murine lymphatic endothelial cell purification

Isolating Endothelial Cells from Murine Dermal Lymphatic Capillaries

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.

Author Spotlight: Magnetic Bead-Based Isolation of Murine Dermal Lymphatic Endothelial Cells

Murine Dermal Lymphatic Endothelial Cell Isolation

Our group's research focuses on delineating the mechanisms regulating endothelial cell physiology. This protocol describes a method for magnetic bead-based isolation of renal endothelial cells, specifically from thermal lymphatic capillaries in facilities where fluorescence-activated cell sorting is not available. The protocol allows the isolation of lymphatic endothelial cells from transgenic mice to study their physiology in vitro.It is particularly suitable where the purity of the lymphatic endothelial cells can be compromised for downstream applications. This process provides a high yield of lymphatic endothelial cells obtained from the lymphatic capillaries and not other collecting vessels. It is also a nice alternative in the case of the absence of cell sorting facilities.

Murine Lymphatic Endothelial Cell Purification

To purify urine lymphatic endothelial cells, use cells isolated from the murine dermis after they reach about 80 to 90%Co-fluency, begin by washing pre-coded magnetic beads in a magnetic separator using one milliliter of magnetic bead coating solution. Resuspend the beads in 150 microliters of DMEM containing 0.1%BSA. Next, wash the cells in the 60 millimeter tissue culture plate twice with PBS.Mix 50 microliters of the antibody conjugated beads with three milliliters of DMEM containing 0.1%BSA and add it to the cells. Seal the dish with biofilm, then incubate the dish on a shaker agitating at 10 RPM at room temperature for exactly 10 minutes. Discard the medium before washing the cells once with PBS.Quickly inspect the cells to verify the presence of LEC colonies on the plate. Add one milliliter of tripsin EDTA to the cells and incubate for three minutes at 37 degrees Celsius to trypsinize the cells. Inspect the cells for successful cell detachment.After neutralizing the solution with two milliliters of complete LEC media. Transfer the cell solution to a five milliliter sterile polypropylene tube. Using a magnetic separator, wash the cells with three milliliters of DMEM containing 0.1%BSA to remove unbound cells.Resuspend the remaining bead bound cells in complete LEC media and plate them onto collagen coated 60 millimeter tissue culture plates. Brightfield and fluorescence imaging easily identified the LYVE1 positive lymphatic endothelial cells after purification and showed several beads attached to them. The cells displayed low-confluency 24 hours after purification, while they were highly cofluent, seven days after purification.

Research

JoVE Journal

Biology

The lymphatic system participates in the regulation of immune surveillance, lipid absorption, and tissue fluid balance. The isolation of murine lymphatic ...