellular physiology. The concentration of hydrocortisone in MV2 media (Promocell) is considerably lower than in other types of microvascular endothelial cell media and its omission led to cell death.
Finally, our method of cell isolation can be applied to other respiratory diseases in which the pulmonary microvasculature may be pivotal such as pulmonary arterial hypertension and idiopathic pulmonary fibrosis. Indeed we have successfully isolated large numbers of cells from these pathologies (data not shown) with higher yields of cells at lower passage than the emphysema model.