Blood Research Institute, BloodCenter of Wisconsin
Sobczak, M., Dargatz, J., Chrzanowska-Wodnicka, M. Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice. J. Vis. Exp. (46), e2316, doi:10.3791/2316 (2010).
Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells (HUVECs) have shown to be convenient, easy to obtain and culture, and thus are the most widely studied endothelial cells. However, for research focused on processes like angiogenesis, permeability or many others, microvascular endothelial cells (ECs) are a much more physiologically relevant model to study 2. Furthermore, ECs isolated from knockout mice provide a useful tool for analysis of protein function ex vivo. Several approaches to isolate and culture microvascular ECs of different origin have been reported to date 3-7, but consistent isolation and culture of pure ECs is still a major technical problem in many laboratories. Here, we provide a step-by-step protocol on a reliable and relatively simple method of isolating and culturing mouse lung endothelial cells (MLECs). In this approach, lung tissue obtained from 6- to 8-day old pups is first cut into pieces, digested with collagenase/dispase (C/D) solution and dispersed mechanically into single-cell suspension. MLECS are purified from cell suspension using positive selection with anti-PECAM-1 antibody conjugated to Dynabeads using a Magnetic Particle Concentrator (MPC). Such purified cells are cultured on gelatin-coated tissue culture (TC) dishes until they become confluent. At that point, cells are further purified using Dynabeads coupled to anti-ICAM-2 antibody. MLECs obtained with this protocol exhibit a cobblestone phenotype, as visualized by phase-contrast light microscopy, and their endothelial phenotype has been confirmed using FACS analysis with anti-VE-cadherin 8 and anti-VEGFR2 9 antibodies and immunofluorescent staining of VE-cadherin. In our hands, this two-step isolation procedure consistently and reliably yields a pure population of MLECs, which can be further cultured. This method will enable researchers to take advantage of the growing number of knockout and transgenic mice to directly correlate in vivo studies with results of in vitro experiments performed on isolated MLECs and thus help to reveal molecular mechanisms of vascular phenotypes observed in vivo.
1. Preparing anti-PECAM-1 antibody-conjugated magnetic beads (Dynabeads)
2. Isolating mouse pulmonary endothelial cells from neonatal mice
Prior to proceeding with the tissue purification protocol, IACUC Committee approval of the procedure is required.
3. Preparing anti-ICAM-2 antibody-conjugated Dynabeads
4. Sorting Mouse Lung Endothelial Cells with anti-ICAM-2 Dynabeads
5. Representative Results
Typically, after 6-7 days from the initial preparation of the cells, we are able to obtain about 1.2 - 1.5 x 106 pulmonary endothelial cells from three 6-8 day old pups. Cells display typical "cobblestone" morphology under light microscopy and show VE-cadherin (CD144) staining at cell-cell junctions, which is characteristic for endothelial cells (Figure 1). Majority of MLECs express VE-cadherin and VEGFR2 as demonstrated by FACS analysis (Figure 2). Usually, we use them for experiments within two weeks after the initial MLEC isolation.
Figure 1. Microscopic analysis of confluent MLEC monolayer. (A) Light microscopy image shows cobblestone morphology of cultured cells typical for endothelial cells. Uniform round structures located in the perinuclear area of many cells are the magnetic beads used for MLEC isolation. (B) Endothelial-specific VE-cadherin is localized at the cell-cell junctions as shown using confocal microscopy. Bar is representative of 20μm.
Figure 2. FACS analysis of cultured MLECs labeled with antibodies specific for endothelial-specific markers: VE-cadherin (CD144) or VEGFR2, as indicted, followed by phycoerythrin-conjugated secondary antibody, confirms endothelial identity of isolated MLECs. Red line indicates isotype-specific control, green line indicates anti -VE-cadherin or anti-VEGFR2 - specific-IgG.
Microvascular ECs have proven to be a useful model in many areas of vascular biology and are believed to be more physiologically relevant to study (e.g. angiogenesis) than widely studied HUVECs 3. Previously, it has been reported that microvascular ECs can be obtained from kidney, heart, skin, retina, brain, gliomas, adipose tissue and also lung 2, 4-7, 10, 11. However, a consistent and reliable method for isolation of microvascular ECs is still required. The procedure presented here is a modification of a previously published protocol 2; it differs from most published procedures in that it uses pups instead of adult animals. This is crucial for the isolation success as cells from young animals have a higher proliferation potential and cultures derived from their tissues tend to yield higher number of cells. One week old animals seem to be optimal, but younger mice (4 day-old) can also be used. Also, higher or lower numbers of pups can be used if required, as we have been successful in isolating MLECs from one pup. However, while scaling up or down the isolation process, cell plating density must remain unchanged, as this is also critical for cell proliferation. The 2-step purification process of MLECs using magnetic beads conjugated first to anti-PECAM-1 and than anti-ICAM-2 antibodies is much more efficient at obtaining pure EC cultures than the previously described single-step purification procedures. This protocol eliminates the need to use very laborious manual techniques, gradient centrifugation and less efficient FACS sorting for discarding contaminating cells such as fibroblasts, blood cells and smooth muscle cells. The resulting MLEC population can be used for in vitro analysis of angiogenic responses, vascular permeability and leukocyte transmigration, wound healing as well as biochemical analysis of signaling pathways. If required, MLECs can be plated on different surfaces such as fibronectin or gelatin-coated coverslips for immunofluorescence or electrode arrays for trans-endothelial resistance (TER) measurements. Obtained results can be directly compared to the phenotypes observed in vivo, which is especially important in the context of the growing number of knockout and transgenic mouse lines. Successful implementation of this method for in vitro analysis of cellular mechanisms underlying defects observed in vivo, has already been presented 12.
Experiments on animals were performed in accordance with the guidelines and regulations set forth by Medical College of Wisconsin IACUC Committee under protocol AUA#00001206.
This research was supported in part by American Heart Association grant 0950118G.to M.C-W.
|Spring scissors||Fine Science Tools||15032-13|
|forceps||Fine Science Tools||11223-20|
|14G cannula||Fisher Scientific||1482516N|
|20 ml syringe||BD Biosciences||309661|
|anti-rat IgG Dynabeads||Invitrogen||110.35|
|Magnetic Particle Concentrator (MPC)||Invitrogen||123-21D|
|anti-mouse PECAM-1 antibody (CD-31)||BD Biosciences||553369|
|anti-mouse ICAM-2 antibody (CD-102)||BD Biosciences||553326|
|Collagenase/Dispase (C/D)||Roche Group||11097113001||100mg/ml stock solution can be prepared and stored at -20°C|
|Bovine Skin Gelatin||Sigma-Aldrich||#G9391-100G|
|Vasculife EnGS-Mv complete kit (VL)||Lifeline Cell Technology||LL0004|
|0.05% Trypsin/EDTA||Mediatech, Inc.||25-052-CI|
|Cell strainer 70 Ám nylon||Falcon BD||352350|
|tissue culture flask 75 cm2||Techno Plastic Products||90076|