Isolation of Human Lymphatic Endothelial Cells by Multi-parameter Fluorescence-activated Cell Sorting

Summary

The goal of this protocol is to isolate lymphatic endothelial cells lining human lymphatic malformation cyst-like vessels and foreskins using fluorescence-activated cell sorting (FACS). Subsequent cell culturing and expansion of these cells permits a new level of experimental sophistication for genetic, proteomic, functional and cell differentiation studies.

Abstract

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity but little is known about their biology. Isolating highly pure human lymphatic endothelial cells (LECs) from diseased and healthy tissue would facilitate studies of the lymphatic endothelium at genetic, molecular and cellular levels. It is anticipated that these investigations may reveal targets for new therapies that may change the clinical management of these conditions. A protocol describing the isolation of human foreskin LECs and lymphatic malformation lymphatic endothelial cells (LM LECs) is presented. To obtain a single cell suspension tissue was minced and enzymatically treated using dispase II and collagenase II. The resulting single cell suspension was then labelled with antibodies to cluster of differentiation (CD) markers CD34, CD31, Vascular Endothelial Growth Factor-3 (VEGFR-3) and PODOPLANIN. Stained viable cells were sorted on a fluorescently activated cell sorter (FACS) to separate the CD34^LowCD31^PosVEGFR-3^PosPODOPLANIN^Pos LM LEC population from other endothelial and non-endothelial cells. The sorted LM LECs were cultured and expanded on fibronectin-coated flasks for further experimental use.

Introduction

A major function of the lymphatic vascular system is to absorb lymph, an excess interstitial fluid containing lipids, proteins and cellular components, and conduct it to the blood venous system. A network of lymphatic capillaries directs lymph to the lymph nodes where it is screened for presence of foreign antigens, an important process in immune surveillance and deployment of white blood cells to neutralize foreign antigens.

The uni-directional lymphatic system starts in tissues with the initial lymphatic capillary, a unique structure with a discontinuous single layer of thin-walled flat endothelial cells with specialized cell junctions th....

Protocol

Ethics statement: Ethical approval for collection of lymphatic malformation and foreskin tissues was obtained from the Human Research Ethics Committees at the Royal Children’s Hospital, Melbourne, Australia. Signed consent was received from patients’ parents prior to surgery. Tissue samples were collected from patients diagnosed with LMs undergoing surgical procedures as part of their clinical management and patients undergoing elective circumcision. All experiments were performed in accordance with guidelines of the National Health and Medical Research Council, Australia.

1. Preparation of Cell Suspension from Foreskin and Lympha....

Results

Following initial tissue digestion, after 24 hours in culture of unfractionated samples, distinct endothelial cell colonies can be observed (Figure 2A) together with fibroblast-like cells and smooth muscle cells. Following sorting and after 24 hours in cell culture, the CD34^LowCD31^PosVEGFR-3^PosPodoplanin^Pos cells attach and show typical cobblestone morphology (Figure 2B and C). Using the FACS method described above, we are able.......

Discussion

LECs play an important role in maintaining fluid homeostasis, immune response to foreign antigens and absorption and transport of some nutrients. LEC homeostasis can be affected by disease processes such as bacterial infections and tumor metastasis, but LECs can also develop somatic mutations that result in formation of dysfunctional lymphatic vessels and significant morbidity for affected patients. To gain more understanding of lymphatic malformation etiology through in vivo implantation and ex vivo ex.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DMEM	Life Technologies (Gibco)	11965-092	Used to collect tissue samples from the operating theatre.
EGM-2 MV Bullet Kit	Lonza	CC-3202	EGM-2 MV Bullet Kit contains 500 ml of EBM-2 media and human EGF, hydrocortisone, gentamycin (GA-1000), fetal bovine serum (FBS), VEGF, human FGF-b, R3-IGF-1 and ascorbic acid.
VEGF-C	R&D Systems	2179-VC-025	Complete endothelial cell media is supplemented with 50 ng/ml VEGF-C
Antibiotic-antimycotic solution (100x)	Life Technologies (Gibco)	15240-062	This solution contains 10,000 U/ml of penicillin, 10,000 mg/ml of streptomycin and 25 mg/ml of Fungizone antimycotic. Use at 1:100 dilution.
Fibronectin from human plasma	Sigma-Aldrich	F2006	Used at 10 mg/ml concentrations. It can be re-used for up to 1 month if kept at 4 °C and maintained sterile. Use 7 ml to coat 150 cm2 flask.
StemPro Accutase Cell dissociation reagent	Life Technologies (Gibco)	A11105-01	StemPro®Accutase® is used at 0.05 ml per cm2 to cover the entire surface area of the flask. Using lesser volumes may result in incomplete cell detachment.
0.4% Trypan Blue dye	Life Technologies (Gibco)	15250-061	Used as a viability stain.
Dispase II	Roche Applied Biosciences	4942078001	Used at 0.04%
Collagenase II	Worthington Lab	4176	Used at 0.25%
DNase I	Roche Applied Biosciences	11284932001	Used at 0.01%
70 mm nylon cell strainers	BD Falcon	352350
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PE-conjugated VEGFR-3 clone 9D9F9, clone WM59	BioLegend	356204	Used at 1:50 dilution
PE-Cy7-conjugated CD34, clone 581	BioLegend	343516	Used at 1:200 dilution
APC-conjugated mouse anti human CD31, clone WM59	BioLegend	303116,	Used at 1:100 dilution
Alexa 488-conjugated rat anti human Podoplanin, clone NC-80.	BioLegend	337006	Used at 1:200 dilution
PE-conjugated mouse IgG1, k isotype, clone MOPC-21	BioLegend	400112	Used at 1:50 dilution
PE-Cy7-conjugated mouse IgG1, k isotype, clone MOPC-21	BioLegend	400126	Used at 1:200 dilution
APC-conjugated mouse IgG1, k isotype, clone MOPC-21	BioLegend	400120	Used at 1:100 dilution
Alexa 488-conjugated mouse IgG2a, k isotype, clone RATK2758	BioLegend	400525	Used at 1:200 dilution