In this article, primary lung endothelial cells were isolated and cultured from neonatal mice.
Endothelial cells play critical roles in the regulation of vascular tone, immunity, coagulation, and permeability. Endothelial dysfunction occurs in medical conditions including diabetes, atherosclerosis, sepsis, and acute lung injury. A reliable and reproducible method to isolate pure endothelial cells from mice is needed to investigate the role of endothelial cells in the pathogenesis of these and other conditions. In this protocol, lung microvascular endothelial cells were prepared from 5-7 day old neonatal mouse pups. Lungs are harvested, minced, and enzymatically digested with collagenase I, and released cells are cultured overnight. Endothelial cells are then selected using anti-PECAM1 (CD-31) IgG conjugated to magnetic beads, and cells again are cultured to confluence. A secondary cell selection then occurs with anti-ICAM2 (CD-102) IgG conjugated to magnetic beads to increase the purity of the endothelial cells, and the cells again are cultured to confluence. The entire process takes approximately 7-10 days before the cells can be used for experimentation. This simple protocol yields highly pure (purity >92%) endothelial cells that can be immediately used for in vitro studies, including the studies focused on endothelial cytokine and chemokine production, leukocyte-endothelial interactions, endothelial coagulation pathways, and endothelial permeability. With many knockouts and transgenic mouse lines available, this procedure also lends itself to understanding the function of specific genes expressed by endothelial cells in healthy and pathologic responses to injury, infection, and inflammation.
Interest in studying the vascular endothelium has grown recently, as dysfunction of microvascular endothelial cells occurs in multiple human diseases, such as stroke, cardiovascular disease, diabetes, acute lung injury, sepsis, and non-infectious injuries1,2,3,4,5. To define the pathogenesis of these conditions and understand the roles of specific genes in protective and dysregulated endothelial cell responses, there is a need for reliable methods to isolate and culture high purity microvascular endothelial cells from mice. While many studies utilize human endothelial cells such as human umbilical vein endothelial cells (HUVEC) or human lung microvascular endothelial cells (HMVEC), there are multiple reasons to use mouse endothelial cells to study endothelial function and dysfunction. First, there is considerable heterogeneity in responses of endothelial cells from different human donors, which leads to variability in results between individual experiments6,7,8. Second, silencing gene targets in primary human cell lines can also lead to variable protein expression levels9,10. In contrast, there is minimal heterogeneity in the responses of mouse endothelial cells11, assuming the genetic background is the same between study groups. Furthermore, large numbers of mouse endothelial cells can be prepared by pooling lungs from several neonatal mice. These factors allow for more consistent and reproducible results between experiments. Finally, the availability of knockout or transgenic mice also provides for the isolation of cell types lacking proteins of interest.
The considerable challenges with isolating healthy and pure populations of mouse lung endothelial cells using published methods12,13,14,15,16 led to developing a more reliable and straightforward method to isolate high-quality mouse lung microvascular endothelial cells. Advantages of the current protocol include using neonatal mice, which are readily available, limiting animal husbandry and housing costs. Additionally, in our experience, the viability of endothelial cells from neonatal mice is substantially higher than endothelial cells prepared from adult mice. Because neonatal mice have small lung tissue, endothelial cells can be harvested by digesting excised lungs into collagenase rather than using tracheal instillation of collagenase, which is recommended for collection from adult mice16. This also reduces the time between euthanizing mice and getting their endothelial cells into cell culture media and the incubator without affecting the purity or yield or the reproducibility of endothelial responses. Lastly, pure cell populations were isolated without flow cytometry, which can damage or lead to lower yields of the endothelial cells14,15,17.
The current modified protocol consistently leads to high purity and viable endothelial cells in 7-10 days that can be used immediately for in vitro experiments. Isolating cells directly from knockout animals also minimizes the manipulation of cells before experimentation. These methods could be used to investigate the role of various proteins in endothelial function to discover endothelial-based therapeutic targets for multiple diseases.
This simple protocol lends itself to high-purity endothelial cells from murine lungs. Although the total time from start to finish is 7-10 days, the hands-on time is around 3-4 h. Compared to other methods13,14,15,16, the current protocol is streamlined, keeping essential steps without compromising yield and purity.
It is worth noting some critical aspects of this protocol. First, it is important to use neonatal pups rather than adult mice. In our experience, endothelial cells from adult mice do not proliferate as easily as cells from neonatal pups, and they are easily overrun by cells with spindle-like morphology consistent with fibroblasts or mesenchymal stem cells15,16. One possible explanation for the difference is that lung development is in the alveolar stage in neonatal mice, characterized by the more rapid proliferation of endothelial cells20. There may also be some degree of senescence with aging21. Enzymatic digestion time also needs to be precise, as underdigestion can lead to a low yield single-cell suspension. In contrast, over digestion can lead to a substantial amount of cell death. We have determined that the optimal time for collagenase digestion is determined to be 45 minutes. This is then followed by mechanical dissociation with a blunt cannula. Instilling intratracheal collagenase during enzymatic digestion has also been reported14,15; however, this can be time-consuming and leads to incomplete digestion during the current work. Lastly, some protocols proceed directly to immunobead isolation immediately following enzymatic digestion13,16. We have found that culturing the cells from the dissociated lung in the medium for a day or two before making the immunobead selection substantially increases the yield of viable and highly pure endothelial cells. This may be related to the speed of getting isolated cells into the tissue culture medium, which leads to less cell death in the early stages after removal from the mice, higher initial seeding density, and time for the endothelial cells to adhere and proliferate before the first immunobead isolation.
Limitations of the preparation and analyses of mouse endothelial cells are as follows. Each mouse only provides a limited number of cells that must be used within a few passages. This issue can be overcome by pooling lung digest from several mice. Unfortunately, we are unsuccessful in our efforts to cryopreserve and thaw the cells for future use. Despite these limitations, mouse endothelial cells have some advantages. They can be helpful, particularly in situations where human endothelial cells either cannot be used (e.g., gene knockout), when complementary studies are required to corroborate results with human cells, or when the heterogeneity of responses of endothelial cells from different human donors makes reproducibility between experiments problematic6. The homogenous endothelial cell population from genetically identical mice allows for reproducibility between experiments and the study of specific genes and pathways under stable background conditions.
Mouse endothelial cells can be used for multiple applications to provide insights into endothelial activation and dysfunction in different disease processes. For example, endothelial cells play an integral role in sepsis, contributing to both beneficial and pathological responses through their roles in activating localized and systemic inflammation, promoting leukocyte trafficking and activation in tissues, regulating coagulation, and modulating endothelial permeability2,22,23. This simple protocol provides viable, functional endothelial cells that can be used in assays for each of these endothelial processes.
The authors have nothing to disclose.
This work was supported by NIH T32GM008440 (JH/EW) and NIH R01AI058106 (JH). We acknowledge the UCSF Parnassus Flow Cytometry Core (RRID:SCR_018206) supported in part by Grant NIH P30 DK063720 and by the NIH S10 Instrumentation Grant S10 1S10OD021822-01.
50 mL conicals | Corning Life Sciences | 430829 | ||
Accutase Cell Detachment Solution | Innovative Cell Technologies, Inc | AT-104 | ||
BD PrecisionGlide Needle 25 G x 5/8" | Becton Disckinson | 305122 | ||
BioRender.com | BioRender | |||
Blunt Cannula 15 G x 1-1/2" | Covidien | 8881202314 | ||
CD-102 Rat anti-mouse (3C4 (MIC2/4)) | BD Biosciences | 553326 | ||
CD-31 Rat anti-mouse (MEC 13.3) | BD Biosciences | 557355 | ||
Collagenase, Type 1 | Worthington Biochemical Corp | LS004194 | ||
DMEM, high glucose | Gibco | 11965092 | ||
Dynabeads Sheep Anti-Rat IgG | Invitrogen | 11035 | ||
Endothelial Cell Growth Supplment | EMD Millipore Corp | 02-102 | ||
Falcon cell strainers (70 µm) | Corning Life Sciences | 352350 | ||
Fetal Bovine Serum, heat inactivated | Gibco | 10082-147 | ||
Fine Scissors | Fine Science Tools | 14060-09 | ||
Fine Scissors | Fine Science Tools | 14060-10 | ||
Fisherbrand Disposable Borosilicate Glass Pasteur Pipets | Fisher Scientific | 13-678-20B | ||
FITC Rat Anti-mouse CD102 (3C4) | BD Biosciences | 557444 | ||
FITC Rat IgG2a, κ Isotype Control (R35-95) | BD Biosciences | 553929 | ||
Graefe Forceps | Fine Science Tools | 11050-10 | ||
HBSS, no calcium, no magnesium, no phenol red | Gibco | 14175095 | ||
Heparin Sodium Injections (1,000 Units/mL) | Medline | 0409-2720-02 | ||
Heparin sodium salt from porcine intestinal mucosa | Sigma | H3393 | ||
HEPES (1 M) | Gibco | 15630106 | ||
Nonessential amino acids (100x) | Gibco | 11140050 | ||
PE Rat Anti-mouse CD31 (MEC 13.3) | BD Biosciences | 553373 | ||
PE Rat IgG2a, κ Isotype Control R35-95) | BD Biosciences | 553930 | ||
Penicillin Streptomycin (10,000 U/mL, 10,000 µg/mL) | Gibco | 15140122 | ||
Recombinant Murine IFN-γ | Peprotech | 315-05 | ||
Recombinant Murine IL-1β | Peprotech | 211-11B | ||
Recombinant Murine TNF-α | Peprotech | 315-01A | ||
Round bottom polystyrene test tubes | Corning Life Sciences | 352058 | ||
Semken Forceps | Fine Science Tools | 11008-13 | ||
Sodium pyruvate (100 mM) | Gibco | 11360070 | ||
Stericup Quick Release-GP Sterile Vacuum Filtration System, 250 mL | Millipore | S2GPU02RE | ||
Steriflip-GP Sterile Centrifuge Tube Top Filter Unit | Milipore | SCGP00525 | ||
Sterile syringe (10 mL) | Fisher Scientific | 14-955-459 | ||
Sterile syringe (20 mL) | Fisher Scientific | 14-955-460 | ||
T-75 cell culture flask with vent cap, CellBIND treated | Corning Life Sciences | 3290 |