Murine Dermal Lymphatic Endothelial Cell Isolation
Summary
This paper describes a method for magnetic bead-based isolation of murine endothelial cells from dermal lymphatic capillaries. The isolated lymphatic endothelial cells can be used for downstream in vitro experiments and protein expression analysis.
Abstract
The lymphatic system participates in the regulation of immune surveillance, lipid absorption, and tissue fluid balance. The isolation of murine lymphatic endothelial cells is an important process for lymphatic research, as it allows the performance of in vitro and biochemical experiments on the isolated cells. Moreover, the development of Cre-lox technology has enabled the tissue-specific deficiency of genes that cannot be globally targeted, leading to the precise determination of their role in the studied tissues. The dissection of the role of certain genes in lymphatic physiology and pathophysiology requires the use of lymphatic-specific promoters, and thus, the experimental verification of the expression levels of the targeted genes.
Methods for efficient isolation of lymphatic endothelial cells from wild-type or transgenic mice enable the use of ex vivo and in vitro assays to study the mechanisms regulating the lymphatic functions and the identification of the expression levels of the studied proteins. We have developed, standardized and present a protocol for the efficient isolation of murine dermal lymphatic endothelial cells (DLECs) via magnetic bead purification based on LYVE-1 expression. The protocol outlined aims to equip researchers with a tool to further understand and elucidate important players of lymphatic endothelial cell functions, especially in facilities where fluorescence-activated cell sorting equipment is not available.
Introduction
The lymphatic system plays a pivotal role in human physiology. It is considered a vital homeostatic factor, that facilitates important functions, such as the maintenance of tissue-plasma fluid balance, immune surveillance, and lipid absorption1, as well as recently-identified functions, such as the repair ability of the heart2 and regenerative capacity of bone and hematopoiesis3. Despite the significant role of the lymphatic system, several aspects of the role of the lymphatics, as well as the molecular mechanisms governing certain physiological parameters and responses remain elusive. Moreover, lymphatic vascular defects are triggering or deteriorating factors in certain pathophysiological conditions. Well-known examples are the primary and secondary lymphedemas, depending on the genetic or non-genetic origin of the disease, respectively, while the role of the lymphatic system on primary tumor growth and metastatic dissemination is pivotal, as it can act as a conduit for metastasis and modulator of immune functions1.
The lag in the study and knowledge of the lymphatic compared to the blood vasculature is mainly due to the later discovery of the lymphatic system in comparison with the blood vascular system and the delay in the identification of lymphatic-specific molecular markers. This has greatly improved in recent decades, leading to the alteration of the conventional picture of the lymphatics at steady state and in diseased conditions1. Similar to angiogenesis, lymphangiogenesis is the formation of new lymphatic vessels from pre-existing ones and plays a pivotal role in the development of a wide spectrum of diseases4,5,6. However, unlike angiogenesis, the investigation of lymphangiogenesis has been limited to mostly in vivo models focusing on developmental vessel formation and structural defects in pathological models. The isolation of the lymphatic endothelial cells is important for in vitro studies, and this can be provided by the presented protocol.
Several protocols for lymphatic endothelial isolation from mice have been developed, which require the use of fluorescence-activated cell sorting7. The advantage of the cell sorter, where available, is the higher degree of purity, contrary to bead-based isolation, with the latter providing a higher yield. The protocol utilizes the expression of lymphatic endothelial hyaluronan receptor 1 (LYVE-1), a lymphatic endothelial marker, important for cell trafficking within the lymphatic vessels4,8. Since LYVE-1 expression is limited to the lymphatic capillaries and not the collecting lymphatic vessels, the technique is suitable for the isolation of lymphatic endothelial cells specifically from the lymphatic capillaries, contrary to other lymphatic markers, such as podoplanin, which are expressed uniformly in all lymphatic endothelial cells9. For the protocol, other important lymphatic markers that were not transmembrane, such as Prox1, were excluded.
As the physiological outcome was lymphangiogenesis, which is usually initiated at the lymphatic capillaries, LYVE-1 was selected as a target due to its selectivity in these cells and because the selected antibody provided a high yield. The enzymes used were collagenase type II, which is generally known to be more effective in collagen dissociation than type I10, and dispase, a broader protease, regularly used for dermis-epidermis separation11; however, other enzymes for tissue separation have been reported12,13 and can be used. The goal of this protocol is to describe a method for dermal lymphatic endothelial cell isolation from lymphatic capillaries of adult mice with high yield using magnetic bead purification, especially in places where fluorescence-activated cell sorting is not readily available. The method is suitable for applications where the purity of lymphatic endothelial cells can be compromised for downstream applications.
Protocol
Representative Results
Discussion
The lymphatic system is an important regulator of the homeostatic function of the body, with the most important functions being the maintenance of fluid plasma, removal of cellular metabolism byproducts and toxic molecules, lipid absorption, and immune cell trafficking1,19. The identification of appropriate markers has provoked a burst in new data in the lymphatics field, revealing novel functions of the lymphatic vasculature, such as their role in the repair and…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
This work was supported by the Hellenic Foundation for Research and Innovation (00376), the National Institutes of Health, the National Cancer Institute (NCI) [Grant R15CA231339], the Texas Tech University Health Sciences Center (TTUHSC) School of Pharmacy Office of the Sciences grant (to C.M.M.), and by the College of Pharmacy, University of Louisiana Monroe start-up funding, the National Institutes of Health (NIH) through the National Institute of General Medical Science [Grant P20 GM103424-21] and the Research Competitiveness Subprogram (RCS) of the Louisiana Board of Regents through the Board of Regents Support Fund (LEQSF(2021-24)-RD-A-23) (to G.M.). The common TTUHSC equipment used was obtained through the Cancer Prevention Research Institute of Texas (CPRIT) Grants RP190524 and RP200572. The funders had no role in the study design, the decision to write, or preparation of the manuscript. The graphical abstract in Figure 1A was created with BioRender.com.
Materials
| 0.2 μm Syringe Filters | Fisher Scientific | 09-719C | |
| 100 mm Tissue Culture Dishes | Fisher Scientific | FB012924 | |
| 60 mm Tissue Culture Dishes | Fisher Scientific | FB012921 | |
| Animal Hair Clipper | Wahl | ||
| Antibiotic-Antimycotic Solution 100x | Fisher Scientific | 15240-062 | |
| Blunt Forceps | Fine Science Tools | 11992-20 | |
| Bovine Serum Albumin | Sigma-Aldrich | A4919 | |
| Cell Strainer 40 μm | Fisher Scientific | 542040 | |
| Cell Strainer 70 μm | Fisher Scientific | 542070 | |
| CO2 Incubator | |||
| Collagen I, High Concentration Rat Tail | Corning | 354249 | dilute in 0.02 M Acetic Acid in H2O |
| Collagenase Type II | Life Technologies | 17101-015 | |
| Dispase | Life Technologies | 17105-041 | |
| DMEM | Fisher Scientific | 11-995-073 | |
| DNAse I Solution (2,500 U/mL) | Thermo Scientific | 90083 | |
| Dynal MPC-L Magnetic Particle Concentrator | Invitrogen | 120-21D | |
| EDTA | Sigma-Aldrich | 3690 | |
| Endothelial Cell Growth Base Medium & Supplement (LEC medium) | R&D Systems | CCM027 | |
| Euthanasia chamber | Euthanex Corporation | ||
| Fine Forceps | Fine Science Tools | 11255-20 | |
| Fine Scissors-Sharp | Fine Science Tools | 14060-10 | |
| FITC anti-mouse F4/80 Antibody | Biolegend | 123107 | |
| Goat Anti-Rabbit IgG Magnetic Beads | New England Biolabs | S1432S | |
| LARC-A E-Z Anesthesia Induction Chamber | Euthanex Corporation | ||
| MagnaBind Goat Anti-Rabbit IgG Beads | Thermo Scientific | 21356 | |
| Paraformaldehyde Solution 4% in PBS | Fisher Scientific | AAJ19943K2 | |
| Phosphate Buffered Saline (PBS) | Fisher Scientific | SH30256FS | |
| Rabbit Anti-Mouse LYVE-1 | ReliaTech GmbH | 103-PA50 | |
| Rotating/Shaking Incubator | |||
| Round-Bottom Polypropylene Tubes | Corning | 352063 | |
| Syringe Filters w 0.2 μm Pores | Fisher Scientific | 09-719C | |
| Trypsin-EDTA | Fisher Scientific | 25-300-120 |
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