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.

Animal studies were performed according to the approved protocols by the Institutional Animal Care and Use Committee (IACUC) of Texas Tech University Health Sciences Center (TTUHSC) for the experiments at TTUHSC and by the Veterinary Administration of the Prefecture of Western Greece according to Directive 2010/63 for the experiments at the University of Patras. Diligently follow waste disposal regulations when disposing of animal waste materials.
1. Isolation of mouse dermis</p...

Isolating Endothelial Cells from Murine Dermal Lymphatic Capillaries

<ol>
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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+lymphatic+vasculature+in+the+21(st)+century:+novel+functional+roles+in+homeostasis+and+disease.">The lymphatic vasculature in the 21(st) century: novel functional roles in homeostasis and disease.</a> Cell. 182 (2), 270-296 (2020).</li><li>Liu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphoangiocrine+signals+promote+cardiac+growth+and+repair.">Lymphoangiocrine signals promote cardiac growth and repair.</a> Nature. 588 (7839), 705-711 (2020).</li><li>Biswas, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphatic+vessels+in+bone+support+regeneration+after+injury.">Lymphatic vessels in bone support regeneration after injury.</a> Cell. 186 (2), 382-397 (2023).</li><li>Tammela, T., Alitalo, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphangiogenesis:+molecular+mechanisms+and+future+promise.">Lymphangiogenesis: molecular mechanisms and future promise.</a> Cell. 140 (4), 460-476 (2010).</li><li>Ji, R. 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(99), e52691 (2015).</li><li>Jackson, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biology+of+the+lymphatic+marker+LYVE-1+and+applications+in+research+into+lymphatic+trafficking+and+lymphangiogenesis.">Biology of the lymphatic marker LYVE-1 and applications in research into lymphatic trafficking and lymphangiogenesis.</a> APMIS: Acta Pathologica, Microbiologica, et Immunologica Scandinavica. 112 (7-8), 526-538 (2004).</li><li>Makinen, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PDZ+interaction+site+in+ephrinB2+is+required+for+the+remodeling+of+lymphatic+vasculature.">PDZ interaction site in ephrinB2 is required for the remodeling of lymphatic vasculature.</a> Genes and Development. 19 (3), 397-410 (2005).</li><li>Wolters, G. H., Vos-Scheperkeuter, G. H., Lin, H. 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Z., Harvey, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prox1+expression+is+negatively+regulated+by+miR-181+in+endothelial+cells.">Prox1 expression is negatively regulated by miR-181 in endothelial cells.</a> Blood. 116 (13), 2395-2401 (2010).</li><li>Lapinski, P. E., King, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+mouse+lymphatic+endothelial+cells+from+lung+tissue.">Isolation and culture of mouse lymphatic endothelial cells from lung tissue.</a> Methods in Molecular Biology. 2319, 69-75 (2021).</li><li>Choi, E. 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The authors declare no competing financial interests.

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...

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.2 &#956;m Syringe Filters</td>
			<td>Fisher Scientific</td>
			<td>09-719C</td>
			<td></td>
		</tr>
		<tr>
			<td>100 mm Tissue Culture Dishes</td>
			<td>Fisher Scientific</td>
			<td>FB012924</td>
			<td></td>
		</tr>
		<tr>
			<td>60 mm Tissue Culture Dishes</td>
			<td>Fisher Scientific</td>
			<td>FB012921</td>
			<td></td>
		</tr>
		<tr>
			<td>Animal Hair Clipper</td>
			<td>Wahl</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic Solution 100x</td>
			<td>Fisher Scientific</td>
			<td>15240-062</td>
			<td></td>
		</tr>
		<tr>
			<td>Blunt Forceps</td>
			<td>Fine Science Tools</td>
			<td>11992-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A4919</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Strainer 40 &#956;m</td>
			<td>Fisher Scientific</td>
			<td>542040</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Strainer 70 &#956;m</td>
			<td>Fisher Scientific</td>
			<td>542070</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 Incubator</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen I, High Concentration Rat Tail</td>
			<td>Corning</td>
			<td>354249</td>
			<td>dilute in 0.02 M Acetic Acid in H2O</td>
		</tr>
		<tr>
			<td>Collagenase Type II</td>
			<td>Life Technologies</td>
			<td>17101-015</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase</td>
			<td>Life Technologies</td>
			<td>17105-041</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Fisher Scientific</td>
			<td>11-995-073</td>
			<td></td>
		</tr>
		<tr>
			<td>DNAse I Solution (2,500 U/mL)</td>
			<td>Thermo Scientific</td>
			<td>90083</td>
			<td></td>
		</tr>
		<tr>
			<td>Dynal MPC-L Magnetic Particle Concentrator</td>
			<td>Invitrogen</td>
			<td>120-21D</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>3690</td>
			<td></td>
		</tr>
		<tr>
			<td>Endothelial Cell Growth Base Medium &#38; Supplement (LEC medium)</td>
			<td>R&#38;D Systems</td>
			<td>CCM027</td>
			<td></td>
		</tr>
		<tr>
			<td>Euthanasia chamber</td>
			<td>Euthanex Corporation</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Forceps</td>
			<td>Fine Science Tools</td>
			<td>11255-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Scissors-Sharp</td>
			<td>Fine Science Tools</td>
			<td>14060-10</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC anti-mouse F4/80 Antibody</td>
			<td>Biolegend</td>
			<td>123107</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat Anti-Rabbit IgG Magnetic Beads</td>
			<td>New England Biolabs</td>
			<td>S1432S</td>
			<td></td>
		</tr>
		<tr>
			<td>LARC-A E-Z Anesthesia Induction Chamber</td>
			<td>Euthanex Corporation</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MagnaBind Goat Anti-Rabbit IgG Beads</td>
			<td>Thermo Scientific</td>
			<td>21356</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde Solution 4% in PBS</td>
			<td>Fisher Scientific</td>
			<td>AAJ19943K2</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>Fisher Scientific</td>
			<td>SH30256FS</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit Anti-Mouse LYVE-1</td>
			<td>ReliaTech GmbH</td>
			<td>103-PA50</td>
			<td></td>
		</tr>
		<tr>
			<td>Rotating/Shaking Incubator</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Round-Bottom Polypropylene Tubes</td>
			<td>Corning</td>
			<td>352063</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe Filters w 0.2 &#956;m Pores</td>
			<td>Fisher Scientific</td>
			<td>09-719C</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Fisher Scientific</td>
			<td>25-300-120</td>
			<td></td>
		</tr>
	</tbody>
</table>

murine dermal lymphatic endothelial cell isolation

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.

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.

The method was developed to identify the protein expression of a small GTPase, RhoA, the coding gene of which was floxed in both alleles and deleted under the effect of the tamoxifen-inducible lymphatic endothelial-specific promoter Prox1-CreERT2 18. The isolated LECs can be cultured for up to four generations, after which they become senescent. They have been frozen, thawed, and successfully stimulated with angiopoietin-2. Due to the limited number of cell passages, we have no...

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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.

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

murine-dermal-lymphatic-endothelial-cell-isolation

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Biology

Isolation of Cells from Murine Dermis

Perform the protocol inside a biosafety cabinet on the euthanized mouse appropriately shaved and disinfected with 70%ethanol. Create a 0.5-inch superficial incision in the animal's abdomen using scissors. Cut longitudinally along the midline towards the neck and lower abdomen.Additionally, make lateral cuts to flap open the skin. Using blunt forceps, gently separate the skin from surrounding tissues, while going around the animal's torso cutting the skin laterally around the neck and the lower abdomen. Carefully place the skin with the dermis side facing down in a 100-millimeter tissue culture plate.And ensure the skin is as stretched as possible. Add six milliliters of Dispase solution to the plate, ensuring the skin is completely covered. Then, cover the plate.Wrap the plate with Parafilm. Incubate the plate at room temperature for 10 minutes to flatten the explant. The next day, using a pipette, remove the liquid from the plate containing the isolated skin.Then, with the aid of forceps, separate the dermis from the epidermis while removing any visible adipose tissue from it. And place the isolated dermis into a clean tissue culture plate. Add one milliliter of DMEM containing 1X antibiotic-antimycotic solution to the dermis in the culture plate.Tilt the plate to accumulate the liquid on one side of the plate. Then, mince the dermis into pieces of one to two square millimeters. And transfer them to a new 50-milliliter conical tube.Add 10 milliliters of collagenase type II solution to the tube. Incubate it for two hours at 37 degrees Celsius with continuous agitation at 30 to 50 rpm to digest the dermis. Filter the cell suspension through a 70-micron cell strainer.And centrifuge it at 250g for five minutes. After discarding the supernatant, resuspend the pellet in 10 millimeters of DMEM containing antibiotic-antimycotic solution. Filter the cell suspension through a 40-micron cell strainer before centrifuging, as demonstrated previously.Resuspend the resulting cell pellet in one milliliter of complete LEC media. Then, plate the cells in complete LEC media in a collagen-coated 60-millimeter tissue culture plate. Replace the media every two days by washing cells with PBS twice and adding fresh LEC media.Low magnification images of cells isolated from the murine dermis showed a mixed cell population.

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.

791 Views.  Texas Tech University Health Sciences Center.  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. 

Isolating Endothelial Cells from Murine Dermal Lymphatic Capillaries

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